Voltage-gated calcium channels in autonomic neuroeffector transmission

Progress in Neurobiology 60 (2000) 181±210

www.elsevier.com/locate/pneurobio

Voltage-gated calcium channels in autonomic neuroe€ector transmission Sally A. Waterman* Department of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia Received 6 April 1999

Abstract Calcium in¯ux through voltage-gated calcium channels (VGCCs) is required for neurotransmitter release. Recent research has characterised several pharmacologically and electrophysiologically distinct VGCC subtypes, some of which are involved in neurotransmitter release. Transmitter release from autonomic neurons can be coupled to calcium entry through N-, P/Q- and/or R-type VGCCs; the precise combination of VGCC subtypes appears to vary according to the neurotransmitter, tissue and species. L-type channels rarely appear to be important in autonomic neurotransmitter release. There does not appear to be a general rule regarding the nature of the VGCCs coupled to release of a particular transmitter in di€erent tissues or species. Release of the same neurotransmitter from di€erent populations of neurons often reveals a di€erent pattern of involvement of VGCCs. Transmitters released from the same population of neurons are sometimes coupled to calcium in¯ux through di€erent VGCC subtypes. However, release of transmitters thought to be co-localised within vesicles is coupled to calcium in¯ux through the same VGCCs. The role of VGCC subtypes in transmitter release can be altered by mode of nerve stimulation. Di€erent VGCC subtypes may be coupled to transmitter release at low versus high electrical stimulation frequencies, or in response to potassium depolarization or chemical stimulation. In certain disease processes, voltage-gated calcium channels on autonomic neurons can be targeted; for example antibodies to P/Q-type VGCCs in Lambert±Eaton myasthenic syndrome downregulate VGCCs, thereby inhibiting autonomic neuroe€ector transmission. # 1999 Elsevier Science Ltd. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

2.

VGCC subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

3.

Role 3.1. 3.2. 3.3.

of VGCC subtypes in transmitter release from autonomic neurons Guinea-pig ileum longitudinal muscle . . . . . . . . . . . . . . . . . . . . Vas deferens longitudinal muscle . . . . . . . . . . . . . . . . . . . . . . . Whole animal studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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185 185 185 186

Abbreviations: ACh, Acetylcholine; ATP, Adenosine triphosphate; CGRP, Calcitonin gene-related peptide; CM, Circular muscle; DMPP, Dimethylphenylpiperazinium; EFS, Electrical ®eld stimulation; EJP, Excitatory junction potential; EPSP, Excitatory postsynaptic potential; FTX, Funnel web spider toxin; GPI, Guinea-pig ileum; HPLC-ECD, High performance liquid chromatography with electrochemical detection; 5-HT, 5-Hydroxytryptamine (serotonin); IJP, Inhibitory junction potential; IPSP, Inhibitory postsynaptic potential; LM, Longitudinal muscle; NA, Noradrenaline; NANC, Non-adrenergic, non-cholinergic; NO, Nitric oxide; NPY, Neuropeptide Y; NSF, N-Ethylmaleimide-sensitive fusion protein; RIA, Radioimmunoassay; SNAP, Soluble NSF attachment protein; SNARE, SNAP receptor; SP, Substance P; TK, Tachykinins; TTX, Tetrodotoxin; VGCC, Voltage-gated calcium channel; VIP, Vasoactive intestinal peptide. * Fax: +61-8-8303-3356. E-mail address: [email protected] (S.A. Waterman) 0301-0082/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 - 0 0 8 2 ( 9 9 ) 0 0 0 2 5 - 8

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4.

Comparison of the role of VGCCs in transmitter release in di€erent species. 4.1. Sympathetic innervation of the atrium . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Mouse atrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Rat atrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3. Guinea-pig atrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Parasympathetic innervation of the bladder detrusor muscle . . . . . . . 4.2.1. Rat detrusor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Guinea-pig detrusor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Rabbit detrusor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4. Mouse detrusor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.5. Human detrusor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Sympathetic innervation of the vas deferens longitudinal muscle . . . .

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186 188 188 188 189 189 189 190 190 190 191 191

5.

Is release of a particular transmitter always coupled to the same VGCC in the autonomic nervous system?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. ATP release from autonomic neurons in the mouse . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Parasympathetic neurons in the bladder . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2. Sympathetic neurons in the vas deferens . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.3. Enteric inhibitory motor neurons in the colon . . . . . . . . . . . . . . . . . . . . . 5.2. NA release from autonomic neurons in the rat . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Sympathetic neurons in the vas deferens . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Sympathetic neurons in the anococcygeus . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3. Sympathetic neurons in the atrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4. Sympathetic neurons in mesenteric resistance arteries . . . . . . . . . . . . . . . . 5.3. Acetylcholine release from autonomic neurons in the guinea-pig . . . . . . . . . . . . . . 5.3.1. Parasympathetic neurons in the atrium . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. Parasympathetic neurons in the trachea . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3. Longitudinal muscle motor neurons in the ileum . . . . . . . . . . . . . . . . . . . 5.3.4. Secretomotor neurons in the ileum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.5. Circular muscle motor neurons in the colon . . . . . . . . . . . . . . . . . . . . . . 5.3.6. Longitudinal muscle motor neurons in the colon . . . . . . . . . . . . . . . . . . . 5.3.7. Motor neurons in the gall bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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191 191 191 191 192 192 192 192 192 193 193 193 193 193 193 193 193 194 194

6.

The role of VGCC subtypes in co-transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Acetylcholine and ATP co-transmission in the mouse urinary bladder . . . . . . . . . . . . 6.2. Noradrenaline and ATP co-transmission in the mouse vas deferens . . . . . . . . . . . . . . 6.3. Nitric oxide and ATP co-transmission in enteric inhibitory motor neurons in the mouse colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Acetylcholine and substance P co-transmission in enteric excitatory motor neurons in guinea-pig colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

194 194 195 195 196

7.

VGCCs and neuropeptide release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 7.1. Neuropeptide release from sensory neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 7.2. Neuropeptide release from autonomic neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

8.

Mode of nerve stimulation and the nature of VGCCs involved in transmitter release . 8.1. Potassium stimulation of transmitter release . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Chemical stimulation of transmitter release . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1. Nicotinic receptor stimulated transmitter release-tetrodotoxin sensitive 8.2.2. Nicotinic receptor stimulated transmitter release-tetrodotoxin resistant 8.3. Electrical stimulation of transmitter release . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4. VGCCs required for non-facilitated and facilitated transmitter release . . . . . . .

9.

Sensitivity of VGCCs to toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

10.

VGCCs in disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 10.1. Mutations in VGCC subunits in mouse and human . . . . . . . . . . . . . . . . . . . . . . . . . 206 10.2. Diseases mediated by antibodies to VGCCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

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11.

Role of VGCCs in other neuronal functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

12.

Concluding points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

1. Introduction Transmitter release is dependent on calcium entry into the nerve terminal through voltage-gated calcium channels (VGCCs). It is now clear that there are several subtypes of VGCCs and that the relative importance of each subtype in neurotransmitter release is di€erent, and varies according to the neurotransmitter, synapse, tissue and species. This review will concen-

trate on the nature of the VGCCs required for neurotransmitter release from autonomic neurons, and will question whether there are any general principles regarding the involvement of particular VGCC subtypes in release of particular transmitters in di€erent tissues and species. Over the last 10 years, enormous advances have been made in the molecular biology of calcium channels, and it is now well established that they contain

Fig. 1. Schematic diagram showing subunit composition of voltage-gated calcium channels and associated SNARE proteins. The a1 subunit forms the pore of the channel and is associated with an intracellular b subunit and an extracellular a2b subunit. The b subunit binds to the a1 subunit in the cytoplasmic linker region between domains I and II [see De Waard et al. (1995) for details]. There is evidence that this interaction occurs speci®cally with the a1A and a1B subunits that form the pores of P/Q- and N-type channels respectively (see Table 1); there is assumed to be a similar interaction with the a1 subunits in L-type channels. T-type channels may be an exception; these channels may be formed by an a1 subunit alone (see Perez-Reyes, 1998). Exocytosis involves the coupling of a rapid increase in cytoplasmic calcium concentration to fusion of synaptic vesicles with the nerve terminal membrane. This process is thought to involve the formation of complexes of target and vesicle membrane proteins (t- and v-SNARES) and calcium channels. Syntaxin and SNAP-25 are t-SNARE proteins that can bind to a1A and a1B subunits via the synprint site on the cytoplasmic linker between domains II and III of the a1 subunit [see Bezprozvanny et al. (1995); Sheng et al. (1998) for details]. Syntaxin probably does not bind to the a1 subunits of L-type calcium channels (see Sheng et al., 1998). Synaptotagmin is a vSNARE protein that interacts with both syntaxin and the a1 subunit of calcium channels [see Sheng et al. (1998) for review].

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multiple subunits (Fig. 1): a pore-forming a1 subunit, together with an intracellular b subunit and a membrane anchored, extracellular a2d subunit. In skeletal muscle, there is an additional intracellular g subunit. A g2 subunit has recently been described in brain (see Dunlap et al., 1995; Letts et al., 1998; Scott et al., 1998), although it is presently unknown whether this subunit is also present in peripheral neuronal calcium channels. The electrophysiological and pharmacological characteristics of the channels depend on the precise combination of subunits present. The a1 subunit is the main determinant of electrophysiological characteristics, and contains the toxin binding site. Ten a1 subunits have been described to date (see Table 1) and four b subunit isoforms, each with splice variants. Voltage-gated calcium channels are divided into high and low voltage-activated types, depending on the degree of membrane potential depolarization required to open the channels. High voltage-activated channels are further divided into L, N, P, Q and R subtypes on the basis of electrophysiological and pharmacological characteristics (Dunlap et al., 1995; Randall and Tsien, 1995). Transmitter release requires calcium in¯ux through high voltage-activated channels. Low voltageactivated, T-type channels appear to be important in cell excitability, rather than transmitter release (Olivera et al., 1994; Perez-Reyes, 1998), and will not be considered further. It is well known that multiple calcium channels coexist in nerve terminals and control transmitter release from central neurons (Dunlap et al., 1995). Recent studies have demonstrated that this is also true of autonomic neurons. This review discusses the role of high voltage-activated calcium channels in transmitter release from mammalian autonomic neurons.

2. VGCC subtypes The identi®cation of VGCC subtypes required for neurotransmitter release has relied on the use of toxins and antagonists that block speci®c channel subtypes. o-Conotoxin GVIA blocks only N-type channels, and its use has provided much information on the role of N-type channels in transmitter release in many tissues. o-Conotoxin MVIIC blocks P- and Q-type channels, and in some tissues, N-type channels. Provided that oconotoxin MVIIC is added after N-type channels have been blocked, it can be used as a speci®c blocker of P/ Q-type channels. R-type channels are resistant to each of these drugs, but are sensitive to SNX-482 (Newcomb et al., 1998) and may be partly sensitive to o-grammotoxin SIA (Lampe et al., 1993). In all tissues studied to date, these toxins do not appear to inhibit responses of the smooth muscle to direct stimulation (e.g. Altiere et al., 1992; Boot, 1994; De Luca et al., 1990; Hong et al., 1996; Maggi, 1991; Maggi et al., 1994Maggi et al., 1988b; Pruneau and Angus, 1990b; Tran and Boot, 1997; Waterman, 1996Waterman, 1997; Zygmunt et al., 1993). An e€ect of the toxin in smooth muscle preparations can therefore be ascribed to an e€ect on neuronal calcium channels. Native P- and Q-type channels were originally described as separate channels in cerebellar Purkinje cells (LlinaÂs et al., 1989) and cerebellar granule cells respectively (Randall and Tsien, 1995). Typical P-type channels are blocked by low nanomolar concentrations of o-agatoxin IVA and have slowly inactivating currents, whereas Q-type channels are sensitive to higher concentrations of o-agatoxin IVA and have a faster rate of inactivation. Expression of the a1A subunit with b subunits in Xenopus oocytes demonstrated that the combination of a1A with b1b or b3 subunits pro-

Table 1 Subtypes of voltage-gated calcium channels Channel subtype

a1 subunit

Threshold for activation

Toxin/antagonist

P Q N

a1A a1A a1B

High High High

L L R ?L T T ?T L

a1C a1D a1E ab1F a1G a1H ac1I a1S

High High High High Low Low Low High

Low nM concentrations of agatoxin IVA o-conotoxin MVIIC High nM concentrations of o-agatoxin IVA o-conotoxin MVIIC o-Conotoxin GVIA o-Conotoxin MVIIC (some N-type VGCCs are resistant, see text) Dihydropyridines Dihydropyridines SNX-482a ?Dihydropyridines Mibefradil Mibefradil Ð Dihydropyridines

a

Newcomb et al. (1998). Bech-Hansen et al. (1998). c Cribbs et al. (1998).

b

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duced a current with characteristics similar to Q-type currents, whereas coexpression of a1A and a2d subunits produced a P-like current (Stea et al., 1994). In contrast, a P-like current was produced in human embryonic kidney (HEK) cells by expression of a1A and a2d subunits with the b1b subunit, and not the b2a or b3 subunits (Moreno et al., 1997). A recent study demonstrated that antibodies speci®c to the a1A subunit inhibit both P-type currents in cerebellar Purkinje cells and Q-type currents in cerebellar granule cells (Pinto et al., 1998). It is therefore likely that the a1A subunit is the pore-forming region of both P- and Q-type VGCCs, and that the di€erence between the currents in some neurons may re¯ect di€erent b subunits, di€erent phosphorylation, or di€erent splice variants of the a1A subunit. For this reason, P- and Q-type channels will be considered in this review as a single channel subtype (P/Q-type) with variable characteristics rather than distinct channel subtypes. 3. Role of VGCC subtypes in transmitter release from autonomic neurons Since the speci®c N-type channel blocker, o-conotoxin GVIA, became widely available in the 1980s, many studies have shown that transmitter release from autonomic neurons is abolished by the toxin. It has therefore become widely accepted that N-type channels are essential for autonomic neurotransmitter release, and even that these are the only channels required for autonomic neurotransmitter release. However, recent studies using a combination of toxins have shown that this is not generally true and that release of some neurotransmitters is independent of N-type VGCCs, or that N-type VGCCs, along with other channel subtypes mediate transmitter release. 3.1. Guinea-pig ileum longitudinal muscle The gastrointestinal tract and vas deferens have been widely used to study transmitter release. The ®rst studies using o-conotoxin GVIA in guinea-pig ileum (GPI) longitudinal muscle (LM) preparations found that electrically evoked twitch responses mediated by acetylcholine (ACh) were abolished by o-conotoxin GVIA (Boot, 1994; Hong et al., 1996; Humphreys and Costa, 1992; Lundy and Frew, 1988Lundy and Frew, 1993, 1994; Tran and Boot, 1997). Enteric neurons were stimulated continuously by electrical ®eld stimulation (EFS) at very low frequencies (0.1±0.2 Hz), and under these stimulation conditions, funnel web spider toxin (FTX, a P/Q-type channel blocker) did not signi®cantly alter twitch responses (Lundy and Frew, 1993). Furthermore the e€ect of the N- and P/Q-type channel blocker, o-conotoxin MVIIC, was not di€er-

185

ent to that of o-conotoxin GVIA, suggesting that only N-type channels are involved in transmitter release in this preparation (Boot, 1994). However stimulating the neurons at higher but still physiological frequencies, revealed a component of the response that was resistant to o-conotoxin GVIA but sensitive to o-conotoxin MVIIC, indicating a role for P/Q-type VGCCs (Hong et al., 1996; Tran and Boot, 1997). At these frequencies, the contractile response is only partly blocked by muscarinic receptor antagonists and thus only partly cholinergic (Hong et al., 1996; Tran and Boot, 1997). The remaining contraction is produced by tachykinins released from enteric LM motor neurons (Holzer and Holzer-Petsche, 1997). In each of the studies using higher stimulation frequencies, the e€ect of the calcium channel toxins was examined on the whole contraction, mediated by the combined actions of ACh and tachykinins. Consequently, it is not clear whether the P/Qtype channels are involved in ACh or tachykinin release, or both. Further studies in which the e€ect of the toxins on responses mediated by ACh alone (in the presence of tachykinin receptor antagonists) or tachykinins alone (in the presence of atropine) need to be undertaken. 3.2. Vas deferens longitudinal muscle The LM of the vas deferens is innervated by postganglionic sympathetic neurons that release noradrenaline (NA), adenosine triphosphate (ATP) and neuropeptide Y (NPY) (Morris and Gibbins, 1992). Electrical stimulation of the neurons produces a contractile response that is inhibited by a combination of antagonists to a1 adrenoceptors and P2X purinoceptors. The predominant e€ect of NPY appears to be presynaptic inhibition of NA and ATP release (Morris and Gibbins, 1992). Although contractile responses are produced by NA and ATP, membrane potential changes in the vas deferens smooth muscle are produced only by ATP (Brock and Cunnane, 1992). Studies using organ bath pharmacological methods and intracellular electrophysiology have investigated the nature of the calcium channels mediating transmitter release from sympathetic neurons in the vas deferens. At stimulation frequencies of 0.1 Hz, twitch responses in the rat vas deferens are abolished by oconotoxin GVIA (Maggi et al., 1988b). Responses in rat, guinea-pig and mouse vas deferens at frequencies of 0.5±50 Hz are reduced and in some reports, abolished by the toxin (Boot, 1994; Brock et al., 1989; De Luca et al., 1990; Maggi et al., 1988b). These studies suggested that N- and non-N-type channels are required for transmitter release from sympathetic neurons, although the relative role of the VGCC subtypes in the release of ATP and NA was not clear,

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since the whole contraction was measured. An electrophysiological study in the guinea-pig vas deferens provided evidence that (purinergic) excitatory junction potentials (EJP) are abolished by o-conotoxin GVIA at stimulation frequencies up to 4 Hz, but that responses can be evoked in the presence of the toxin at higher stimulation frequencies (Brock et al., 1989), suggesting a role for N- and non-N-type channels in ATP release. The nature of the non-N-type channels has now been investigated in some species. Studies in the mouse vas deferens in which noradrenergic and purinergic responses were studied separately, showed that at stimulation frequencies below 5 Hz, contractions produced by either transmitter were abolished by oconotoxin GVIA. At higher stimulation frequencies, the o-conotoxin GVIA-resistant contractions were abolished by o-agatoxin IVA and o-conotoxin MVIIC, demonstrating that P/Q-type channels are involved in the release of NA and ATP from sympathetic neurons in the mouse vas deferens (Waterman, 1997). Intracellular electrophysiological recordings from mouse vas deferens have con®rmed that ATP release from this tissue is dependent on Ntype channels at low stimulation frequencies and P/Qtype channels at higher frequencies (Fig. 2, Fig. 3). Further evidence for a role of P/Q-type channels in transmitter release from sympathetic neurons in mouse vas deferens has been provided by Wright and Angus (1996), although the role of the channel subtypes in release of NA and ATP separately was not investigated. Although some studies found no e€ect of o-agatoxin IVA or of o-conotoxin MVIIC alone on contractile responses to 0.1±0.2 Hz stimulation in rat vas deferens (Boot, 1994; Lundy and Frew, 1994), more recent studies in which these toxins have been added after blockade of N-type channels show that P/Q-type channels are indeed involved in transmitter release at higher stimulation frequencies in this preparation (Tran and Boot, 1997; Wright and Angus, 1996). Transmitter release was abolished by blocking N- and P/Q-type channels, although the relative role of the channel subtypes in the release of NA and ATP was not investigated. In contrast to studies in the mouse and rat vas deferens in which N- and P/Q-type channels are sucient to mediate maximal transmitter release, a component of transmitter release coupled to non-N- and non-P/Qtype channels was reported in guinea-pig vas deferens (Smith and Cunnane, 1996). This component was sensitive to o-grammotoxin SIA, which blocks a component of R-type currents, in addition to other channels. In guinea-pig vas deferens, Smith and Cunnane (1996) found no evidence for a role of P/Qtype channels.

3.3. Whole animal studies Most of our current understanding of the role of VGCCs in autonomic neuroe€ector transmission comes from studies in vitro. There have, however, been a few studies investigating the e€ect of blocking N-type VGCCs in vivo. Treatment of rabbits with 3 or 10 mg/kg o-conotoxin GVIA intravenously results in a selective abolition of sympathetic control of the heart and blood vessels (Pruneau and Angus, 1990c). Decreased heart contractility results in a decreased cardiac output and together with the loss of sympathetically mediated vasoconstriction, causes a drop in blood pressure; the subsequent parasympathetic withdrawal produces a tachycardia. The sympathetic component of the baroreceptor re¯ex is lost, but not the parasympathetic component (Pruneau and Angus, 1990c). These results are in keeping with in vitro studies in which N-type VGCCs are required for NA release from sympathetic neurons in the heart, but not for ACh release from parasympathetic neurons in the heart (see Sections 5.1 and 6.3). As in the rabbit, treatment of pithed rats in vivo with 1.6 or 3.2 mg/kg oconotoxin GVIA intravenously inhibited the responses to sympathetic nerve stimulation but not the responses to intravascular NA (Pruneau and Angus, 1990a). The e€ects of a single dose of o-conotoxin GVIA (10 mg/kg intravenously) have also been examined over longer periods. After 2 h, the toxin caused a reduction in blood pressure, increased heart rate, attenuation of the barore¯ex curve and postural hypotension (Wright and Angus, 1997), as was reported previously after 30 min (Pruneau and Angus, 1990c). However 24 h after administration of the toxin, blood pressure and heart rate were normal, although the barore¯ex curves was still attenuated and postural hypotension was evident (Wright and Angus, 1997). By 96 h, there was a steady decrease in blood pressure and heart rate, and a normal response to head-up tilt. After 186 h, blood pressure, heart rate and barore¯ex responses were similar to controls (Wright and Angus, 1997). Wright and Angus (1997) concluded that the sympatholytic and vagolytic e€ects of the toxin occurred during the ®rst 48 h and were followed by a second phase of hypotension and bradycardia, presumably due to indirect mechanisms. 4. Comparison of the role of VGCCs in transmitter release in di€erent species Experiments to identify the nature of calcium channels controlling autonomic neurotransmitter release have been undertaken in a range of species, and it is now clear that there are species di€erences in the subtypes of VGCC subserving release of a particular transmitter in a speci®ed tissue. As examples, studies

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187

Fig. 2. E€ect of calcium channel toxins on sympathetic transmission in the mouse vas deferens. Traces show membrane potential recorded using intracellular microelectrodes. Sympathetic neurons were stimulated electrically with trains of 10 pulses. ., Stimulus artifact; , spontaneous transmitter release (spontaneous excitatory junction potential). Traces in (a) and (b) were recorded in the absence of calcium channel toxins. oConotoxin GVIA (30 nM) was present in (c)±(e) and o-conotoxin MVIIC (300 nM) was present in (f). Stimulus parameters were (a) 1 Hz, amplitude 6 V, pulse duration 0.15 msec, (b) 5 Hz, 5 V, 0.15 msec, (c) 5 Hz, 10 V, 0.3 msec; (d) 20 Hz, 10 V, 0.3 msec, (e) 50 Hz, 20 V, 0.3 msec, (f) 20 Hz, 10 V, 0.3 msec.

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Fig. 3. Amplitude of excitatory junction potentials (mediated by ATP acting at P2X purinoceptors) recorded in mouse vas deferens in the absence and presence of 30 nM o-conotoxin GVIA to block N-type voltage-gated calcium channels. Control responses are shown by ®lled symbols, responses in the presence of toxin are shown by open symbols. Q, Responses to 1 Hz stimulation (10 V, 0.15 msec); q, responses to 50 Hz stimulation (10 V, 0.15 msec); r, responses to 20 Hz stimulation (10 V, 0.3 msec); , responses to 10 Hz stimulation (10 V, 0.3 msec); w, responses to 1 Hz stimulation (10 V, 0.15 msec).

on the atrium, bladder and vas deferens will be reviewed. 4.1. Sympathetic innervation of the atrium The atrium of the heart is innervated by postganglionic sympathetic neurons that contain NA and a variety of co-transmitters including NPY and the opioid peptides, enkephalin and dynorphin (Morris and Gibbins, 1992). Stimulation of these ®bres increases heart rate, contractility and force of atrial contraction; these responses can be largely abolished by the b-adrenoceptor antagonist, propranolol. 4.1.1. Mouse atrium Noradrenergic positive chronotropic and inotropic responses in the spontaneously beating mouse right atrium are abolished by low (10 nM) concentrations of o-conotoxin GVIA at stimulation frequencies of <2 Hz (De Luca et al., 1990), demonstrating a clear role for N-type channels in NA release. Over¯ow of ‰3 HŠNA was inhibited in parallel by the toxin (De Luca et al., 1990). However, the same concentration of o-conotoxin GVIA produces only 55% inhibition of the response if stimulation frequency is increased to 8 Hz (De Luca et al., 1990). These authors did not investigate e€ects of higher concentrations of the toxin. A more recent study on spontaneously beating mouse right atrium found responses to stimulation with up to four pulses at 90 Hz during the atrial refractory period were abolished by 10±100 nM o-conotoxin GVIA (Wright and Angus, 1996). However, responses to stimulation with up to 64 pulses were reduced by only

50% (Wright and Angus, 1996). Although N-type channels are therefore important in NA release, nonN-type channels must also play a role. The same investigators found that 1000 nM o-conotoxin MVIIC inhibited responses to stimulation with 64 pulses by nearly 80%. The signi®cantly greater inhibition produced by o-conotoxin MVIIC compared to o-conotoxin GVIA under identical stimulation conditions suggests that P/Q-type channels are also involved in NA release. The e€ect of cumulative addition of the two toxins on noradrenergic responses in the atria was not investigated. 4.1.2. Rat atrium In spontaneously beating rat atria, noradrenergic responses to low frequency electrical stimulation are abolished by 10 nM o-conotoxin GVIA, although at a frequency of 8 Hz, the toxin reduces the response by only 35% (De Luca et al., 1990). Higher concentrations of the toxin were not tested. Wright and Angus (1996) tested the e€ect of up to 100 nM o-conotoxin GVIA on noradrenergic responses evoked by stimulation during the atrial refractory period in spontaneously beating right atria. Responses evoked by up to four pulses were abolished by the toxin, con®rming the role of N-type channels in NA release in rat atrium. However, 30% of the positive chronotropic response to 64 pulses remained in the presence of the toxin. Since this concentration of toxin is considered maximal for blocking N-type channels, the resistant responses imply a role for non-N-type VGCCs in NA release. In separate experiments, o-conotoxin MVIIC caused signi®cantly less inhibition of the responses

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than had o-conotoxin GVIA. Thus in response to 64 pulse stimulation, 1000 nM o-conotoxin MVIIC decreased the positive chronotropic response by only 50% (Wright and Angus, 1996). The observation that the N-type channel blocker had a larger e€ect than the N- and P/Q-type channel blocker is similar to a ®nding reported by Boot (1994) in rat vas deferens, and suggests that there is a population of N-type VGCCs that is insensitive to o-conotoxin MVIIC (see Section 9). Although NA release in the rat atrium is only partly inhibited by o-conotoxin GVIA under some stimulation conditions, the identity of the channel subtype(s) coupled to the remaining transmitter release is uncertain. The small e€ect of o-conotoxin MVIIC demonstrated by Wright and Angus (1996) may indicate that P/Q-type channels play a role, although the de®nitive experiment of adding the two toxins in combination remains to be performed. 4.1.3. Guinea-pig atrium Hong and Chang (1995) investigated the role of calcium channel subtypes in transmitter release in paced guinea-pig atria. Stimulation of the sympathetic nerves in bursts of four pulses at 200 Hz per pacing pulse increased contractile force. This response was abolished by o-conotoxin GVIA with an IC50 value of 420 250 nM, indicating that N-type channels alone are sucient to mediate NA release. Nevertheless, the IC50 in this preparation is considerably greater than that reported for other tissues (comparable values in the rat and mouse atrium are an order of magnitude less). Whether this represents a true species di€erence or a di€erence in methods used, is unclear. o-Conotoxin MVIIC also signi®cantly inhibited noradrenergic responses in the guinea-pig atrium, although 9% of the response remained in the presence of 5 mM toxin. This toxin was not added in the presence of o-conotoxin GVIA, so it is not possible to deduce whether the action of o-conotoxin MVIIC in this tissue is due to blockade of N-type and/or P/Q-type VGCCs. Nevertheless, since o-conotoxin MVIIC caused less inhibition than o-conotoxin GVIA, it is likely that this tissue contains N-type channels that are insensitive to o-conotoxin MVIIC; if all N-type channels on sympathetic nerve terminals in the guinea-pig atrium were sensitive to both toxins, one would expect the toxins to cause the same degree of inhibition. A study on guinea-pig perfused heart in situ (Haass et al., 1990) con®rmed the ®nding that N-type channels are crucial for NA release; stimulation of the sympathetic ®bres to the heart at 12 Hz evoked over¯ow of ‰3 HŠNA (and NPY) that was virtually abolished by 100 nM o-conotoxin GVIA. In contrast to the e€ect of the toxins on electrically evoked NA release, Hong and Chang (1995) found

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that responses evoked by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) were relatively insensitive to either o-conotoxin GVIA or oconotoxin MVIIC. Noradrenergic responses were inhibited only by 29% by 10 mM o-conotoxin GVIA and/or 3 mM o-conotoxin MVIIC. Signi®cant NA release can thus occur in the presence of N- and P/Qtype channel blockade. Since responses were also insensitive to the L-type channel blocker, nifedipine, it unlikely that L-type channels are involved. The nature of the channels mediating this NA release is uncertain. The e€ect of di€erent modes of nerve stimulation on the roles of calcium channel subtypes in transmitter release is discussed in Section 6. In summary, the importance of N-type VGCCs in sympathetic neurotransmitter release clearly varies between species, playing a large role in guinea-pig and progressively smaller roles in mouse and rat. P/Q-type VGCCs play a role in sympathetic neurotransmission in mouse atrium, but there is currently no evidence that they play a role in the rat or guinea-pig. 4.2. Parasympathetic innervation of the bladder detrusor muscle The detrusor muscle of the bladder is innervated by postganglionic parasympathetic neurons that release ACh and ATP (Morris and Gibbins, 1992). These neurons also contain peptides such as NPY, substance P and somatostatin, the precise combination varying between species. The peptides do not appear to mediate neuroe€ector transmission in the bladder, rather they act pre- or postjunctionally to modify the release of ACh and ATP (Morris and Gibbins, 1992). To date, studies have investigated the role of calcium channels subtypes only in ACh and ATP release in the bladder. 4.2.1. Rat detrusor The ®rst study in rat detrusor showed that combined cholinergic and purinergic responses to low frequency (0.1 Hz) nerve stimulation are only inhibited 25% by o-conotoxin GVIA (Maggi et al., 1988b). o-conotoxin GVIA caused greater inhibition at a higher stimulation frequency of 5 Hz (50% inhibition), however, further increases in stimulation frequency coincided with less inhibition (10% inhibition at 50 Hz stimulation). A similar pattern was reported by De Luca et al. (1990). ACh over¯ow from parasympathetic nerves rat bladder in response to 20 Hz stimulation was also only partly (50%) blocked by o-conotoxin GVIA and was insensitive to nifedipine (Somogyi et al., 1997). Thus the majority of transmitter release in this preparation is not coupled to calcium in¯ux through nerve terminal N-type VGCCs, and there is no role for L-type channels under these stimulation conditions.

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Maggi et al. (1988b) tested the e€ect of o-conotoxin GVIA on the purinergic component of transmission (in the presence of atropine). The toxin signi®cantly inhibited purinergic responses at stimulation frequencies up to 5 Hz, but not at higher stimulation frequencies. At all stimulation frequencies, a signi®cant component of the response remained in the presence of the toxin. Thus, N-type channels partly mediate ATP release at low stimulation frequencies; non-N-type channels are coupled to ATP release at all stimulation frequencies, but play a greater role at frequencies >5 Hz. Together with a later study (Maggi, 1991), Maggi and colleagues have demonstrated that cholinergic neurotransmission in rat detrusor is more sensitive to o-conotoxin GVIA than the purinergic component, and that ACh release is coupled to calcium in¯ux through non-N-type channels only at stimulation frequencies above 5 Hz (Maggi et al., 1988b). The nature of the non-N-type channels was not investigated in this study, as o-conotoxin MVIIC was not available at the time. A recent study by Frew and Lundy (1995) investigated the nature of the calcium channels involved in the combined response to ACh and ATP in rat bladder evoked by 5 Hz stimulation. Consistent with previous ®ndings (Maggi, 1991; Maggi et al., 1988b), o-conotoxin GVIA alone inhibited responses by only 43%. Subsequent addition of o-conotoxin MVIIC (3 mM) or o-agatoxin IVA (3 mM) caused further signi®cant inhibition, leaving only 18±20% of the control response. Thus P/Q-type VGCCs mediate a large component of transmitter release from parasympathetic neurons in the rat bladder. Frew and Lundy did not investigate the role of the channel subtypes in release of ACh and ATP separately. Nevertheless, 80% of the response in their experiments was purinergic and since o-conotoxin MVIIC inhibited responses by >40%, part of that e€ect must have been due to inhibition of ATP release. It is tempting to predict that the component of cholinergic transmission coupled to non-N-type channels at stimulation frequencies >5 Hz described by Maggi et al. (1988b) also involves P/Q-type VGCCs. However, experiments to test this have not yet been performed. In the rat detrusor therefore, N-type channels have a relatively small role in ACh and ATP release. P/Qtype channels are involved in ATP release, and possibly in ACh release. The relative importance of the channel subtypes varies with stimulation frequency and is di€erent for the two transmitters. 4.2.2. Guinea-pig detrusor Stimulation of strips of guinea-pig detrusor muscle evokes a contraction which is largely atropine-resistant, and probably mediated by ATP (Maggi et al., 1988b). Purinergic responses evoked by 0.1 Hz stimulation were decreased 70±80% by o-conotoxin GVIA.

Thus N-type VGCCs are the principal channels required for ATP release in the guinea-pig bladder, with a relatively small component involving non-Ntype channels whose identity remains unknown. The nature of the VGCCs required for ACh release in this preparation and at a range of stimulation frequencies has not been investigated. 4.2.3. Rabbit detrusor Stimulation of parasympathetic nerves in the rabbit detrusor at 10 Hz evokes a response that is ca 70% cholinergic and 30% purinergic (Zygmunt et al., 1993). o-conotoxin GVIA inhibited the combined contractile responses and purinergic responses. The proportion of the response that was resistant to o-conotoxin GVIA increased with stimulation frequency, such that at 30 Hz stimulation, 47% of the response remained. o-conotoxin GVIA caused the same maximal inhibition of the total and purinergic responses at 10 Hz stimulation (85-88%), although the IC50 values varied signi®cantly (2 nM for combined response and 6 nM for purinergic response). 4.2.4. Mouse detrusor The role of VGCC subtypes in mediating transmitter release from parasympathetic neurons in the mouse bladder has been studied more thoroughly than in other species. Contraction of the mouse bladder dome produced by ACh and ATP release from parasympathetic nerves is signi®cantly decreased by o-conotoxin GVIA, demonstrating an important role for N-type channels (Waterman, 1996). The inhibitory e€ect of oconotoxin GVIA decreased as stimulation frequency was increased such that at 50 Hz stimulation, 55% of the response remained. o-conotoxin MVIIC alone also inhibited the combined cholinergic and purinergic responses, but the maximal inhibition was signi®cantly greater than that produced by o-conotoxin GVIA, suggesting that P/Q-type VGCC may be involved in transmitter release. The IC50 values for inhibition by oconotoxin MVIIC (0200 nM) were signi®cantly greater than those for o-conotoxin GVIA (030 nM), except at a stimulation frequency of 50 Hz (IC50 for GVIA >100 nM; IC50 for MVIIC 470 nM). Sequential addition of o-conotoxin GVIA, o-agatoxin IVA and oconotoxin MVIIC con®rmed that the component of transmitter release not coupled to N-type VGCCs involved P/Q-type VGCCs (Waterman, 1996). To investigate whether the N- and P/Q-type channels had di€erential roles in the release of ACh and ATP, identical experiments were performed in which responses to ACh or ATP were isolated in the presence of antagonists to P2X purinoceptors or muscarinic receptors, respectively (Waterman, 1996). Cholinergic responses were inhibited by o-conotoxin GVIA by 32± 56% at stimulation frequencies from 1±50 Hz. The

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remaining responses were largely blocked by o-agatoxin IVA and o-conotoxin MVIIC. This suggests that ACh release depends primarily on calcium in¯ux through N-type VGCCs, and to a lesser extent, on P/ Q-type VGCCs. In contrast, purinergic responses were decreased by only 7±15% at stimulation frequencies up to 5 Hz and by 25±41% at stimulation frequencies of 10±50 Hz by o-conotoxin GVIA. The large resistant responses were abolished by the combination of o-agatoxin IVA and o-conotoxin MVIIC. Thus ATP release depends primarily on calcium in¯ux through P/Q-type channels, with a relatively small role for N-type VGCCs (Waterman, 1996). 4.2.5. Human detrusor Parasympathetic responses in human bladder di€er from those in many other species in that they are largely atropine-sensitive (Maggi et al., 1989; Morris and Gibbins, 1992). Electrically evoked cholinergic responses in the bladder dome were abolished by 100 nM o-conotoxin GVIA, indicating that N-type channels are involved in ACh release from human parasympathetic nerve terminals (Maggi et al., 1989). 4.3. Sympathetic innervation of the vas deferens longitudinal muscle The release of NA and ATP from sympathetic neurons in the vas deferens of rat, guinea-pig and mouse has been reviewed above. In each species, contractile responses mediated by the combined actions of NA and ATP are inhibited by o-conotoxin GVIA, demonstrating a role for N-type channels. In mouse vas deferens, there is clear electrophysiological and pharmacological evidence that the remaining release of NA and ATP is coupled to calcium in¯ux through P/ Q-type VGCCs (Waterman, 1997; Wright and Angus, 1996). These channels mediate release of transmitter sucient to cause 50% of the maximal response. Nand P/Q-type VGCCs are also involved in sympathetic responses in rat vas deferens, although the N-type VGCCs appear to play a relatively a larger role than in the mouse [80-90% of responses at 20 Hz in rat versus 50% in mouse; Tran and Boot (1997); Waterman (1997); Wright and Angus (1996)], and P/Q-type channels a smaller role. In contrast to mouse and rat, the component of ATP release in guinea-pig vas deferens resistant to o-conotoxin GVIA is also resistant to oconotoxin MVIIC. Thus P/Q-type channels are not involved in ATP release in this species. 5. Is release of a particular transmitter always coupled to the same VGCC in the autonomic nervous system? The evidence reviewed above indicates that there is

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signi®cant species variation in the role of speci®c calcium channel subtypes in mediating release of neurotransmitter from particular neuronal populations. However, within a single species, is there a consistent pattern in the nature of VGCC subtypes required for release of a particular transmitter from di€erent neuronal populations? The VGCCs required for the release of ATP, NA and ACh will be reviewed. 5.1. ATP release from autonomic neurons in the mouse ATP is a neurotransmitter widely distributed in autonomic neurons. It is colocalised with ACh in postganglionic parasympathetic neurons innervating the bladder detrusor and with NA in postganglionic sympathetic neurons innervating the vas deferens. In each of these tissues, ATP acts on P2X purinoceptors and causes contraction of the target organ. ATP is also present in enteric inhibitory motor neurons innervating the circular muscle (CM) of the intestine and colon, where it acts on P2Y receptors to cause relaxation of the smooth muscle. 5.1.1. Parasympathetic neurons in the bladder ATP release from parasympathetic neurons in the mouse bladder in response to electrical stimulation is predominantly coupled to calcium in¯ux through P/Qtype channels (see Section 3.2). The role of P/Q-type channels in ATP release is greatest at moderate frequencies of nerve stimulation (50±65% inhibition of responses to 2±10 Hz) and less at low and high stimulation frequencies [35±41% inhibition of responses to 1 and 20±50 Hz stimulation; Waterman (1996)]. N-type channels play a signi®cantly less important role in ATP release from these neurons, although their role increases with increasing frequency of nerve stimulation. N-type channel blockade produces only 7±15% inhibition of responses to 1±5 Hz stimulation, and 25± 41% inhibition at stimulation frequencies of 10±50 Hz (Waterman, 1996). 5.1.2. Sympathetic neurons in the vas deferens ATP release from postganglionic sympathetic neurons in the mouse vas deferens is coupled to calcium in¯ux through N- and P/Q-type channels (Waterman, 1997). At stimulation frequencies less than 10 Hz, contractile responses mediated by ATP are abolished by o-conotoxin GVIA. The role of N-type channels is less important as stimulation frequency is increased; at 50 Hz stimulation, o-conotoxin GVIA reduces ATPmediated responses by only 37% (Waterman, 1997). This pattern is the opposite to that observed for ATP release from parasympathetic neurons in the bladder. The role of P/Q-type channels in ATP release in the vas deferens increases with stimulation frequency, such that >40% of the response to 50 Hz stimulation is

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inhibited by P/Q-type channel blockade. This pattern of involvement of P/Q-type channels is also di€erent to that observed in the bladder. Furthermore, the relative roles of N- vs P/Q-type channels is the opposite for ATP release in each tissue. 5.1.3. Enteric inhibitory motor neurons in the colon The release of ATP from enteric inhibitory motor neurons in mouse colon is also dependent on calcium in¯ux through N- and P/Q-type channels (Waterman and Nichols, 1999). As in the vas deferens, the role of N-type channels in ATP release decreases with increasing stimulation frequency, and the role of P/Q-type channels increases with increasing stimulation frequency. Thus at 5 Hz stimulation, the inhibitory e€ect of ATP is decreased 60% by o-conotoxin GVIA and abolished by subsequent o-conotoxin MVIIC; at 20 Hz stimulation, o-conotoxin GVIA reduces the response by 30% and o-conotoxin MVIIC inhibits the remaining response (Waterman and Nichols, 1999). Although the pattern of involvement of N- vs P/Qtype channels is therefore similar, P/Q-type channels mediate a signi®cantly larger component of transmitter release at all stimulation frequencies in the colon, compared to the vas deferens. The proportion of ATPmediated responses in the colon that is sensitive to P/ Q-type channel blockade is, however, similar to that in the bladder. In three di€erent tissues in the same species, the role of VGCC subtypes in mediating ATP release clearly di€ers. The e€ect of stimulation frequency on the calcium channel subtypes required for transmitter release di€ers, as does the relative importance of Nand P/Q-type channels. Furthermore, there is no correlation between VGCC subtypes involved in ATP release and the subsequent excitatory or inhibitory action of ATP on the target tissue. This is not surprising; whether or not the transmitter is excitatory or inhibitory depends on the postjunctional receptors and is not a feature of the transmitter per se. One would therefore not necessarily anticipate that di€erent VGCCs would be required for ATP release where it had a postjunctional excitatory e€ect vs inhibitory e€ect. 5.2. NA release from autonomic neurons in the rat 5.2.1. Sympathetic neurons in the vas deferens Transmitter release in the rat vas deferens is predominantly coupled to N-type VGCCs at low stimulation frequencies (Boot, 1994; De Luca et al., 1990; De Potter et al., 1997; Lundy and Frew, 1994; Maggi et al., 1988b; Tran and Boot, 1997; Wright and Angus, 1996), but a role for N- and P/ Q-type VGCCs has been demonstrated at 20 Hz (Tran and Boot, 1997; Wright and Angus, 1996).

Approximately 10±20% of the response to 20 Hz stimulation is sensitive to blockade of P/Q-type channels. 5.2.2. Sympathetic neurons in the anococcygeus As in the vas deferens, noradrenergic responses to low frequency stimulation (<10 Hz) in the anococcygeus muscle are abolished by o-conotoxin GVIA (De Luca et al., 1990; Lundy and Frew, 1994; Mudumbi and Leighton, 1994; Smith and Cunnane, 1997). Responses that are resistant to o-conotoxin GVIA can be evoked at higher stimulation frequencies; at 20±50 Hz stimulation 80±100% of the contractile response remains (Mudumbi and Leighton, 1994), compared to 10±20% of the response under similar conditions in the rat vas deferens (Tran and Boot, 1997; Wright and Angus, 1996). An electrophysiological study in rat anococcygeus (Smith and Cunnane, 1997) con®rmed that o-conotoxin GVIA does not abolish noradrenergic EJPs at stimulation frequencies above 10 Hz. The remaining EJPs were reduced in amplitude by o-agatoxin IVA and could be abolished by o-conotoxin MVIIC, demonstrating that P/Q-type VGCCs are crucial for NA release at high stimulation frequencies. Interestingly, o-agatoxin IVA and o-conotoxin MVIIC each signi®cantly decreased EJP amplitude at low stimulation frequencies in the absence of N-type channel blockade (Smith and Cunnane, 1997). This is good evidence that P/Q-type VGCCs are coupled to NA release in sympathetic neurons in the rat anococcygeus muscle at all stimulation frequencies. EJPs evoked at high stimulation frequencies in the presence of P/Q-type channel blockade were abolished by o-conotoxin GVIA, con®rming that N-type VGCCs are also required for transmitter release at all stimulation frequencies in this preparation. Nifedipine had no e€ect on transmitter release (Smith and Cunnane, 1997), demonstrating again that L-type VGCCs are not involved in action potential-evoked autonomic neurotransmitter release. 5.2.3. Sympathetic neurons in the atrium NA release from sympathetic neurons in the rat atrium is somewhat similar to that described in vas deferens and anococcygeus; responses to low frequency stimulation are blocked by o-conotoxin GVIA (De Luca et al., 1990; Wright and Angus, 1996). The response (ca 30%) that remains at higher frequencies or longer train durations (64 pulses) may be due to calcium in¯ux through P/Q-type channels, since o-conotoxin MVIIC has an e€ect in this tissue; however, the two toxins have not been tested cumulatively in this preparation to ensure that the e€ect of o-conotoxin MVIIC is not due to an action at Ntype VGCCs.

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5.2.4. Sympathetic neurons in mesenteric resistance arteries NA release from sympathetic ®bres innervating mesenteric resistance vessels appears to be under similar control to that in vas deferens and atrium. N-type channels alone are sucient to mediate maximal transmitter release at low stimulation frequencies, but 20% of the response remains at 24 Hz stimulation (Pruneau and Angus, 1990b; Wright and Angus, 1996). The VGCC subtype coupled to the remaining transmitter release is unknown, since o-conotoxin MVIIC has not been tested after N-type channel blockade. As in the atrium, o-conotoxin MVIIC alone inhibited part of the noradrenergic response (Wright and Angus, 1996), although this may have been due to its e€ect on Ntype VGCCs. Unlike its e€ect in rat anococcygeus muscle, o-agatoxin IVA alone did not signi®cantly alter NA release in the mesenteric vessels (Wright and Angus, 1996). In these four tissues in the rat, there is evidence that N-type channels are sucient to mediate maximal NA release at low stimulation frequencies. At higher stimulation frequencies, a variable proportion of the neurogenic response (from 10±20% in rat vas deferens and mesenteric vessels and 30% in the atrium to 80±100% in the anococcygeus) remains after N-type channels have been blocked. In vas deferens and anococcygeus, P/Q-type channels mediate this component of transmitter release, but the identity of the VGCC subtypes responsible for this component in the atrium and mesenteric resistance vessels is unknown. In the anococcygeus, but apparently not in the vas deferens, atrium or mesenteric resistance vessels, P/Q-type channels are involved in transmitter release at low as well as high stimulation frequencies. 5.3. Acetylcholine release from autonomic neurons in the guinea-pig 5.3.1. Parasympathetic neurons in the atrium Release of ACh from vagal parasympathetic neurons innervating the guinea-pig atrium is only partly sensitive to o-conotoxin GVIA (Hong and Chang, 1995). Approximately half of the response to bursts of four pulses at 200 Hz per pacing pulse remained after Ntype channels had been blocked. o-conotoxin MVIIC alone (500 nM) abolished the cholinergic responses (Hong and Chang, 1995). o-conotoxin MVIIC also abolished cholinergic responses in the heart evoked by nicotinic agonist receptor stimulation. The increased inhibition produced by o-conotoxin MVIIC compared to o-conotoxin GVIA suggests that the former is blocking P/Q-type VGCCs, as well as N-type VGCCs. Thus N- and P/Q-type VGCCs are required for ACh release in this preparation. In the stimulation conditions used in this study, the N- and P/Q-type chan-

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nels mediate a similar proportion of transmitter release. 5.3.2. Parasympathetic neurons in the trachea Acetylcholine release from postganglionic parasympathetic neurons in the rat trachea is abolished by oconotoxin GVIA at stimulation frequencies below 5 Hz. At higher stimulation frequencies, large cholinergic responses remain (60±85% of control at 30±50 Hz) (Altiere et al., 1992). The nature of the channels mediating this component of transmitter release has not been investigated. 5.3.3. Longitudinal muscle motor neurons in the ileum Acetylcholine release from motor neurons innervating the LM of the GPI is coupled to calcium in¯ux through N-type channels at low stimulation frequencies (Boot, 1994; Hong et al., 1996; Lundy and Frew, 1988; Lundy and Frew, 1993; Lundy and Frew, 1994; Tran and Boot, 1997), but requires P/Q-type channels as well as stimulation frequencies above 3 Hz (Hong et al., 1996; Tran and Boot, 1997). At a stimulation frequency of 30 Hz for example, blockade of N- and P/ Q-type channels inhibits responses by 26 and 28%, respectively (Hong et al., 1996). The nature of the channel coupled to the remaining 46% of the response is unknown. 5.3.4. Secretomotor neurons in the ileum A subpopulation of secretomotor neurons in the GPI is cholinergic, and can be preferentially activated by 5-hydroxytryptamine (5-HT) in the presence of the 5-HT2 receptor antagonist, ketanserin (Vremec et al., 1997). ACh release from these neurons is relatively insensitive to o-conotoxin GVIA (33% inhibition of response), although transmitter release from noncholinergic secretomotor neurons is reduced by 75% by the toxin. The nature of the VGCCs coupled to the remaining transmitter release has not yet been studied. 5.3.5. Circular muscle motor neurons in the colon Acetylcholine release from CM motor neurons in the guinea-pig proximal colon depends on calcium in¯ux through N-type channels at stimulation frequencies below 3 Hz, and on N- and non-N-type VGCCs at higher stimulation frequencies (De Luca et al., 1990; Maggi et al., 1994). Non-N-type VGCCs mediate transmitter release to produce 49% of the maximal response at 30 Hz stimulation (Maggi et al., 1994). The nature of the non-N-type channel involved in ACh release in this tissue has not been studied. 5.3.6. Longitudinal muscle motor neurons in the colon Cholinergic responses in the LM of the guinea-pig colon are inhibited by 75±80% by o-conotoxin GVIA at all stimulation frequencies from 1±10 Hz (De Luca

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et al., 1990). Unlike the ®ndings in the CM (see above), the degree of inhibition did not vary with stimulation frequency (De Luca et al., 1990). The VGCCs mediating the remaining transmitter release have not been identi®ed. 5.3.7. Motor neurons in the gall bladder Contraction of gall bladder smooth muscle mediated by ACh is reduced 22% by o-conotoxin GVIA (Parkman et al., 1997), demonstrating a relatively small role for N-type VGCCs in ACh release from these enteric neurons. 5.3.8. Summary N-type VGCCs play a highly variable role in ACh release from neurons in the guinea-pig. The proportion of the cholinergic response inhibited by o-conotoxin GVIA varies from <33% in gall bladder and secretomotor neurons in the ileum (and in trachea and LM motor neurons of the ileum at high stimulation frequencies), to 50% of the response in atrium and 75± 80% of the response in LM of the colon. In many preparations (trachea, ileum LM and colon CM), Ntype channels are more important in transmitter release at low stimulation frequencies. However, in colon LM, for example, o-conotoxin GVIA produces similar inhibition at all stimulation frequencies tested. P/Q-type VGCCs play an equally important role with N-type VGCCs in ACh release in the atrium and CM of the colon, and a signi®cant role in the LM of the ileum. P/Q-type channels may also be important in the trachea, secretomotor neurons in the ileum, LM of the colon and motor neurons in the gall bladder; however, this hypothesis has not yet been tested. 6. The role of VGCC subtypes in co-transmission Autonomic neurons contain and have the potential to release multiple neurotransmitters (Morris and Gibbins, 1992). Many organ bath pharmacological experiments record whole contractions that are due to the combined actions of two or more transmitters. To identify the VGCC subtypes required for release of a particular transmitter, the e€ect of one transmitter needs to be isolated by inhibiting the release or action of the co-transmitters. Alternatively, over¯ow of a speci®c transmitter can be measured. In some studies such approaches have been taken. These studies give some insight into the general question of whether release of multiple neurotransmitters present in the same neurons is coupled to the same VGCCs. If the transmitters are stored in the same vesicle, one assumes that the same VGCCs must be required for exocytosis. However, in the case of the transmitters that are stored in entirely di€erent vesicles or only

partly overlapping vesicle populations in the same neuron, the possibility exists that di€erent VGCC subtypes may be involved in exocytosis. Location of di€erent vesicle populations in di€erent regions of the nerve terminal near di€erent VGCCs may thus allow di€erential coupling of exocytosis to VGCC subtype. To date studies of co-transmission in the bladder, vas deferens and colon have been performed and are reviewed here. 6.1. Acetylcholine and ATP co-transmission in the mouse urinary bladder Contractions in the mouse bladder are mediated by ACh and ATP, each transmitter mediating ca 50% of the response at a range of stimulation frequencies up to 20 Hz. At 50 Hz stimulation, 70% of the response is cholinergic (Waterman, 1996). Cholinergic responses in the bladder are highly sensitive to oconotoxin GVIA, and are reduced by ca 50% at all stimulation frequencies. Blockade of P/Q-type channels reduces the responses by a further 15±30% (Waterman, 1996). In contrast, o-conotoxin GVIA has little e€ect on purinergic responses, causing only 7±15% inhibition at stimulation frequencies below 10 Hz and 25±41% inhibition at 10±50 Hz stimulation (Waterman, 1996). P/Q-type channels play a major role in ATP release; blocking these channels reduces the purinergic response by 50±62% at stimulation frequencies of 2±10 Hz and by 37±41% at 1 and 20± 50 Hz. The striking di€erence in the importance of N- and P/Q-type VGCCs in release of ATP and ACh from these neurons suggests that the transmitters can be differentially released. Although the transmitters are thought to be present in the same neurons, it is possible that each transmitter is actually released from a di€erent neuron, and that di€erent VGCC subtypes are involved in this process. Alternatively the transmitters might be released from the same neuron, presumably from di€erent populations of vesicles, and the exocytosis of each is coupled to di€erent VGCCs (Fig. 4). Such a mechanism would require preferential docking of ACh-containing vesicles near N-type VGCCs and ATP-containing vesicles near P/Q-type VGCCs. Synaptic vesicles dock through interactions between vesicle proteins (v-SNARES, e.g. synaptotagmin) and target proteins in the nerve terminal membrane [tSNARES, e.g. syntaxin, which also binds to N- and P/ Q-type VGCCs by binding the a1B and a1A subunits, respectively; Figs. 1, Sheng et al. and 1998]. Whether di€erent isoforms of the v-SNARES allow di€erent vesicle populations to interact preferentially with di€erent t-SNARES and hence dock near di€erent VGCC subtypes is not known.

S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210

195

Fig. 4. Release of co-transmitters that are not co-stored. ACh and ATP may be released from separate vesicles in parasympathetic nerve terminals in the bladder. ACh release in mouse bladder is coupled preferentially to calcium in¯ux through N-type VGCCs, whereas ATP release is preferentially coupled to P/Q-type VGCCs. Two possible arrangements whereby this may be achieved are shown diagrammatically. In (a), AChand ATP-containing vesicles are preferentially docked in the vicinity of N-and P/Q-type VGCCs respectively. For clarity, the vesicles have been shown spatially separated in the nerve terminal, however they may be distributed heterogeneously. Speci®c protein±protein interactions between v-SNAREs and t-SNAREs could provide a mechanism whereby di€erent synaptic vesicle populations dock near di€erent VGCC subtypes (see Section 6.1). In (b), ACh and ATP are released from di€erent nerve terminals and each nerve terminal expresses di€erent proportions of VGCC subtypes (see Section 6.1).

6.2. Noradrenaline and ATP co-transmission in the mouse vas deferens Responses mediated by NA and ATP in the mouse vas deferens are equally sensitive to o-conotoxin GVIA, o-agatoxin IVA and to o-conotoxin MVIIC (Waterman, 1997). Noradrenergic and purinergic responses are both abolished by o-conotoxin GVIA at stimulation frequencies below 10 Hz, and signi®cantly inhibited at high frequencies. Blockade of P/Q-type channels reduces both noradrenergic and purinergic responses by 60% at 50 Hz stimulation. These results are consistent with the belief that NA and ATP are stored in the same vesicles. Exocytosis of these transmitters is therefore coupled to the same VGCCs (Fig. 5). 6.3. Nitric oxide and ATP co-transmission in enteric inhibitory motor neurons in the mouse colon Enteric inhibitory motor neurons in the intestine and colon produce inhibition of the intestinal smooth muscle via the actions of nitric oxide (NO) and ATP. NO is synthesised by NO synthase on demand and is

not believed to be stored in vesicles. NO synthase requires bound calcium-calmodulin, hence NO is only synthesised when cytosolic calcium concentrations in the vicinity of the enzyme are high (Bredt and Snyder, 1994). The calcium dependency of NO `release' is thus a feature of the enzyme activation, rather than of exocytosis. ATP is thought to be stored conventionally in vesicles, and the calciumdependence of its release relates to the calcium dependency of exocytosis. In mouse colon, both NO and ATP release depends on calcium in¯ux through N- and P/Q-type VGCCs, although the pattern of involvement of the channels varies. Blockade of N-type channels reduced NOmediated responses by 32±38% at low and high stimulation frequencies, whereas purinergic responses were inhibited 60% at a low stimulation frequency and 30% at high frequency. The remaining responses in all cases were predominantly mediated by calcium in¯ux through P/Q-type VGCCs. As expected given their di€erent modes of release, these co-transmitters can be di€erentially released and this process is di€erentially coupled to VGCC subtypes.

196

S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210

6.4. Acetylcholine and substance P co-transmission in enteric excitatory motor neurons in guinea-pig colon Cholinergic and tachykininergic responses in the guinea-pig distal colon are both abolished by o-conotoxin GVIA at low stimulation frequencies (<3 Hz) and greatly decreased at higher frequencies (Maggi et al., 1994). Both responses are inhibited by ca 50% at 100 Hz stimulation. The nature of the VGCC mediating the component of transmitter release resistant to oconotoxin GVIA has not yet been identi®ed. If the same VGCC mediates the remaining component of both cholinergic and tachykininergic responses, then together with the parallel e€ect o-conotoxin GVIA, it is suggested that the two transmitters might be coreleased. It is assumed that tachykinins are stored in large-dense cored vesicles, and ACh in small synaptic vesicles. If the transmitters are indeed released from separate vesicle populations, it is interesting that these vesicles may be dependent on calcium in¯ux through the same VGCC subtypes. 7. VGCCs and neuropeptide release It has been postulated that release of classical neurotransmitters stored in small synaptic vesicles is coupled to calcium in¯ux through non-L-type VGCCs and exocytosis of neuropeptides stored in large dense core vesicles is coupled to calcium in¯ux through L-type VGCCs (Hirning et al., 1988). Neuropeptides are present in many autonomic and sensory neurons, and in some cases, the nature of the VGCCs involved in their release has been studied. These studies provide no evidence that L-type channels are preferentially involved in the release of neuropeptides. 7.1. Neuropeptide release from sensory neurons Small diameter, capsaicin-sensitive primary a€erent nerve ®bres have dual sensory and e€erent functions, and are now sometimes considered part of the autonomic nervous system (Maggi, 1995). These neurons commonly contain the peptides substance P (SP) and calcitonin gene-related peptide (CGRP). CGRP release from sensory ®bres in the guinea-pig bronchus evoked by electrical stimulation of the vagus or by low concentrations of capsaicin is inhibited by o-conotoxin GVIA but not by nifedipine (Lou and Lundberg, 1992). Tachykininergic responses evoked by EFS in the guinea-pig bronchus are also inhibited by o-conotoxin GVIA (Altiere et al., 1992; Maggi et al., 1988a), demonstrating a role for N-type VGCCs in the release of both peptides. Similarly, bradykinin-evoked CGRP release from sensory ®bers in guinea-pig atrium is una€ected by nifedipine but reduced 52% by o-cono-

Fig. 5. Release of co-stored co-transmitters. NA and ATP are thought to be stored in the same vesicles in sympathetic nerve terminals in the vas deferens. Release of these transmitters is coupled to calcium in¯ux through the same VGCC subtypes: N- and P/Q-type channels (see Section 6.2).

toxin GVIA (Geppetti et al., 1990; Maggi et al., 1988a). Thus N- but not L-type channels are required for CGRP release in this tissue. Tachykinin release evoked by low concentrations of potassium from sensory ®bres in rabbit iris sphincter is inhibited by oconotoxin GVIA but not by o-agatoxin IVA, demonstrating a role for N-type not P-type VGCCs (Kageyama et al., 1997). Nicardipine also reduced responses to potassium depolarization in this tissue, although it is unknown whether nicardipine also blocks responses to action potential-evoked transmitter release (see Section 9). Substance P and CGRP release from rat ureter is decreased by 57±71% by o-conotoxin GVIA (Maggi et al., 1990) and SP and CGRP release in the guinea-pig renal pelvis is abolished at low frequency nerve stimulation by o-conotoxin GVIA and decreased by ca 45% at 10 Hz (Maggi et al., 1992). Experiments on isolated dorsal root ganglion neurons also demonstrated that SP and CGRP release evoked by bradykinin involves N- not L-type VGCCs (Evans et al., 1996). Together these studies demonstrate that N-type

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197

Fig. 6. Mode of stimulation of neuron and nature of VGCCs required for transmitter release. (a) Electrical stimulation of neurons triggers action potentials and subsequent neurotransmitter release from the readily releasable pool that is usually coupled to calcium in¯ux through N- and/or P/Q-type VGCCs. This transmitter release is TTX-sensitive. (b) Prolonged, direct depolarization of the nerve terminal by veratridine or a high potassium concentration results in the opening of L-type VGCCs a well as other VGCC subtypes (see Section 8.1). Transmitter may be released from the readily releasable and reserve pools. (c) The nicotinic receptor agonist, DMPP, can act at nerve terminals to trigger transmitter release in a TTX-insensitive manner. Release of transmitter in this case may also be resistant to VGCC blockers (see Section 8.2.2). The calcium required to trigger exocytosis may enter the nerve terminal through the nicotinic receptors themselves, or through other receptor-operated calcium channels. (d) Stimulation of nicotinic receptors on the cell soma can trigger action potentials and transmitter release in a manner similar to electrical stimulation. Such transmitter release is sensitive to both TTX and VGCC blockers (see Section 8.2.1).

Cat

Atrium

NA

Transmitter(s)

Sympathetic Sympathetic Sympathetic Sympathetic

Snall mesenteric arteries Rat

NA Guinea-pig ATP Rabbit NA Rabbit NA

Pulmonary artery

Ear artery

Sympathetic

NA

Dog

Splenic artery

Sympathetic

NO NA

NA

Sheep

Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Parasympathetic Sensory Sensory Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic inhibitory

Sympathetic

Nerve type or action

Middle cerebral artery

Guinea-pig NA NA NA NA NPY ACh CGRP CGRP Human NA Mouse NA NA Rat NA NA

Species

Tissue

Table 2 VGCCs required for neurotransmitter release in cardiovascular tissues

N, non-N N N, non-N N, non-N

N, non-N

(Not N) N

N

N (not L) N (not L, T) N N N (not L) P/Q (not L, N, T) N, non-N (not L) N N (not L) N, P/Q N, non-N N, non-N N, non-N

(Not N)

EFS EFS EFS EFS

EFS

EFS EFS

EFS

Spontaneous release in vivo EFS EFS EFS EFS EFS EFS Bradykinin EFS EFS EFS EFS EFS EFS

VGCC subtype Mode of mediating transmitter stimulation release

Yamazaki et al. (1997)

Reference

Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology, intracellular electrophysiology Organ bath pharmacology Intracellular electrophysiology Organ bath pharmacology Organ bath pharmacology

Organ bath pharmacology

Wright and Angus (1996) Morris et al. (1998) De Luca et al. (1990) Lundy and Frew (1994)

Pruneau and Angus (1990b)

Matthew and Wadsworth (1997) Ren et al. (1994)

Matthew and Wadsworth (1997)

HPLC (over¯ow) Haass et al. (1990) Organ bath pharmacology Hong and Chang (1995) Organ bath pharmacology Vega et al. (1995) Organ bath pharmacology Houzen et al. (1998) RIA (over¯ow) Haass et al. (1990) Organ bath pharmacology Hong and Chang (1995) RIA (over¯ow) Geppetti et al. (1990) Organ bath pharmacology Maggi et al. (1988a) ‰3 HŠNA over¯ow Gothert and Molderings (1997) Organ bath pharmacology Wright and Angus (1996) Organ bath pharmacology, ‰3 HŠNA over¯ow De Luca et al. (1990) Organ bath pharmacology Wright and Angus (1996) Organ bath pharmacology De Luca et al. (1990)

NA dialysis, HPLC-ECD

Methods

198 S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210

Dog Pig Guinea-pig

Guinea-pig

Small intestine CM

Small intestine LM

Dog

Guinea-pig

Ileocolonic junction CM

Colon CM

Mouse

Guinea-pig

Small intestine secretomotor neurons

Mouse Rabbit Rat

Rat Rat

Gastric fundus Duodenum

Small intestine myenteric plexus

Species

Tissue

Enteric Enteric Enteric Enteric Enteric Enteric excitatory Enteric excitatory Enteric excitatory Enteric excitatory Enteric excitatory Enteric excitatory Enteric excitatory Sympathetic Enteric excitatory Enteric inhibitory Sympathetic Excitatory Excitatory Enteric inhibitory Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric

ACh SOM ACh ACh ACh ACh ACh ACh ACh ACh/TK ACh/TK ACh NA ACh NANC NA ACh NANC NO NO NO ACh Tachykinins NO NO ATP ATP ACh ACh/TK Tachykinins NO NO/ATP

inhibitory inhibitory excitatory excitatory inhibitory inhibitory inhibitory inhibitory excitatory excitatory excitatory inhibitory inhibitory

excitatory excitatory excitatory excitatory excitatory

Enteric inhibitory Enteric inhibitory Sensory Inhibitory Enteric inhibitory Enteric excitatory

Nerve type or action

NO NANC NANC NO NANC ACh

Transmitter(s)

Table 3 VGCCs required for neurotransmitter release in gastrointestinal tissues

non-N non-N non-N non-N non-N

P/Q

non-N (not L)

non-N (not L, T) non-N (not L, T) P/Q

N, non-N (not L) (not N, L) N, non-N N, non-N (not N) (Not N) N, non-N N, non-N N, P/Q N P/Q N, P/Q N, non-N

N, non-N N, non-N (not L)

N N N N, N N N N, N, N, N, N,

N, N, N, N N,

N, non-N (not N) (not N) N N (not P/Q, L) N, non-N (not L)

VGCC subtype mediating transmitter release

EFS DMPP EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS

EFS EFS EFS EFS EFS VIP EFS EFS EFS EFS EFS DMPP, 5-HT + ketanserin DMPP, 5-HT EFS

EFS EFS Capsaicin EFS EFS Senktide, neurokinin B DMPP DMPP EFS DMPP EFS

Mode of stimulation

Superfusion bioassay Superfusion bioassay Organ bath pharmacology Organ bath pharmacology Sucrose gap electrophysiology Organ bath pharmacology Organ bath pharmacology Sucrose gap electrophysiology Organ bath pharmacology Intracellular electrophysiology Organ bath pharmacology Organ bath pharmacology Intracellular electrophysiology

Secretion (Ussing chamber) Organ bath pharmacology

Takahashi et al. (1992) Takahashi et al. (1992) Hong et al. (1996) Hong et al. (1996) Lundy and Frew (1988)

[3H]ACh over¯ow RIA Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology, chemiluminscence Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Secretion (Ussing chamber)

Boeckxstaens et al. (1993) Boeckxstaens et al. (1993) Maggi et al. (1994) Maggi et al. (1994) Zagorodnyuk and Maggi (1994) Humphreys and Costa (1992) Humphreys and Costa (1992) Zagorodnyuk and Maggi (1994) Waterman and Nichols (1999) Watson et al. (1991) Waterman and Nichols (1999) Waterman and Nichols (1999) Watson et al. (1991) (continued on next page)

Vremec et al. (1997) Boeckxstaens et al. (1993)

Lundy and Frew (1994) Boot (1994) Lundy and Frew (1993) Tran and Boot (1997) Humphreys and Costa (1992) Katsoulis et al. (1992) De Luca et al. (1990) De Luca et al. (1990) De Luca et al. (1990) Lundy and Frew (1994) De Luca et al. (1990) Vremec et al. (1997)

De Luca et al. (1990) Maggi et al. (1988b) Maggi et al. (1988b) Cayabyab et al. (1997) Borderies et al. (1997) Yau et al. (1992)

Reference

Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Intracellular electrophysiology Intracellular electrophysiology [3H]ACh over¯ow

Methods

S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210 199

Guinea-pig

Guinea-pig Mouse Rat

Taenia caeci

Gall bladder Anoccygeus

Rabbit Guinea-pig

Guinea-pig Guinea-pig

Colon CM/LM Colon LM

Caecum submucous plexus

Species

Tissue

Table 3 (continued )

Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric Enteric excitatory Sympathetic Enteric excitatory Enteric excitatory Enteric inhibitory Enteric inhibitory Enteric inhibitory Enteric excitatory Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Inhibitory Inhibitory Inhibitory

ATP ACh ACh/TK Tachykinins NO ATP ACh/TK ACh NANC NA ACh ACh ATP ATP ATP ACh NA NA NA NA NA NA NO NO NO

inhibitory excitatory excitatory excitatory inhibitory inhibitory excitatory excitatory

Nerve type or action

Transmitter(s)

N N, non-N N, non-N N N, non-N

N (not P/Q) N (not P/Q) N, non-N N, P/Q N, non-N N, non-N N, non-N N, non-N N, non-N N, non-N N, P/Q, R (not L) N, non-N

N, P/Q N, non-N, L N, non-N N, non-N (not N) N, non-N N, non-N N, P/Q

VGCC subtype mediating transmitter release

EFS EFS EFS EFS EFS

EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS

EFS EFS EFS EFS EFS EFS EFS EFS

Mode of stimulation

Intracellular electrophysiology Intracellular electrophysiology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Intracellular electrophysiology Organ bath pharmacology, [3H]NA over¯ow Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology

Organ bath pharmacology Bioassay on GPI Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Intracellular electrophysiology

Methods

Lundy and Frew (1994) De Luca et al. (1990) De Luca et al. (1990) Mudumbi and Leighton (1994) Lundy and Frew (1994)

Cunningham et al. (1998) Cunningham et al. (1998) De Luca et al. (1990) Houzen et al. (1998) Lundy and Frew (1994) Humphreys and Costa (1992) De Luca et al. (1990) Parkman et al. (1997) De Luca et al. (1990) Mudumbi and Leighton (1994) Smith and Cunnane (1997) Lundy and Frew (1993)

Waterman and Nichols (1999) Marino et al. (1993) De Luca et al. (1990) De Luca et al. (1990) Humphreys and Costa (1992) Humphreys and Costa (1992) De Luca et al. (1990) Cunningham et al. (1998)

Reference

200 S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210

Rat

Guinea-pig

Ureter

Renal pelvis

NA/ATP NA/ATP ATP ATP ATP NA NA/ATP ATP ATP NA NA/ATP NA/ATP NA/ATP NA/ATP NA/ATP NA/ATP NPY ATP Tachykinins ACh ACh ATP ACh ATP ACh ACh ACh/ATP ACh/ATP ACh/ATP ACh/ATP ATP NA Tachykinins NANC NA NO NO NO Tachykinins CGRP Tachykinins

Transmitter(s) Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Sympathetic Parasympathetic Sensory Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Parasympathetic Sympathetic Sensory Inhibitory Excitatory Inhibitory Inhibitory Inhibitory Sensory Sensory Sensory

Nerve type or action

Except for facilitated transmitter release (see Section 8.4 of the text).

Rabbit

Urethral lamina propria

a

Pig Rabbit

Rat

Rabbit

Human Mouse

Guinea-pig

Rat

Urethra CM

Bladder dome

Guinea-pig

Vas deferens

Mouse

Species

Tissue

Table 4 VGCCs required for neurotransmitter release in genitourinary tissues

N N N, non-N N N,R (not P/Q) N, P/Q N, P/Q N, P/Q N, P/Q (not L) N, non-N N N, non-N N N N, P/Q N, P/Q N, non-N N, non-N (Not N) N N, P/Q N, P/Q N, non-N N, non-N N, non-N N, non-N (not L)a N, P/Q N, non-N N, non-N N, non-N N, non-N N, non-N (not L)a (not N) N, non-N N, non N N, non-N N, non-N (not L, T) N, P/Q N, non-N N, non-N N, non-N

VGCC subtype mediating transmitter release EFS EFS EFS EFS EFS EFS EFS EFS EFS 120 mM K+ EFS EFS EFS EFS EFS EFS 120 mM K+ EFS Capsaicin EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS EFS Capsaicin EFS, latrotoxin, K+ EFS EFS EFS EFS EFS EFS EFS

Mode of stimulation Organ bath pharmacology Organ bath pharmacology Intracellular electrophysiology Extracellular electrophysiology Intracellular electrophysiology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Intracellular electrophysiology Over¯ow Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Over¯ow Organ bath pharmacology Organ bath pharmacology, RIA Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology [3H]ACh over¯ow Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology [3H]NA over¯ow Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology Organ bath pharmacology RIA RIA Organ bath pharmacology

Methods

Maggi et al. (1988b) Brock et al. (1989) Brock et al. (1989) Brock et al. (1989) Smith and Cunnane (1996) Waterman (1997) Wright and Angus (1996) Waterman (1997) Waterman, unpublished De Potter et al. (1997) Boot (1994) De Luca et al. (1990) Lundy and Frew (1994) Maggi et al. (1988b) Tran and Boot (1997) Wright and Angus (1996) De Potter et al. (1997) Maggi et al. (1988b) Maggi et al. (1988b) Maggi et al. (1989) Waterman (1996) Waterman (1996) Zygmunt et al. (1993) Zygmunt et al. (1993) Maggi (1991) Somogyi et al. (1997) Frew and Lundy (1995) Maggi et al. (1988b) Lundy and Frew (1994) De Luca et al. (1990) Maggi (1991) Somogyi et al. (1997) Maggi et al. (1988b) Werkstrom et al. (1997) Zygmunt et al. (1993) Zygmunt et al. (1993) Zygmunt et al. (1993) Zygmunt et al. (1995) Maggi et al. (1990) Maggi et al. (1990) Maggi et al. (1992)

Reference

S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210 201

pharmacology pharmacology pharmacology pharmacology

Kageyama et al. (1997) Organ bath pharmacology

Organ bath Organ bath Organ bath Organ bath RIA Over¯ow Over¯ow

Rabbit

Guinea-pig

Guinea-pig

Dog

Iris sphincter

Trachea

Bronchus

Splenic nerve

ACh NANC Tachykinins Tachykinins CGRP NA NPY

Parasympathetic Inhibitory Sensory Sensory Sensory Sympathetic Sympathetic

N, N, N, N, N, N, N,

non-N non-N non-N non-N non-N (not L) non-N non-N

45.9 mM K+, 10 mM veratridine EFS EFS EFS EFS EFS, capsaicin EFS EFS N, non-N, L (not P)

Mode of stimulation VGCC subtype mediating transmitter release Nerve type or action Transmitter(s) Species

In the majority of studies reviewed here, transmitter release was evoked by electrical stimulation of neurons; transmitter release occurs following invasion of the nerve terminal by an action potential, and the re-

Tissue

8. Mode of nerve stimulation and the nature of VGCCs involved in transmitter release

Table 5 VGCCs required for neurotransmitter release in other tissues

The tachykinins, substance P and neurokinin A, are present in excitatory motor neurons throughout the small and large intestine of many species. Electrically evoked tachykininergic responses in the guinea-pig colon CM are abolished by o-conotoxin GVIA at low stimulation frequencies and reduced by 50% at frequencies of 50 Hz and above (Maggi et al., 1994), demonstrating a role for N-type and non-N-type VGCCs in tachykinin release. De Luca et al. (1990) also reported that the non-cholinergic (tachykininergic) responses in the guinea-pig colon were signi®cantly inhibited by o-conotoxin GVIA. Vasoactive intestinal peptide (VIP) induced tachykininergic contractions of the GPI LM were abolished by o-conotoxin GVIA (Katsoulis et al., 1992). N-type VGCCs clearly play a major role in tachykinin release in these tissues. Nicotinic receptor agonist-evoked release of somatostatin in guinea-pig small intestine myenteric ganglia is inhibited by o-conotoxin GVIA and una€ected by nifedipine (Takahashi et al., 1992), demonstrating that N- and not L-type VGCCs are required for its release. Neuropeptide Y is present in sympathetic neurons innervating the heart, and its release in the guinea-pig heart is abolished by o-conotoxin GVIA (Haass et al., 1990, 1991). Similarly, NPY release from sympathetic neurons in the rat vas deferens is inhibited by o-conotoxin GVIA (De Potter et al., 1997). The studies reviewed above demonstrate that peptide release from autonomic neurons depends on calcium in¯ux through N- and non-N-type VGCCs and not Ltype VGCCs, and is therefore indistinguishable from the VGCCs required for release of non-peptide transmitters from these neurons. These studies do not support the hypothesis that L-type VGCCs are preferentially involved in the release of peptides (Hirning et al., 1988).

Methods

7.2. Neuropeptide release from autonomic neurons

Sensory

Reference

VGCCs are the most important in release of SP and CGRP from sensory ®bres in a number of tissues. However, there is evidence from one study (Kageyama et al., 1997) that L-type VGCCs may be involved, although potassium depolarization rather than electrical stimulation was used. Whether P/Q-type VGCCs also play a role in peptide release from sensory ®bres has yet to be investigated.

Altiere et al. (1992) Altiere et al. (1992) Altiere et al. (1992) Maggi et al. (1988a) Lou and Lundberg (1992) De Potter et al. (1997) De Potter et al. (1997)

S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210

Tachykinins

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sponses are abolished by tetrodotoxin. This mode of stimulation presumably evokes release of transmitter from the readily releasable pool that is closely associated with active zones and has been described in many neuronal populations (Burgoyne and Morgan, 1995). Such studies demonstrate the role of VGCCs in action potential-evoked transmitter release. Other studies have investigated the role of VGCC subtypes in transmitter release evoked by alternative means: sustained potassium depolarization or by chemical stimulation. Potassium depolarizes the nerve terminal directly, and action potentials are not evoked. This mode of stimulation may release transmitter from a reserve pool, as well as from the readily releasable pool (GonzalesBurgos et al., 1995; Prado et al., 1992). Chemical stimulation commonly refers to the use of nicotinic receptor agonists to activate neurons and evoke transmitter release, and is discussed later. The role of VGCCs in transmitter release evoked by these di€erent means is reviewed (see Fig. 6 and Table 2Table 3Table 4Table 5). 8.1. Potassium stimulation of transmitter release Relatively few studies have investigated the nature of the VGCCs required for potassium-evoked transmitter release. Nevertheless, it seems clear that the channels involved are di€erent to those used when the nerve terminal is invaded by an action potential. Peripheral endings of sensory ®bres in the rabbit iris sphincter muscle release tachykinins which cause contraction of the iris smooth muscle (Kageyama et al., 1997). Tachykinin release can be evoked by electrical stimulation, or by sustained potassium depolarization or veratridine (a sodium channel opener). L-type VGCCs are required for transmitter release evoked by potassium depolarization and by veratridine stimulation, and N-type VGCCs play a role when the nerves are stimulated by low (15.9 mM) but not high (45.9 mM) concentrations of potassium (Kageyama et al., 1997). Potassium-induced release of ACh from superior cervical ganglia is decreased by o-conotoxin GVIA, FTX and o-agatoxin IVA, but not by the L-type VGCCs blocker, nitrendipine (Gonzales-Burgos et al., 1995). Thus N- and P/Q-type VGCCs are involved in ACh release when evoked by sustained depolarization. In contrast, action potential-evoked ACh release in this tissue is coupled to calcium in¯ux through P/Qtype VGCCs, with no apparent role for N- or L-type VGCCs (Gonzales-Burgos et al., 1995). 8.2. Chemical stimulation of transmitter release Transmitter release can be activated by stimulation of nicotinic receptors on cell bodies or nerve terminals

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of autonomic neurons. Depending on the location of the nicotinic receptors, a nicotinic receptor agonist may evoke action potentials that invade the nerve terminal and trigger exocytosis in a tetrodotoxin-sensitive manner, or if nicotinic receptors are present on the nerve terminal, stimulation can evoke transmitter release independent of action potentials (and insensitive to tetrodotoxin). This process may depend on calcium in¯ux through the nicotinic receptor itself, or through other receptor operated channels (Ren et al., 1994). A limited number of studies has investigated the role of VGCCs in nicotinic receptor stimulated transmitter release. 8.2.1. Nicotinic receptor stimulated transmitter releasetetrodotoxin sensitive Nicotinic receptor stimulation of cholinergic ®bres by DMPP in the guinea-pig isolated heart produces a decrease in heart rate and contractile strength which is inhibited by o-conotoxin MVIIC. Electrically evoked responses are also sensitive to this toxin but not to oconotoxin GVIA, and are presumably tetrodotoxin (TTX)-sensitive, suggesting that ACh release is therefore coupled to calcium in¯ux through P/Q-type VGCCs (Hong and Chang, 1995). In contrast, DMPPevoked noradrenergic responses in the guinea-pig isolated heart are largely resistant to o-conotoxin GVIA and MVIIC (Hong and Chang, 1995). It is possible that the noradrenergic responses are TTX-resistant and that DMPP stimulated transmitter release through an action at the nerve terminal, triggering exocytosis by calcium entry through nicotinic receptors. In contrast to these ®ndings, nicotinic receptor stimulation in the perfused guinea-pig heart in situ evokes the release of NA and NPY in a o-conotoxin GVIA-sensitive manner (Haass et al., 1990; Haass et al., 1991). This di€erence presumably re¯ects the di€erent preparation used; in the perfused heart in situ with intact sympathetic innervation, nicotinic receptor agonists may act at receptors on the postganglionic sympathetic neuronal cell bodies, evoking transmitter release in a TTX-sensitive manner; the cell bodies of sympathetic neurons are not present in the isolated heart preparations used by Hong and Chang (1995). 8.2.2. Nicotinic receptor stimulated transmitter releasetetrodotoxin resistant Calcitonin gene-related peptide (CGRP) can be released from sensory ®bres in the guinea-pig bronchi in response to electrical stimulation of the vagus nerve, capsaicin and by nicotinic receptor stimulation. Whereas vagal stimulation and low concentrations of capsaicin evoke transmitter release in a TTX-sensitive manner and require the involvement of N-type VGCCs, nicotinic receptor agonist-evoked release is largely resistant to TTX and o-conotoxin GVIA (Lou

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and Lundberg, 1992). Similarly, noradrenergic vasoconstrictor responses evoked by electrical stimulation in the canine isolated, perfused arteries are sensitive to both TTX and o-conotoxin GVIA, whereas nicotinic agonist-evoked responses are not (Ren et al., 1994). At the canine ileocolonic junction, electrically evoked NO release is sensitive to TTX and to o-conotoxin GVIA but nicotinic receptor agonist-evoked release is not (Boeckxstaens et al., 1993). These ®ndings are consistent with the coupling of transmitter release to calcium in¯ux through receptor-operated channels (i.e. the nicotinic receptor in these examples) rather than through VGCCs. 8.3. Electrical stimulation of transmitter release Action potential-evoked transmitter release may be coupled to calcium in¯ux through several di€erent VGCC subtypes (see above sections), although in the majority of studies, L-type channels do not appear to be involved in transmitter release. The subtype of VGCC required for action potential-evoked transmitter release usually varies with the frequency of nerve stimulation. In many preparations, N-type channels alone appear to be sucient for transmitter release at low stimulation frequencies (<5 Hz), and P/Q-type channels also play a role at higher frequencies (e.g. in GPI LM, rat and mouse vas deferens LM). However, this is not always the case. ACh release in mouse bladder and NA release in rat anococcygeus depend on calcium in¯ux through N-type and P/Q-type channels at all stimulation frequencies from 1±50 Hz (Smith and Cunnane, 1997; Waterman, 1996). The frequency dependence of VGCC subtype involvement in transmitter release in some tissues may indicate that di€erent channel subtypes are activated at di€erent stimulation frequencies. Di€erent channel subtypes clearly have di€erent electrophysiological properties; P-type currents activate above ÿ50 mV, peak at ÿ30 to ÿ10 mV (in 5±25 mM Ba2+) and exhibit little inactivation during depolarising pulses to ÿ30 and 0 mV. In contrast, N-type currents are activated above ÿ30 mV, peak at ca ÿ10 mV and partially inactivate during depolarising pulses [for review see Nooney et al. (1997)]. It is not clear however, whether di€erences in these properties translate into preferential opening of di€erent VGCC subtypes in response to nerve stimulation at di€erent frequencies. The behaviour of these channels in di€erent stimulation conditions is also likely to be in¯uenced by other channels in the vicinity. Electrophysiological studies on the cell somata of autonomic neurons have demonstrated that there is selective coupling between VGCCs and subtypes of Ca2+-activated K+ channel; for example calcium entry through N-type VGCCs selectively activates small conductance Ca2+-activated K+ chan-

nels (Davies et al., 1996). Similar selective coupling of channels in the nerve terminal may provide a means whereby N-type channels play a lesser role at high stimulation frequencies if activation of the nearby Ca2+-activated K+ channels causes these channels, but not P/Q-type channels to close at high stimulation frequencies. Calcium in¯ux through P/Q-type channels is required for transmitter release in some tissues at low and high stimulation frequencies (e.g. in rat anococcygeus, mouse bladder) and in other tissues at only high frequencies (e.g. mouse and rat vas deferens). Since the calcium channels are presumably located close to the site of exocytosis (as at the skeletal neuromuscular junction and in central synapses), whenever the channels open one would predict that the resultant calcium in¯ux would trigger exocytosis of docked vesicles. The frequency dependence of P/Q-type VGCC involvement in transmitter release in some but not all tissues may therefore re¯ect di€erences in the channel properties between tissues. There are numerous reports of di€erences within channel subtypes between tissues [see Nooney et al. (1997) and Section 9]. Alternatively, technical limitations may have limited our ability to detect a role for each channel subtype at a range of stimulation frequencies. Several studies have shown that P/Q-type channel blockers do not have signi®cant e€ects on autonomic neuroe€ector transmission in some tissues when tested alone (Boot, 1994; Lundy and Frew, 1994; Waterman, 1996Waterman, 1997). However, a signi®cant e€ect of the toxins can be demonstrated after N-type channels have been blocked (Frew and Lundy, 1995; Tran and Boot, 1997; Waterman, 1996Waterman, 1997). Thus P/Q-type channels are involved in autonomic neurotransmitter release, but their role can sometimes only be revealed after N-type channel blockade and/or when a range of stimulation frequencies is used. 8.4. VGCCs required for non-facilitated and facilitated transmitter release Transmitter release from autonomic nerve terminals is not commonly coupled to calcium in¯ux through Ltype VGCCs. However, under some conditions, a role for L-type channels can be demonstrated. Thus in rat urinary bladder, ACh release in response to intermittent electrical stimulation is resistant to nifedipine, but facilitated ACh release in response to continuous stimulation is inhibited (Somogyi et al., 1997). In this tissue, activation of M1 muscarinic receptors on cholinergic nerve terminals enhances transmitter release; activation of the receptors occurs in response to long trains of high frequency (5±20 Hz) nerve stimulation (Somogyi et al., 1997). Activation of the M1 receptors may enhance L-type currents indirectly

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via activation of protein kinase C (Somogyi et al., 1997). The authors propose that activation of the otherwise silent L-type channels most likely occurs during micturition, producing massive transmitter release and promoting complete bladder emptying. 9. Sensitivity of VGCCs to toxins There is good evidence that the detailed pharmacological characteristics of VGCC subtypes vary between tissues. In particular, N-type VGCCs in some tissues are sensitive to o-conotoxin MVIIC, whereas in other tissues they are insensitive. In GPI LM, electrically evoked contractions are inhibited by both o-conotoxins GVIA and MVIIC (Boot, 1994). At the stimulation frequencies used in this study, o-conotoxin MVIIC had no e€ect when added after o-conotoxin GVIA, indicating that it was acting only at N-type channels (Boot, 1994). In rat vas deferens, o-conotoxin MVIIC did not cause a signi®cant inhibition of responses, suggesting that the o-conotoxin GVIA-sensitive Ntype channels in this tissue are insensitive to o-conotoxin MVIIC (Boot, 1994). Sympathetic inotropic responses in the rat and mouse heart are abolished by oconotoxin GVIA (Wright and Angus, 1996); in the mouse, these responses are also abolished by 1000 nM o-conotoxin MVIIC, whereas in the rat, responses are inhibited only 50% by 1000 nM o-conotoxin MVIIC (Wright and Angus, 1996). In the mouse atrium, N-

205

type VGCCs are thus sensitive to both o-conotoxins GVIA and MVIIC. In the rat atrium, however, a proportion of the N-type VGCCs is either insensitive to o-conotoxin MVIIC or sensitive only to very high concentrations (>1000 nM). In rat mesenteric vessels, contractile responses that are abolished by o-conotoxin GVIA are inhibited by 50% by 1000 nM o-conotoxin MVIIC (Wright and Angus, 1996). As in the rat atrium, this suggests there may be a population of Ntype VGCCs that is insensitive to o-conotoxin MVIIC or sensitive to high concentrations. Some variation between tissues has also been reported in the concentrations of toxins required to inhibit responses by 50% (IC50) (Table 6). Some of this variability can be accounted for by variations in stimulus parameters; increasing the frequency of stimulation appears to increase the IC50. For example in mouse vas deferens, the IC50 for o-conotoxin MVIIC varies from ca 20 nM at 5±20 Hz to 250 nM at 50 Hz stimulation (Waterman, 1997). Similar values have been reported for o-conotoxin MVIIC in GPI LM (26 nM) at low stimulation frequencies (Boot, 1994). However higher concentrations of o-conotoxin MVIIC were required to inhibit contractions in the mouse bladder using identical stimulation frequencies [IC50s from 200 nM at 5 Hz to 470 nM at 50 Hz; Waterman (1996)]. The reason for the di€erences between channel subtypes in di€erent tissues is not known with certainty, although it is assumed that di€erences in the associ-

Table 6 Concentrations of o-conotoxins GVIA and MVIIC producing 50% inhibition of responsesa Tissue

Stimulation frequency (Hz)

IC50

Guinea-pig atrium (PS)

200

>10 mM

280

Hong and Chang (1995)

Guinea-pig atrium (S) Guinea-pig ileum LM (enteric motor neurons) Mouse bladder (PS, whole contraction)

200 0.1

420 21

490 26

Hong and Chang (1995) Boot (1994)

5 10 20 50 5 10 20 50 10 10

30 30 125 >1000 < 10 < 10 25 < 300 2 6.2

200 200 225 470 20 30 16 250 ND

Waterman (1996)

25 10 0.05 Single pulses

1.3 12.6 20 2.5

ND ND >1 mM 173

Zygmunt et al. (1993) Zygmunt et al. (1993) Boot (1994) Wright and Angus (1996)

Mouse vas deferens (S, whole contraction)

Rabbit detrusor (PS, whole contraction) Rabbit detrusor (PS, non-cholinergic contraction) Rabbit urethra (PS, whole contraction) Rabbit urethra (PS, relaxation) Rat vas deferens (S, whole contraction)

for GVIA (nM)

IC50

for MVIIC (nM) References

Waterman (1997)

Zygmunt et al. (1993) Zygmunt et al. (1993)

a GVIA, o-conotoxin GVIA; MVIIC; o-conotoxin MVIIC; ND, not done; PS, parasympathetic; S, sympathetic. `Whole contraction' indicates that the combined contraction produced by two or more co-transmitters was measured.

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ated subunits, di€erent splice variants or di€erential phosphorylation may be at least partly responsible. 10. VGCCs in disease 10.1. Mutations in VGCC subunits in mouse and human Recently, several mutations in calcium channel subunits have been described in diseases a€ecting the central nervous system of humans and mice. Loss-offunction mutations in the a1A subunit are present in episodic ataxia type 2 (EA-2) and in absence epilepsy in tottering and leaner mice (Fletcher et al., 1996; Opho€ et al., 1996). Glutamine expansions occur in the a1A gene in spinocerebellar ataxia type 6 (SCA6) and may cause constitutive channel activation (Zhuchenko et al., 1997). Recently, a loss-of-function mutation has been described in a1F channel subunits in the retina, causing night blindness (Bech-Hansen et al., 1998). Gain-of-function mutations occur in the a1A subunit in familial hemiplegic migraine (Opho€ et al., 1996). Mutations in the g2 subunit, which associates with the a1A subunit, have been described in the stargazer mouse which has a form of absence epilepsy (Letts et al., 1998). The lethargic mouse, which has ataxia and absence epilepsy, has a mutation in the b4 subunit (Burgess et al., 1997). The a1A subunit probably forms the pore of P/Q-type channels, and although these channels are clearly involved in autonomic neurotransmission, it is not known whether any of the mouse mutants described above show autonomic dysfunction. Whether the a1F, b4 or g2 subunits are expressed in autonomic neurons is also unknown; if they are present one might predict abnormal autonomic neurotransmission in individuals with congenital stationary night blindness and in stargazer and lethargic mice. 10.2. Diseases mediated by antibodies to VGCCs Antibodies to P/Q-type VGCCs are produced by patients with the autoimmune disease, Lambert±Eaton myasthenic syndrome (LEMS). The antibodies decrease the number of functional channels, rather than alter the kinetics of individual channels (Lang and Newsom-Davis, 1995), and thereby inhibit release of transmitter at autonomic and motor nerve terminals. Patients with LEMS exhibit a range of autonomic symptoms including constipation, dry mouth, impotence in males and diculty emptying the bladder. Around one third of LEMS patients also produce antibodies to N-type VGCCs, although evidence suggests that these antibodies do not have pathological signi®cance (reviewed in Waterman et al., 1997). At least 80% of LEMS patients have autonomic symp-

toms, irrespective of the presence of antibodies to Ntype VGCCs. Passive transfer of LEMS to mice by injecting patient IgG interferes with parasympathetic and sympathetic transmission from neurons in the bladder and vas deferens, respectively, by downregulating the component of transmitter release coupled to P/ Q-type channels (Waterman et al., 1997). Similarly, exposure of guinea-pig taenia caeci to LEMS IgG in vitro for 6 h inhibits nerve-mediated responses by downregulating P/Q-type channels (Houzen et al., 1998). Noradrenergic responses in the guinea-pig atrium depend on calcium in¯ux through N-type but not P/Q-type VGCCs; exposure of the atrium to LEMS IgG in vitro does not signi®cantly alter noradrenergic transmission (Houzen et al., 1998). Together these studies provide good evidence that P/ Q-type channels play a very important role in autonomic neuroe€ector transmission and that downregulation of these channels produces clinically signi®cant autonomic dysfunction. 11. Role of VGCCs in other neuronal functions This review has concentrated on the role of VGCCs in autonomic neuroe€ector transmission. Calcium channels play many other roles in neurons however, particularly those channels located in the cell soma. Calcium in¯ux through VGCCs activates nearby calcium-activated potassium channels (Davies et al., 1996; Marrion and Tavalin, 1998; Sah, 1995); currents through the latter channels are involved in action potential repolarization and in afterhyperpolarizations. T-type channels are important in generating rhythmic ®ring of neurons and cardiac myocytes (Perez-Reyes, 1998; Tsien, 1998). Calcium channels are also important in cell metabolism and proliferation, gene expression and cytoskeletal function (Fisher and Bourque, 1996; Miljanich and Ramachandran, 1995). 12. Concluding points N-type VGCCs have long been considered the most important or only channels involved in neurotransmitter release from autonomic neurons. The research reviewed here indicates that this is not true and that multiple VGCCs are required for autonomic neurotransmitter release. The importance of N-type VGCCs is highly variable; in parasympathetic neurons in the atrium there is no signi®cant role for N-type VGCCs in ACh release; in sympathetic neurons in the atrium, N-type VGCCs mediate the majority of calcium in¯ux required for NA release. The transmitter release occurring in the presence of N-type channel blockade has been referred to as `residual release' by Smith and

S.A. Waterman / Progress in Neurobiology 60 (2000) 181±210

Cunnane (1996, 1997). Such a term implies that only a small component of transmitter release remains, which is the case at low stimulation frequencies in the guinea-pig vas deferens where the term was ®rst applied. Since transmitter release coupled to non-N-type channels in some tissues exceeds that coupled to N-type VGCCs, the term residual release can be misleading. The author suggests that in the light of recent studies on a range of tissues and transmitters, it is now more accurate to de®ne components of transmitter release according to the name of the calcium channel subtype involved. Since multiple VGCC subtypes are required for release of a variety of autonomic neurotransmitters from di€erent neuronal populations in di€erent tissues and species, it is natural to look for general patterns in the nature of VGCCs coupled to transmitter release. Few patterns appear to exist. The studies reviewed here demonstrate that the release of the same neurotransmitter from di€erent populations of neurons may require di€erent VGCC subtypes (see Tables 2±5). Furthermore, release of the same neurotransmitter in the same tissue in di€erent species may require di€erent VGCC subtypes. In the majority of tissues, it is clear that the nature of VGCCs required for neurotransmitter release varies with stimulation frequency and with the type of stimulation. It appears generally to be true that L-type channels have no role in action potential-evoked transmitter release (e.g. Hong and Chang, 1995; Smith and Cunnane, 1997; Somogyi et al., 1997; S.A. Waterman, unpublished observations on mouse vas deferens). However when neurons are subject to sustained depolarization (e.g. Kageyama et al., 1997) or facilitation of transmitter release by activation of presynaptic receptors (Somogyi et al., 1997), L-type VGCCs can be important in transmitter release. In many tissues, the combination of N- and/or P/Q type channels appears sucient to mediate maximal transmission. A variable component of transmitter release that remains may involve R-type or other, as yet undescribed, channels. Autonomic neurons use multiple transmitters, although the role of VGCC subtypes in the release of co-transmitters has not been widely studied. The evidence to date suggests that transmitters that are believed to be released from the same population of neurons may depend on calcium in¯ux through di€erent VGCCs. However, where the transmitters are thought to be stored in the same vesicles, as for NA and ATP in the sympathetic neurons in the vas deferens, the same VGCCs are required for release of each transmitter. Although much is now known about the role of VGCC subtypes in the release of autonomic neurotransmitters, many questions remain. The role of VGCCs in release of co-transmitters needs to be investigated thoroughly. The pharmacological, biophysical

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and molecular properties of VGCCs at nerve terminals needs to be studied in greater detail and compared to the properties of the channels in somata and dendrites. Our understanding of the role and pharmacology of VGCCs in the autonomic nervous system will be greatly facilitated by anatomical studies mapping the cellular and subcellular localization of VGCC subunits in autonomic neurons.

Acknowledgements The author's work has been assisted by funds from the National Health and Medical Research Council of Australia and The Australian Research Council.

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