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Protein kinase C, transmitter release and α2-autoinhibition in the guinea pig vas deferens
174
not attributable to noradrenaline reaches about 80% for 5 stimuli at 50V, yet is around 50% for 2 stimuli at 20V.
Long term effects of small numbers of stimuli Freshly dissected vasa were mounted in an organ bath, and one train of 10 stimuli applied. After 30 min one train of two stimuli was followed after 5 min by 6 trains of 5 stimuli at 1 min intervals, all at the same voltage. Various times after the last train of 5 a single train of 2 was applied. The response to 5 stimuli declines rapidly from train to train, at a rate dependent on stimulus voltage. Soon after 6 trains of 5 stimuli the response to a train of 2 stimuli is greatly reduced, an inhibition which recovers exponentially with a time constant of about 11 minutes. Depression of the response is not associated with a reduction in the sensitivity of the preparation to either exogenous noradrenaline or c~/~ methylene ATP and it is therefore assumed to be prejunctional. Prazosin (10-7 M) reduces the response to both control and inhibited trains by a more or less constant decrement of tension. The long lasting stimulus-induced inhibition therefore acts more
upon the component of the tension response w'hich is not due to noradrenaline. Conditions which favour a higher fraction of prazosin resistant response lead to a greater stimulus-induced inhibition of total tension response, suggesting that the development of tile long lasting stimulus-induced inhibition is associated with the release of a transmitter other than noradrenaline. This long lasting inhibition by very small numbers of stimuli is unaffected by the **2-adrenoreceptor antagonist yohimbine (10 7 M), but is also largely unaffected by 8-phenyl theophylline (10 6 M), suggesting that Pt purinoceptors are not involved. As the prazosin sensitive and resistant components can, to some extent, be both separately activated by different patterns of stimulation, and separately modulated by long lasting stimulus-induced inhibition, the transmitters responsible for each do not necessarily come from the same nerve terminals. Reference 1 Kurokawa, M. and Tsunoo, A., Parasympathetic depression of vas deferens contraction in the guinea-pig involves adenosine receptors, J. Physiol., 407 (1988) 135-153.
Protein kinase C, transmitter release and a2vas deferens
in the
.pig
C.F. Wardell and T.C. Cunnane University Department of Pharmacology, South Parks Road, Oxford, OX1 3QT, U.K.
Surprisingly little is known about the processes involved in depolarization-secretion coupling in sympathetic nerve terminals. Protein kinase C (PKC) has been identified within nerve terminals [5] and may play a role in transmitter release. Activation of P K C by phorbol esters enhances [3H]-noradrenaline release from both central [3] and peripheral [4] sympathetic neurones. The mechanisms involved in inhibition of transmitter release following stimulation of prejunctional a2-adrenoceptors are also unclear but
may involve effects on PKC a n d / o r calcium entry into nerve terminals [2]. In the present study, electrophysiotogical methods have been used to investigate the possible role of P K C and calcium on transmitter release mechanisms and az-autoinh_ibition in the guinea p i g vas deferens. Changes in m e m b r a n e potentials were measured using conventional intracellular recording techniques. Focal extracellular recordings of the nerve terminal impulse and excitatory junction currents to measure transmitter release were also
175
i~u,,~,v,
i
I0mV
/
t
, I se¢
Fig. 1A. Effects of 0.3 ~ M p h o r b o l d i b u t y r a t e ( P D B u ) o n E J P s in the g u i n e a p i g vas d e f e r e n s . L I P s w e r e e v o k e d b y s t i m u l a t i o n o f the h y p o g a s t r i c n e r v e t r u n k w i t h t r a i n s of 7 stimuli at 1 Hz. N o t e t h a t the a m p l i t u d e o f the first E J P in a t r a i n w a s increased after PDBu and action potentials were initiated by E J P s later in the train. R e s t i n g m e m b r a n e p o t e n t i a l w a s a b o u t - 70 mV. Control
Control
(0-CTX 0.1 ~M (10 mins)
Cloaidine 0.1 ~tM (10 mins)
~CTX + 0.1 p.M PDBu (20 mins) C10nidine + 0.1 p.M PDBu (20 mins)
5o ~tv 20 ms Fig. lB. F o c a l e x t r a c e l l u l a r r e c o r d i n g s s h o w i n g the n e r v e t e r m i n a l i m p u l s e a n d a s s o c i a t e d t r a n s m i t t e r release. Left h a n d p a n e l s s h o w the i n a b i l i t y of p h o r b o l d i b u t y r a t e ( P D B u ) to reverse the i n h i b i t i o n o f t r a n s m i t t e r release p r o d u c e d b y ¢oc o n o t o x i n (¢o-CTX). R i g h t h a n d p a n e l s s h o w reversal b y P D B u of the i n h i b i t i o n o f t r a n s m i t t e r release p r o d u c e d b y c l o n i d i n e . In all cases t h e r e w a s n o a p p a r e n t c h a n g e in the n e r v e t e r m i n a l impulse. T r a c e s r e p r e s e n t s u p e r i m p o s e d r e c o r d s elicited b y 25 stimuli at l Hz.
made using the method described by Brock and Cunnane [1]. Drugs were applied either inside the focal suction electrode or outside the electrode to the whole tissue. Phorbol 12,13 dibutyrate (PDBu), an activator of PKC, produced a concentration-dependent (0.03 0.3/LM) increase in the amplitude of excitatory junction potentials (EJPs) evoked by trains of stimuli at 1 Hz (Fig. 1A). A particular point to note is that the amplitude of the first E3P in a train was greatly increased following the application of PDBu (0.3 /~M). There was no apparent effect on the amplitude of spontaneous EJPs. Focal extracellular recording was used to determine whether the increase in EJP amplitude was due to an effect on action potential propagation in sympathetic nerve terminals. This technique allows changes in both the nerve terminal impulse and the associated transmitter release from a small population of varicosities to be studied on an impulse-to-impulse basis. Local application of PDBu (0.1 FzM) within the electrode produced an increase in evoked transmitter release with no detectable effect on the nerve terminal impulse. When PDBu (0.1 ~M) was applied externally to the whole tissue there was still no effect of PDBu on the nerve terminal impulse. Spontaneous transmitter release was apparently unaffected. The effects of PDBu on the inhibition of evoked transmitter release produced by the calcium channel blocker ~0-conotoxin (~-CTX) and the a:adrenoceptor agonist clonidine were also compared (Fig. 1B). Local application of ~0-CTX (0.1 /~M) abolished evoked transmitter release but PDBu (0.1 /zM) did not reverse the block. In contrast, the inhibition of transmitter release produced by the local application of clonidine (0.1 /~M) was fully reversed by PDBu (0.1/~M). Several conclusions may be drawn from these studies: (1) P K C appears to play a role in depolarizationsecretion coupling in sympathetic nerve terminals and may be involved in the development of facilitation; (2) entry of calcium through voltage-dependent calcium channels is necessary for PDBu to enhance evoked transmitter release; and (3) clonidine and ~-conotoxin apparently inhibit transmitter release through different mechanisms. One interesting possibility raised by the present
176
findings is that clonidine may not modulate calcium entry directly through c~2-adrenoceptors as presently believed but may act at some step involved in depolarization-secretion coupling. Clearly there are other possible explanations and further studies are required to establish whether PDBu leads to, for example, phosphorylation of the c~2-adrenoceptor which then uncouples the a2-adrenoceptor from calcium channels and hence alters calcium entry indirectly.
References 1 Brock. J.A. and Cunnane, T.('., J. Physiol.. 39t9 (1988) 602 632. 2 Lipscombe, D., Kongsamut, S. and Tsien, R.W.. N~ture 340 (1989) 639-642. 3 Malenka, R.C., Madison, D.V. and Nicoll, R.A., Nature 321 (1986) 175-177. 4 Musgrave, I.F. and Majewski, H., Naunyn-Schmiedeberg's Arch. Pharm., 339 (1989) 48-53. 5 Wood, J.G.. Girad, P.R., Mazzei, O.J. and Kuo. J.F...I. Neurosci., 6 (1986) 2571-2577.
C.F.W. is a Squibb Research Scholar.
Contribution of acetyicholine and vasoactive intestinal pe~ypeptide in controlling c a t e c h o t a m ~ secretion Arun R. Wakade Department of Pharmacology, School of Medicine, Wayne State University, Detroit, M I 48201, U.S.A.
Our physiological and pharmacological studies have shown that secretion of adrenal medullary hormones is regulated by multiple neurotransmitters released from splanchnic nerve terminals of the rat [1,2]. From these and other studies we proposed that, in addition to the classical neurotransmitter acetylcholine (ACh), vasoactive intestinal polypeptide (VIP) may contribute to the stimulation of chromaffin cells and induce secretion of catecholamines (CA). The purpose of the present investigation was to determine the relative contribution of ACh and VIP at different levels of neuronal activity in regulating the secretion of CA. Field stimulation of splanchnic neurones caused release of VIP-like immunoreactivity (VIPLI), determined by radioimmunoassay, in the perfusate of the isolated adrenal gland of the rat. The release was inversely related to the frequency of stimulation (8.6 x 1 0 - 3 , 4.9 x 1 0 - 3 and 4.0 x 10 3 f m o l / p u l s e at 1, 3 and 10 Hz, respectively). Release of ACh was determined by loading the gland with 3H-choline and separating 3H-ACh from the total tritium in the perfusate. Field stimulation produced a significant increase in the release of
3H-ACh over the spontaneous release. The stimulation-evoked fractional release of 3H-ACh was directly related to the frequency of stimulation ( 4 . 5 x 1 0 2 at 1 Hz for 5 min and 7 . 4 x i 0 - 2 at 10 Hz for 30 sec). Release of VIP-LI and 3H-ACh was abolished by omission of C a C I : from the perfusing medium and by Ca + +-channel blockers. After chronic ablation of splanchnic nerves, field stimulation of the adrenal gland did not evoke release of either VIP. 3H-ACh or CA, although infusion of exogenous ACh or VIP produced secretion of CA. These results provide direct evidence for the release of ACh and VIP from the splanchnic nerve endings in the rat adrenal medulla. Of additional importance is the finding that ACh release predominates at higher frequencies whereas release of VIP declines with increasing neural activity.
References 1 Malhotra, R.K. and Wakade, A.R.. J. Physiol., 383 ~19871 639-652. 2 Wakade. A.R.. J Neurochem. 50. (1988~ 1302-1308.
not attributable to noradrenaline reaches about 80% for 5 stimuli at 50V, yet is around 50% for 2 stimuli at 20V.
Long term effects of small numbers of stimuli Freshly dissected vasa were mounted in an organ bath, and one train of 10 stimuli applied. After 30 min one train of two stimuli was followed after 5 min by 6 trains of 5 stimuli at 1 min intervals, all at the same voltage. Various times after the last train of 5 a single train of 2 was applied. The response to 5 stimuli declines rapidly from train to train, at a rate dependent on stimulus voltage. Soon after 6 trains of 5 stimuli the response to a train of 2 stimuli is greatly reduced, an inhibition which recovers exponentially with a time constant of about 11 minutes. Depression of the response is not associated with a reduction in the sensitivity of the preparation to either exogenous noradrenaline or c~/~ methylene ATP and it is therefore assumed to be prejunctional. Prazosin (10-7 M) reduces the response to both control and inhibited trains by a more or less constant decrement of tension. The long lasting stimulus-induced inhibition therefore acts more
upon the component of the tension response w'hich is not due to noradrenaline. Conditions which favour a higher fraction of prazosin resistant response lead to a greater stimulus-induced inhibition of total tension response, suggesting that the development of tile long lasting stimulus-induced inhibition is associated with the release of a transmitter other than noradrenaline. This long lasting inhibition by very small numbers of stimuli is unaffected by the **2-adrenoreceptor antagonist yohimbine (10 7 M), but is also largely unaffected by 8-phenyl theophylline (10 6 M), suggesting that Pt purinoceptors are not involved. As the prazosin sensitive and resistant components can, to some extent, be both separately activated by different patterns of stimulation, and separately modulated by long lasting stimulus-induced inhibition, the transmitters responsible for each do not necessarily come from the same nerve terminals. Reference 1 Kurokawa, M. and Tsunoo, A., Parasympathetic depression of vas deferens contraction in the guinea-pig involves adenosine receptors, J. Physiol., 407 (1988) 135-153.
Protein kinase C, transmitter release and a2vas deferens
in the
.pig
C.F. Wardell and T.C. Cunnane University Department of Pharmacology, South Parks Road, Oxford, OX1 3QT, U.K.
Surprisingly little is known about the processes involved in depolarization-secretion coupling in sympathetic nerve terminals. Protein kinase C (PKC) has been identified within nerve terminals [5] and may play a role in transmitter release. Activation of P K C by phorbol esters enhances [3H]-noradrenaline release from both central [3] and peripheral [4] sympathetic neurones. The mechanisms involved in inhibition of transmitter release following stimulation of prejunctional a2-adrenoceptors are also unclear but
may involve effects on PKC a n d / o r calcium entry into nerve terminals [2]. In the present study, electrophysiotogical methods have been used to investigate the possible role of P K C and calcium on transmitter release mechanisms and az-autoinh_ibition in the guinea p i g vas deferens. Changes in m e m b r a n e potentials were measured using conventional intracellular recording techniques. Focal extracellular recordings of the nerve terminal impulse and excitatory junction currents to measure transmitter release were also
175
i~u,,~,v,
i
I0mV
/
t
, I se¢
Fig. 1A. Effects of 0.3 ~ M p h o r b o l d i b u t y r a t e ( P D B u ) o n E J P s in the g u i n e a p i g vas d e f e r e n s . L I P s w e r e e v o k e d b y s t i m u l a t i o n o f the h y p o g a s t r i c n e r v e t r u n k w i t h t r a i n s of 7 stimuli at 1 Hz. N o t e t h a t the a m p l i t u d e o f the first E J P in a t r a i n w a s increased after PDBu and action potentials were initiated by E J P s later in the train. R e s t i n g m e m b r a n e p o t e n t i a l w a s a b o u t - 70 mV. Control
Control
(0-CTX 0.1 ~M (10 mins)
Cloaidine 0.1 ~tM (10 mins)
~CTX + 0.1 p.M PDBu (20 mins) C10nidine + 0.1 p.M PDBu (20 mins)
5o ~tv 20 ms Fig. lB. F o c a l e x t r a c e l l u l a r r e c o r d i n g s s h o w i n g the n e r v e t e r m i n a l i m p u l s e a n d a s s o c i a t e d t r a n s m i t t e r release. Left h a n d p a n e l s s h o w the i n a b i l i t y of p h o r b o l d i b u t y r a t e ( P D B u ) to reverse the i n h i b i t i o n o f t r a n s m i t t e r release p r o d u c e d b y ¢oc o n o t o x i n (¢o-CTX). R i g h t h a n d p a n e l s s h o w reversal b y P D B u of the i n h i b i t i o n o f t r a n s m i t t e r release p r o d u c e d b y c l o n i d i n e . In all cases t h e r e w a s n o a p p a r e n t c h a n g e in the n e r v e t e r m i n a l impulse. T r a c e s r e p r e s e n t s u p e r i m p o s e d r e c o r d s elicited b y 25 stimuli at l Hz.
made using the method described by Brock and Cunnane [1]. Drugs were applied either inside the focal suction electrode or outside the electrode to the whole tissue. Phorbol 12,13 dibutyrate (PDBu), an activator of PKC, produced a concentration-dependent (0.03 0.3/LM) increase in the amplitude of excitatory junction potentials (EJPs) evoked by trains of stimuli at 1 Hz (Fig. 1A). A particular point to note is that the amplitude of the first E3P in a train was greatly increased following the application of PDBu (0.3 /~M). There was no apparent effect on the amplitude of spontaneous EJPs. Focal extracellular recording was used to determine whether the increase in EJP amplitude was due to an effect on action potential propagation in sympathetic nerve terminals. This technique allows changes in both the nerve terminal impulse and the associated transmitter release from a small population of varicosities to be studied on an impulse-to-impulse basis. Local application of PDBu (0.1 FzM) within the electrode produced an increase in evoked transmitter release with no detectable effect on the nerve terminal impulse. When PDBu (0.1 ~M) was applied externally to the whole tissue there was still no effect of PDBu on the nerve terminal impulse. Spontaneous transmitter release was apparently unaffected. The effects of PDBu on the inhibition of evoked transmitter release produced by the calcium channel blocker ~0-conotoxin (~-CTX) and the a:adrenoceptor agonist clonidine were also compared (Fig. 1B). Local application of ~0-CTX (0.1 /~M) abolished evoked transmitter release but PDBu (0.1 /zM) did not reverse the block. In contrast, the inhibition of transmitter release produced by the local application of clonidine (0.1 /~M) was fully reversed by PDBu (0.1/~M). Several conclusions may be drawn from these studies: (1) P K C appears to play a role in depolarizationsecretion coupling in sympathetic nerve terminals and may be involved in the development of facilitation; (2) entry of calcium through voltage-dependent calcium channels is necessary for PDBu to enhance evoked transmitter release; and (3) clonidine and ~-conotoxin apparently inhibit transmitter release through different mechanisms. One interesting possibility raised by the present
176
findings is that clonidine may not modulate calcium entry directly through c~2-adrenoceptors as presently believed but may act at some step involved in depolarization-secretion coupling. Clearly there are other possible explanations and further studies are required to establish whether PDBu leads to, for example, phosphorylation of the c~2-adrenoceptor which then uncouples the a2-adrenoceptor from calcium channels and hence alters calcium entry indirectly.
References 1 Brock. J.A. and Cunnane, T.('., J. Physiol.. 39t9 (1988) 602 632. 2 Lipscombe, D., Kongsamut, S. and Tsien, R.W.. N~ture 340 (1989) 639-642. 3 Malenka, R.C., Madison, D.V. and Nicoll, R.A., Nature 321 (1986) 175-177. 4 Musgrave, I.F. and Majewski, H., Naunyn-Schmiedeberg's Arch. Pharm., 339 (1989) 48-53. 5 Wood, J.G.. Girad, P.R., Mazzei, O.J. and Kuo. J.F...I. Neurosci., 6 (1986) 2571-2577.
C.F.W. is a Squibb Research Scholar.
Contribution of acetyicholine and vasoactive intestinal pe~ypeptide in controlling c a t e c h o t a m ~ secretion Arun R. Wakade Department of Pharmacology, School of Medicine, Wayne State University, Detroit, M I 48201, U.S.A.
Our physiological and pharmacological studies have shown that secretion of adrenal medullary hormones is regulated by multiple neurotransmitters released from splanchnic nerve terminals of the rat [1,2]. From these and other studies we proposed that, in addition to the classical neurotransmitter acetylcholine (ACh), vasoactive intestinal polypeptide (VIP) may contribute to the stimulation of chromaffin cells and induce secretion of catecholamines (CA). The purpose of the present investigation was to determine the relative contribution of ACh and VIP at different levels of neuronal activity in regulating the secretion of CA. Field stimulation of splanchnic neurones caused release of VIP-like immunoreactivity (VIPLI), determined by radioimmunoassay, in the perfusate of the isolated adrenal gland of the rat. The release was inversely related to the frequency of stimulation (8.6 x 1 0 - 3 , 4.9 x 1 0 - 3 and 4.0 x 10 3 f m o l / p u l s e at 1, 3 and 10 Hz, respectively). Release of ACh was determined by loading the gland with 3H-choline and separating 3H-ACh from the total tritium in the perfusate. Field stimulation produced a significant increase in the release of
3H-ACh over the spontaneous release. The stimulation-evoked fractional release of 3H-ACh was directly related to the frequency of stimulation ( 4 . 5 x 1 0 2 at 1 Hz for 5 min and 7 . 4 x i 0 - 2 at 10 Hz for 30 sec). Release of VIP-LI and 3H-ACh was abolished by omission of C a C I : from the perfusing medium and by Ca + +-channel blockers. After chronic ablation of splanchnic nerves, field stimulation of the adrenal gland did not evoke release of either VIP. 3H-ACh or CA, although infusion of exogenous ACh or VIP produced secretion of CA. These results provide direct evidence for the release of ACh and VIP from the splanchnic nerve endings in the rat adrenal medulla. Of additional importance is the finding that ACh release predominates at higher frequencies whereas release of VIP declines with increasing neural activity.
References 1 Malhotra, R.K. and Wakade, A.R.. J. Physiol., 383 ~19871 639-652. 2 Wakade. A.R.. J Neurochem. 50. (1988~ 1302-1308.