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Electrophysiology of noradrenaline release from smooth muscle organs
335
TIPS -August 1983 affinity binding site of these ligands was affected in vivo by the administration of naloxazone to rats and mice (see Ref. 13); this means that only a part of the binding was really relevant pharmacologically, the other part being labelling on recognition or binding sites, unrelated to analgesic effects or to any other effect. It is no exaggeration to state that the origin of most of the biphasic and curvilinear Scatchard plots is mainly due to the presence of non-specific binding in addition to specific binding; moreover sometimes a linear Scatchard may only be an indication of non-specific displaceable sites; [3H]cimetidine, [3H]ranitidine, [3H]desipfamine, [3H]imipramine, [3H]histamine etc. are ligands for such non-specific sites. Recently [aH]tamoxifen was found to label the estrogen receptor and distinct antiestrogen binding sitest4; nevertheless the authors pertinently concluded that these binding sites may not he directly involved in mediation of the classically recognized estrogen antagonism of anti-estrogens; but they might influence the action of antiestrogens in vivo, possibly in a passive manner, by altering the distribution of these
drugs and, hence, their biological potency. We are now returning towards a more unitary concept of the opiate receptor, thus deviating from the original proposal of multiple neurotransmitter receptors~5; the euphoric dedication of researchers to exposing more and more subtypes of the opiate receptor seems to have reached its peak. Let us hope that this multiplicatory tendency will be only a short-lived fashion as was the case for the dopamine receptor. Obviously, the multiple receptor concept does not contribute to the development of new drugs or to the exploration of the nature of brain receptors.
Reading list 1 Guth, P. S. (1982) Trends PharmacoL So. 3, ,167 2 llmn, B., Gonssen, H. and Laduron, P. (1982) Mol. Pharmacol. 22, 243-249 3 Ladtwon, P. (1982) mAdvances in Dopamme Research (Koksaka, M. etal., eds), Vol. 37, pp. 71--82, Pergamon Press, Oxford and New Yodt 4 Seeman, P. (1980)Pharmacol. Rev. 32, 229-313 5 Leysen, J. E. and Gomn~ren, W. (1981) J. Neurochem. 36, 201-219 6 Sokoloff, P., Martres, M.-P, and Schwartz, J -C.
(1980) Naunyn-Schmiedebergs Arch. Pharmakol. 315, 89-102 7 Lazareno, S. and Nakorskt, S R. (1982)Eur. J. Pharmacol. 81,273-285 8 Bevdacqua, M , Vago, T , Scorza, D. and Norbtato, G. (1982) Btochera. Biophys. Res. Commun. 108, 1661-1669 9 Huff, R M. and Molinoff, P, B. (1982) Proc. Natl Acad. Sci. USA 79, 7561-7565 10 De 12an, A , Kilpamck, B F and Caron, M. C. (1982) Mol. Pharmacol. 22,290-297 II Gillan, M . G . C . and Kostedgz, H. W. (1982)Br. J. Pharmacol. 77,461--469 12 Pfetffer, A., PaSl, A., Mehraem, P and Herz, A. (1982) Brain Res. 248, 87-94 13 Laduton, P. (1982) Trends Pharmacol. Sci. 3, 351-352 14 Sudo, K., Monsma, F. J. and Katzenellenbogen, B. S. (1983) Endocrinology l 12,425.-434 15 Snyder, S. H., and Goodman, R. R. 0 9 8 0 ) J . Neurochem. 35, 5-15
The author was born in 1936 in Naraur, Belgmm and graduated in medicine at the Umverstty of Louvain in 1961. After having worked with A. F. De Schaepdryver at the Heymans Institute of Pharmacology in Ghent frorn 1964to 1968, he obtamed the degree o f Agr~g~ at the Universityo f Louvain in 1969. At that time he Ioined the Janssen Pharmaceutica Research Laboratories m Beerse where he ~sHead o f the pharmacology Research Group.
i
Electrophysiology of noradrenaline r e l e a s e from s m o o t h m u s c l e organs P. Illes Department of Pharmacology, Umversity o/ Freiburg, Hermann-Herder-Strasse 5, D- 7800 Freiburg ~.Br. FRG.
In the last decade much interest has been focused on the mechanism of noradrenaline release from sympathetic nerve terminals of smooth-muscle organs. Preparations used include the vasa deferentia of various species, the cat nictitating membrane and various vascular preparations. The vas deferens and the nictitating membrane serve as suitable models for studying noradrenergic synapses with possible implications for similar structures in the central nervous system. Experiments on vascular preparations have the additional benefit of offering insight into the local control of circulation. In smooth-muscle organs, a
wide range of endogenous substances are able to modulate the nerve stimulationinduced release of noradrenaline. The majority of our knowledge concerning these mechanisms derives from the biochemical measurement of transmitter release. The procedure most commonly employed is to incorporate radiolahelled noradrenaline into the transmitter pool and subsequently measure the outflow of radioactivity in response to stimulation. More refined biochemical methods have been used less frequently to evaluate the minute amounts of endogenous noradrenaline released.
The approach of the electrophysiologist is different ~'2. He impales the smooth muscle cell with a fine, tipped microelectrode containing a concentrated solution of a potassium salt (Fig. 1). With this electrode the membrane potential of individual smooth-muscle ceils can be continuously recorded. Nerve stimulation evokes transient depolarizing responses called excitatory junction potentials (e.j.ps), which are caused by the post-junctional action of noradrenaline. The transmitter is released from varicose nerve terminals, occupies adrenergic receptors situated on the smooth-muscle membrane and initially triggers an inward, followed by an outward, ionic current resulting in an e.j.p. The amplitude of the e.j.p, is a measure of the conductance change induced by the transmitter, and, thus, a measure of noradrenaline release per impulse. S ~ muscle cells are electrically coupled to each other, with only a fraction of the cells being directly innervated by noradrenergic nerves. However, depolarization caused by the transmitter in an innervated cell, is decrementally conducted to its noninnervated counterparts. Therefore, it is possible to register e.j.ps at any point in the smooth-muscle bundle, although these e.j.ps are the result of transmitter release
© 1983El~vx=~
Publtd~'~B V , Ams~h~l~n 0165- 6147/83/$01(30
336
TIPS - August 1983
1
Fig. 1. lntracellular reeordmg o f excttatory luncUon potentials (e4.ps) from a smooth muscle cell o f the mouse vas deferens. The cell is impaled by a glass microelectrode filled wtth a concentrated solutton o f a potasstum salt. Electrical field stimulatton excites intramural varicose nerve termmals ( varicosities are indicated by open circles). Noradrenaline released on samulanon evokes an e j p. by causing a temporary conductance mcrease o f the post-junctional membrane. When sttmulation voltage ts increased, more nerve fibers are recruited and a htgher amplitude o f e4.ps reflects an increase in the quantity o f noradrenahne released. The black dots between the smooth muscle cells represent the sites where these cells are electrically coupled to each other
from a large number of varicose nerve endings distributed over the organ.
Evidence for the quantal nature of evoked noradrenaline release In most smooth-muscle organs, even in the absence of nerve stimulation, spontaneous depolarizations, resembling e.j.ps occur at random intervals. It is generally believed that noradrenaline is released from the varicose nerve endings in discrete packets or quanta, and that spontaneous e.j.ps (s.e.j.ps) represent the spontaneous quantal release of transmitter. The amplitude histogram of s.e.j.ps is skewed because of electric coupling between smooth-muscle cells, with most of the small amplitude potentials being masked by the noise of the recording electrode. Thus it is impossible to determine a preferred value of s.e.j.p, amplitude, as would be the case with a Gaussian distribution. This in turn excludes the possibility of proving that the e.j.p, is composed of the simultaneous occurrence of several spontaneous potentials. Fortunately, Blakeley and Cunnane 3 noticed that the rising phase of the e.j.p, in the guinea-pig or mouse vas deferens is discontinuous. It was rational to assume, that these discontinuities separated distinct depolarizing responses occurring with different latencies, and represented the quantal release of transmitter from individual varicosities. A statistical evaluation of their data confirmed this assumption. A more direct proof of the quantal nature of transmitter release in noradrenergically innervated organs was provided by Hirst and Neild4. These authors used short isolated segments of arteries taken from the intestinal submucosa of guinea-pigs. These short pieces of arteries were shown to
remain isopotential during current injection, and no attenuation of s.e,j.p, amplitude occurred with distance from the junctional current source. According to the postulates of the quantal theory of transmitter release, the s.e.j.p, amplitudes were unimodally distributed, and the amplitude of s.e.j.ps was similar to that of the smallest e.j.p.
Postjunctional adrenoceptors Smooth-muscle organs have been shown to contain both pre-junctional (predominantly a~-) and post-junctional (at- and a~-) adrenoceptors. These 2 subtypes of receptors have different pharmacological characteristics and can easily be differentiated by means of specific ligands. In order to study post-junctional adrenoceptors, noradrenaline can be locally applied to a small area of the smoothmuscle membrane by use of a fine-tipped micropipette, filled with a strong solution of the amine. When an anodic current is passed through the pipette a depolarizing response can be recorded with a second microelectrode, positioned in close proximity to the drug application site. Prazosin, an al-adrenoceptor antagonist, abolished noradrenaline-evoked depolarizations in the mouse vas deferens, without affecting nerve-stimulation evoked e.j.ps or spontaneous e.j.ps 5. Since iontophoretic pulses of short duration failed to evoke a detectable depolarization, noradrenaline had to be ejected by pulses of rather long duration. Thus, the amine may have reached extrajunctional receptors of the at-subtype, which are, in contrast to junctional receptors, blocked by prazosin. As a possible explanation of the well known resistance of e.j.ps and s.e.j.ps to a-adrenoceptor block-
ade, the existence of a second motor transmitter in addition to noradrenaline, has been considered. For approximately 20 years the inability of a-adrenoceptor antagonists to inhibit e.j.ps in the vas deferens, was attributed to a peculiarity of noradrenergic transmission in this organ. However, similar results have recently been acquired with a series of small arteries6. Although, in some of these preparations a-blockade by phentolamine abolished nerve stimulation-induced constriction, the e.j.p, amplitude was in all cases unaffected. It was suggested that phentolamine depresses the contractile response in view of its direct effect on the smooth muscle, possibly by uncoupling some step in the excitation-contraction coupling mechanism. In contrast to the results obtained with the vas deferens, in submucosal arteries of the guinea-pig intestine it was possible to mimic the amplitude and shape of an e.j.p. by iontophoretic application of noradrenaline from high resistance micropipettes7. Apparently, in this case responses to exogenous and endogenous noradrenaline were evoked via the same adrenoceptor population (junctional receptors). A visualization of catecholamine fibers after iontophoresis revealed that a depolarizing response to noradrenaline could be elicited only at regions close to the sympathetic nerves. At a non-innervated region, noradrenaline application evoked only a constriction in the absence of a detectable depolarization, possibly by an action on extra-junctional receptors. Depolarizing responses to nerve stimulation or noradrenaline application to junctional sites were insensitive to phentolamine, whereas extra-junctional receptors were blocked by this antagonist. It was suggested, that extra-junctional receptors are of the classical a-type, while junctional receptors represent a new class of adrenoceptor. These findings, if extended to the vas deferens, may render unnecessary hypotheses which assume that this organ is non-adrenergically innervated. It may be suggested that, generally, in organs where individual adrenergic varicosities are close to the muscle, the formation of junctional adrenoceptors is favoured. In large arteries, having a wide junctional cleft, no e.j.ps can be recorded. However, contractions in response to nerve stimulation of bath-applied noradrenaline are specifically blocked by a-adrenoceptor antagonists in a specific manner.
Prejunctional adrenoceptors An activation of pre-junctional a2receptors was shown to inhibit the nerve stimulation induced release of nor-
337
TIPS -August 1983
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Fig. 2. Amplitudes of e.j.ps elicited by trams of field pulses at 3 Hz m the mouse vas deferens. In A e.j.ps were ehcued before (C) and after ( Pra.), the addition of 0.5 prazosin. Inset shows a typical record before the addition of prazosin. In B e.j.ps were elicited before (CO and after the addition of euher O.1 ~ ( Yoh. 0.1) or 1 I.*M()'oh. 1) yohimbine or no drug (C O. From Ref. 5.
adrenaline and in a consequence also e.j.p. amplitudes in various smooth muscle preparations. TMs inhibitory action can be readily antagonized by ~2-adrenoceptor antagonists. Electrical stimulation of the intramural nerves in the mouse vas deferens with trains of 15 pulses at 3Hz elicited e.j.ps which were subject to facilitations (Fig. 2). All e.j.ps in a train were depressed by the c~2agonist clonidine. The otl-antagonistprazosin did not significantlyalter the e.j.p, amplitudes (Fig, 2A), whereas the ot2antagonist yohimbine clearly increased the response to the 4th, and later, pulses in a train (Fig. 2B). In vasa deferentia from reserpine-treated mice, the e.j.p, amplitudes were altered in much the same way as by yohimbine. Yohimbine did not further increase the e.j.p, amplitudes in these organs, whereas clonidine caused a marked inhibition. These data are in good agreement with the prmjunctional autoreceptor concept, which claims that activation of ot2receptors by previously released noradrenaline leads to an inhibition of subsequent action potential evoked transmitter release. The fact that e.j.ps elicited by the first 2-3 pulses were not enhanced by yohimbine may be due to the necessity for a certain minimal concentration of released noradrenaline to accumulate in the vicinity of the prejunctional receptor. When the tissue noradrenaline stores were depleted by
reserpine, the normal a-adrenergic inhibition was presumably interrupted by the lack of noradrenaline. The post-junctionaleffect of the transmitter was only reduced, since in the narrow junctional cleft even the low' quantities of noradrenaline released from partly depleted varicosities probably sufficed to elicit a small post-junctional response. Both the inhibitory action of ot~agonists and the facilitatory action of a~antagonists have been recently demonstrated in the guinea-pig mesenteric, and ear artery preparations by electrophysiological methods. In addition to noradrenaline, which acts on prejunctional ~-adrenoceptors, a number of other substances (e.g. prostaglandins, muscarinic cholinergic agonists, opiates and opioid peptides) also depress noradrenaline release by an action at their respective receptors. In certain vascular preparations endogenous prostaglandins (PGs) may function as regulator substances in neuromuscular transmissio#. Inhibition of PG synthesis by indorr;etacin enhanced the amplitude of e.j.ps in the guinea-pig mesenteric artery, possibly by eliminating a negative feed-back mechanism. In the guinea-pig vas deferens, PGs interfered with neuromuscular transmission, however, indometacin had no effect on e.j.p, amplitudes produced by repetitive stimulation. Thus, a PG-mediated regulatory mechanism does not seem to be operative.
The effect of opiates and opioid peptides has been thoroughly studied in the mouse vas deferens preparation (see Ref. 9). These ligands depress the e.j.p, amplitude by acting in a naloxone reversible and stereospecific fashion at a pre-junctional opiate receptor. On the other hand the frequency and amplitude of s.e.j.ps is not influenced by these substances. Chronic treatment of mice by morphine rendered the nerve terminals in their vasa tolerant to normorphine. A cross-tolerance with leucine-enkephalin did not occur, suggesting that opiates and certain opioid peptides act at separate receptors. Post-receplor mechanimns The possible mechanisms by which the activation of pre-junctional receptors leads to a depression of noradrenaline release has been discussed at length. The 3 basic alternatives are shown in Fig. 3. Opiates or a~-agonists failed to alter the action potential recorded extracellularly from small nerve bundles of the vas deferens, in concentrations clearly depressing ej.p. amphtudes. Thus a local anaesthetic action of these agents is unlikely, however, the possibility of a specific blockade of action potential propagation from terminal axons to varicosities must be taken into consideration (see Ref. 10). Whereas nerve stimulation-induced transmitter release is directly proportional to the concentration of C#+-ions in the
338
TIPS -August 1983
[Ca2÷]o 2~
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noradrenaline at the sub-junctional membrane is certainly a most reliable indicator for transmitter effects. The registration of e.j.ps supplies us with information about the amount of transmitter released per impulse and the s.e.j.p, frequency is the equivalent of spontaneous quantal transmitter release. Thus, in a number of cases electrophysiology is the method of choice for studying transmitter release in smoothmuscle organs.
Reading list
Fig 3. An action potential invades the varicostty and causes an increase in the axoplasmic concentration o f free calctum ([Ca~+]O wa a temporary permeability increase o f the varicosity membrane, resulting in an inward flux o f extracellular calcium ([Ca ~+]o). The mcreased axoplasmic calctum level trtggers the release o f vestcular noradrenahne (NA). Noradrenaline crosses the juncttonal cleft, occupies post-juncttonal adrenoceptors, andevokesane.j.p Possiblemechanismsbywhichtheactivationofpre-tunctionalreceptors leads to a depression o f noradrenaline release. 1: Specific blockade o f action potential propagation to the varicostty. 2: Interference wtth the mflux o f Ca 2+through voltage-dependent calctum channels. 3: Interference with the sequestratton o f Ca 2+into an mtraceUular pool other than the mttochondrium.
medium, the depression of e.j.p, amplitudes by prostaglandins and opiates shows an inverse dependence on external Ca z+. In general, manipulations designed to increase intracellular free calcium enhanced the e.j.p, amplitude and prevented the inhibitory action of these agents n. Therefore, transmitter release may be inhibited by a reduction in the supply of Ca2+-ions to the stimuhls-release coupling mechanism. Mg: m~d Ca 2+ were suggested to compete for a pre-junctional site X which is responsible for the influx of Ca ~÷ through voltagedependent channels. By contrast, normorphine and PGE~ inhibited nerve stimulation-induced transmitter release by a different mechanism, as shown by their non-competitive interference with the effect of Ca 2÷. The s.e.j.p, frequency was reduced by Mg 2., implying that a significant portion of spontaneous noradrenaline release is the consequence of calcium entry into the varicosity. Both normorphine and PGE1 failed to affect the s.e.j.p, frequency, again in favour of a different mode of action from that of Mg 2+. A third alternative is that some of these agents may inhibit the binding of calcium to an intracellular site. Electrophysiological
evidence obtained in enteric ganglia and locus coeruleus neurons favours the existance of such a site, which may be important for buffering increased intracellular free Ca2+-concentrations during neuronal activityTM. Inhibition of the Ca 2÷sequestration would prolong the period of increased intracellular Ca 2+, and in a consequence would reduce the concentration gradient between extra- and intra-cellular Ca ~÷. This would result in a reduced driving force for Ca 2+- influx into the cell and also in a depression of stimulus-release coupling. Moreover, the enhanced free calcium concentration could activate a calcium-dependent potassium conductance, which in turn, would hyperpolarize the resting membrane and lead to a block of action potential propagation.
1 Holman, M. E. (1970) inSmooth Muscle (Biilbrrag, E., Brading, A. F. and Jones, A. W., eds), pp. 244-288, Edward Arnold, London 2 Burnstock, G. and Bell, G. (1974) in The Peripheral Nervous System (Hubbard, J. 1., ed.), pp. 277-327, Plenum Press, New York, London 3 Blakeley, A. G. H and Cunnane, T. C. (1979)J. Physiol (London) 296, 85-96 4 Hirst, G. D. S. and Neild, T. O. (1980)J. Phystol. (London) 303, 43-60 5 Illes, P. and Starke, K. (1983) Br. J. Pharmacol. 78,365-373 6 Holman, M. E. and Surprenant, A. (1980) Br. J. Pharmacol. 71,651-661 7 Hirst, G. D. S and Neild, T. O. (1981)J. Physiol. (London) 313, 343-350 8 Kuriyama, H and Makita, Y. (1982)J. Physiol. (London) 327, 431-448 9 flies, P. (1982) m Regulatory Peptides: From Molecular Biology to Function (Costa, E. and Trabucchi, M,, eds), pp. 347-352, Raven Press, New York 10 files, P. and North, R. A. (1982) Br. J. Pharmacol. 75, 599--604 11 Illes, P., Zieglgansberger, W. and Herz, A. (1980) Brain Res. 191,511-522 12 Williams, J. T., Egan, T. M. and North, R. A. (1982)Nature (London) 299, 74--77
Conclusion The investigation of mechanisms that modulate the release of noradrenaline requires a multi-disciplinary approach. Although biochemical and contraction measurements have yielded a wealth of information, it is important to supplement them with electrophysiological methods. The conductance increase induced by
Peter llles received his M D. and Ph.D degrees from the University o f Budapest under Professor J. Knoll. He worked as an Assistant Professor o f Pharmacology at the Untverstty untd 1978. He subsequently spent a year at the Universtty o f Lund, Sweden, and 2 years at the Max.Planck Institute o f Psychiatry, Munich. In 1981 he moved to the Department o f Pharmacology, University o f Fretburg. His main interest is in neurotransmission.
TIPS -August 1983 affinity binding site of these ligands was affected in vivo by the administration of naloxazone to rats and mice (see Ref. 13); this means that only a part of the binding was really relevant pharmacologically, the other part being labelling on recognition or binding sites, unrelated to analgesic effects or to any other effect. It is no exaggeration to state that the origin of most of the biphasic and curvilinear Scatchard plots is mainly due to the presence of non-specific binding in addition to specific binding; moreover sometimes a linear Scatchard may only be an indication of non-specific displaceable sites; [3H]cimetidine, [3H]ranitidine, [3H]desipfamine, [3H]imipramine, [3H]histamine etc. are ligands for such non-specific sites. Recently [aH]tamoxifen was found to label the estrogen receptor and distinct antiestrogen binding sitest4; nevertheless the authors pertinently concluded that these binding sites may not he directly involved in mediation of the classically recognized estrogen antagonism of anti-estrogens; but they might influence the action of antiestrogens in vivo, possibly in a passive manner, by altering the distribution of these
drugs and, hence, their biological potency. We are now returning towards a more unitary concept of the opiate receptor, thus deviating from the original proposal of multiple neurotransmitter receptors~5; the euphoric dedication of researchers to exposing more and more subtypes of the opiate receptor seems to have reached its peak. Let us hope that this multiplicatory tendency will be only a short-lived fashion as was the case for the dopamine receptor. Obviously, the multiple receptor concept does not contribute to the development of new drugs or to the exploration of the nature of brain receptors.
Reading list 1 Guth, P. S. (1982) Trends PharmacoL So. 3, ,167 2 llmn, B., Gonssen, H. and Laduron, P. (1982) Mol. Pharmacol. 22, 243-249 3 Ladtwon, P. (1982) mAdvances in Dopamme Research (Koksaka, M. etal., eds), Vol. 37, pp. 71--82, Pergamon Press, Oxford and New Yodt 4 Seeman, P. (1980)Pharmacol. Rev. 32, 229-313 5 Leysen, J. E. and Gomn~ren, W. (1981) J. Neurochem. 36, 201-219 6 Sokoloff, P., Martres, M.-P, and Schwartz, J -C.
(1980) Naunyn-Schmiedebergs Arch. Pharmakol. 315, 89-102 7 Lazareno, S. and Nakorskt, S R. (1982)Eur. J. Pharmacol. 81,273-285 8 Bevdacqua, M , Vago, T , Scorza, D. and Norbtato, G. (1982) Btochera. Biophys. Res. Commun. 108, 1661-1669 9 Huff, R M. and Molinoff, P, B. (1982) Proc. Natl Acad. Sci. USA 79, 7561-7565 10 De 12an, A , Kilpamck, B F and Caron, M. C. (1982) Mol. Pharmacol. 22,290-297 II Gillan, M . G . C . and Kostedgz, H. W. (1982)Br. J. Pharmacol. 77,461--469 12 Pfetffer, A., PaSl, A., Mehraem, P and Herz, A. (1982) Brain Res. 248, 87-94 13 Laduton, P. (1982) Trends Pharmacol. Sci. 3, 351-352 14 Sudo, K., Monsma, F. J. and Katzenellenbogen, B. S. (1983) Endocrinology l 12,425.-434 15 Snyder, S. H., and Goodman, R. R. 0 9 8 0 ) J . Neurochem. 35, 5-15
The author was born in 1936 in Naraur, Belgmm and graduated in medicine at the Umverstty of Louvain in 1961. After having worked with A. F. De Schaepdryver at the Heymans Institute of Pharmacology in Ghent frorn 1964to 1968, he obtamed the degree o f Agr~g~ at the Universityo f Louvain in 1969. At that time he Ioined the Janssen Pharmaceutica Research Laboratories m Beerse where he ~sHead o f the pharmacology Research Group.
i
Electrophysiology of noradrenaline r e l e a s e from s m o o t h m u s c l e organs P. Illes Department of Pharmacology, Umversity o/ Freiburg, Hermann-Herder-Strasse 5, D- 7800 Freiburg ~.Br. FRG.
In the last decade much interest has been focused on the mechanism of noradrenaline release from sympathetic nerve terminals of smooth-muscle organs. Preparations used include the vasa deferentia of various species, the cat nictitating membrane and various vascular preparations. The vas deferens and the nictitating membrane serve as suitable models for studying noradrenergic synapses with possible implications for similar structures in the central nervous system. Experiments on vascular preparations have the additional benefit of offering insight into the local control of circulation. In smooth-muscle organs, a
wide range of endogenous substances are able to modulate the nerve stimulationinduced release of noradrenaline. The majority of our knowledge concerning these mechanisms derives from the biochemical measurement of transmitter release. The procedure most commonly employed is to incorporate radiolahelled noradrenaline into the transmitter pool and subsequently measure the outflow of radioactivity in response to stimulation. More refined biochemical methods have been used less frequently to evaluate the minute amounts of endogenous noradrenaline released.
The approach of the electrophysiologist is different ~'2. He impales the smooth muscle cell with a fine, tipped microelectrode containing a concentrated solution of a potassium salt (Fig. 1). With this electrode the membrane potential of individual smooth-muscle ceils can be continuously recorded. Nerve stimulation evokes transient depolarizing responses called excitatory junction potentials (e.j.ps), which are caused by the post-junctional action of noradrenaline. The transmitter is released from varicose nerve terminals, occupies adrenergic receptors situated on the smooth-muscle membrane and initially triggers an inward, followed by an outward, ionic current resulting in an e.j.p. The amplitude of the e.j.p, is a measure of the conductance change induced by the transmitter, and, thus, a measure of noradrenaline release per impulse. S ~ muscle cells are electrically coupled to each other, with only a fraction of the cells being directly innervated by noradrenergic nerves. However, depolarization caused by the transmitter in an innervated cell, is decrementally conducted to its noninnervated counterparts. Therefore, it is possible to register e.j.ps at any point in the smooth-muscle bundle, although these e.j.ps are the result of transmitter release
© 1983El~vx=~
Publtd~'~B V , Ams~h~l~n 0165- 6147/83/$01(30
336
TIPS - August 1983
1
Fig. 1. lntracellular reeordmg o f excttatory luncUon potentials (e4.ps) from a smooth muscle cell o f the mouse vas deferens. The cell is impaled by a glass microelectrode filled wtth a concentrated solutton o f a potasstum salt. Electrical field stimulatton excites intramural varicose nerve termmals ( varicosities are indicated by open circles). Noradrenaline released on samulanon evokes an e j p. by causing a temporary conductance mcrease o f the post-junctional membrane. When sttmulation voltage ts increased, more nerve fibers are recruited and a htgher amplitude o f e4.ps reflects an increase in the quantity o f noradrenahne released. The black dots between the smooth muscle cells represent the sites where these cells are electrically coupled to each other
from a large number of varicose nerve endings distributed over the organ.
Evidence for the quantal nature of evoked noradrenaline release In most smooth-muscle organs, even in the absence of nerve stimulation, spontaneous depolarizations, resembling e.j.ps occur at random intervals. It is generally believed that noradrenaline is released from the varicose nerve endings in discrete packets or quanta, and that spontaneous e.j.ps (s.e.j.ps) represent the spontaneous quantal release of transmitter. The amplitude histogram of s.e.j.ps is skewed because of electric coupling between smooth-muscle cells, with most of the small amplitude potentials being masked by the noise of the recording electrode. Thus it is impossible to determine a preferred value of s.e.j.p, amplitude, as would be the case with a Gaussian distribution. This in turn excludes the possibility of proving that the e.j.p, is composed of the simultaneous occurrence of several spontaneous potentials. Fortunately, Blakeley and Cunnane 3 noticed that the rising phase of the e.j.p, in the guinea-pig or mouse vas deferens is discontinuous. It was rational to assume, that these discontinuities separated distinct depolarizing responses occurring with different latencies, and represented the quantal release of transmitter from individual varicosities. A statistical evaluation of their data confirmed this assumption. A more direct proof of the quantal nature of transmitter release in noradrenergically innervated organs was provided by Hirst and Neild4. These authors used short isolated segments of arteries taken from the intestinal submucosa of guinea-pigs. These short pieces of arteries were shown to
remain isopotential during current injection, and no attenuation of s.e,j.p, amplitude occurred with distance from the junctional current source. According to the postulates of the quantal theory of transmitter release, the s.e.j.p, amplitudes were unimodally distributed, and the amplitude of s.e.j.ps was similar to that of the smallest e.j.p.
Postjunctional adrenoceptors Smooth-muscle organs have been shown to contain both pre-junctional (predominantly a~-) and post-junctional (at- and a~-) adrenoceptors. These 2 subtypes of receptors have different pharmacological characteristics and can easily be differentiated by means of specific ligands. In order to study post-junctional adrenoceptors, noradrenaline can be locally applied to a small area of the smoothmuscle membrane by use of a fine-tipped micropipette, filled with a strong solution of the amine. When an anodic current is passed through the pipette a depolarizing response can be recorded with a second microelectrode, positioned in close proximity to the drug application site. Prazosin, an al-adrenoceptor antagonist, abolished noradrenaline-evoked depolarizations in the mouse vas deferens, without affecting nerve-stimulation evoked e.j.ps or spontaneous e.j.ps 5. Since iontophoretic pulses of short duration failed to evoke a detectable depolarization, noradrenaline had to be ejected by pulses of rather long duration. Thus, the amine may have reached extrajunctional receptors of the at-subtype, which are, in contrast to junctional receptors, blocked by prazosin. As a possible explanation of the well known resistance of e.j.ps and s.e.j.ps to a-adrenoceptor block-
ade, the existence of a second motor transmitter in addition to noradrenaline, has been considered. For approximately 20 years the inability of a-adrenoceptor antagonists to inhibit e.j.ps in the vas deferens, was attributed to a peculiarity of noradrenergic transmission in this organ. However, similar results have recently been acquired with a series of small arteries6. Although, in some of these preparations a-blockade by phentolamine abolished nerve stimulation-induced constriction, the e.j.p, amplitude was in all cases unaffected. It was suggested that phentolamine depresses the contractile response in view of its direct effect on the smooth muscle, possibly by uncoupling some step in the excitation-contraction coupling mechanism. In contrast to the results obtained with the vas deferens, in submucosal arteries of the guinea-pig intestine it was possible to mimic the amplitude and shape of an e.j.p. by iontophoretic application of noradrenaline from high resistance micropipettes7. Apparently, in this case responses to exogenous and endogenous noradrenaline were evoked via the same adrenoceptor population (junctional receptors). A visualization of catecholamine fibers after iontophoresis revealed that a depolarizing response to noradrenaline could be elicited only at regions close to the sympathetic nerves. At a non-innervated region, noradrenaline application evoked only a constriction in the absence of a detectable depolarization, possibly by an action on extra-junctional receptors. Depolarizing responses to nerve stimulation or noradrenaline application to junctional sites were insensitive to phentolamine, whereas extra-junctional receptors were blocked by this antagonist. It was suggested, that extra-junctional receptors are of the classical a-type, while junctional receptors represent a new class of adrenoceptor. These findings, if extended to the vas deferens, may render unnecessary hypotheses which assume that this organ is non-adrenergically innervated. It may be suggested that, generally, in organs where individual adrenergic varicosities are close to the muscle, the formation of junctional adrenoceptors is favoured. In large arteries, having a wide junctional cleft, no e.j.ps can be recorded. However, contractions in response to nerve stimulation of bath-applied noradrenaline are specifically blocked by a-adrenoceptor antagonists in a specific manner.
Prejunctional adrenoceptors An activation of pre-junctional a2receptors was shown to inhibit the nerve stimulation induced release of nor-
337
TIPS -August 1983
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Fig. 2. Amplitudes of e.j.ps elicited by trams of field pulses at 3 Hz m the mouse vas deferens. In A e.j.ps were ehcued before (C) and after ( Pra.), the addition of 0.5 prazosin. Inset shows a typical record before the addition of prazosin. In B e.j.ps were elicited before (CO and after the addition of euher O.1 ~ ( Yoh. 0.1) or 1 I.*M()'oh. 1) yohimbine or no drug (C O. From Ref. 5.
adrenaline and in a consequence also e.j.p. amplitudes in various smooth muscle preparations. TMs inhibitory action can be readily antagonized by ~2-adrenoceptor antagonists. Electrical stimulation of the intramural nerves in the mouse vas deferens with trains of 15 pulses at 3Hz elicited e.j.ps which were subject to facilitations (Fig. 2). All e.j.ps in a train were depressed by the c~2agonist clonidine. The otl-antagonistprazosin did not significantlyalter the e.j.p, amplitudes (Fig, 2A), whereas the ot2antagonist yohimbine clearly increased the response to the 4th, and later, pulses in a train (Fig. 2B). In vasa deferentia from reserpine-treated mice, the e.j.p, amplitudes were altered in much the same way as by yohimbine. Yohimbine did not further increase the e.j.p, amplitudes in these organs, whereas clonidine caused a marked inhibition. These data are in good agreement with the prmjunctional autoreceptor concept, which claims that activation of ot2receptors by previously released noradrenaline leads to an inhibition of subsequent action potential evoked transmitter release. The fact that e.j.ps elicited by the first 2-3 pulses were not enhanced by yohimbine may be due to the necessity for a certain minimal concentration of released noradrenaline to accumulate in the vicinity of the prejunctional receptor. When the tissue noradrenaline stores were depleted by
reserpine, the normal a-adrenergic inhibition was presumably interrupted by the lack of noradrenaline. The post-junctionaleffect of the transmitter was only reduced, since in the narrow junctional cleft even the low' quantities of noradrenaline released from partly depleted varicosities probably sufficed to elicit a small post-junctional response. Both the inhibitory action of ot~agonists and the facilitatory action of a~antagonists have been recently demonstrated in the guinea-pig mesenteric, and ear artery preparations by electrophysiological methods. In addition to noradrenaline, which acts on prejunctional ~-adrenoceptors, a number of other substances (e.g. prostaglandins, muscarinic cholinergic agonists, opiates and opioid peptides) also depress noradrenaline release by an action at their respective receptors. In certain vascular preparations endogenous prostaglandins (PGs) may function as regulator substances in neuromuscular transmissio#. Inhibition of PG synthesis by indorr;etacin enhanced the amplitude of e.j.ps in the guinea-pig mesenteric artery, possibly by eliminating a negative feed-back mechanism. In the guinea-pig vas deferens, PGs interfered with neuromuscular transmission, however, indometacin had no effect on e.j.p, amplitudes produced by repetitive stimulation. Thus, a PG-mediated regulatory mechanism does not seem to be operative.
The effect of opiates and opioid peptides has been thoroughly studied in the mouse vas deferens preparation (see Ref. 9). These ligands depress the e.j.p, amplitude by acting in a naloxone reversible and stereospecific fashion at a pre-junctional opiate receptor. On the other hand the frequency and amplitude of s.e.j.ps is not influenced by these substances. Chronic treatment of mice by morphine rendered the nerve terminals in their vasa tolerant to normorphine. A cross-tolerance with leucine-enkephalin did not occur, suggesting that opiates and certain opioid peptides act at separate receptors. Post-receplor mechanimns The possible mechanisms by which the activation of pre-junctional receptors leads to a depression of noradrenaline release has been discussed at length. The 3 basic alternatives are shown in Fig. 3. Opiates or a~-agonists failed to alter the action potential recorded extracellularly from small nerve bundles of the vas deferens, in concentrations clearly depressing ej.p. amphtudes. Thus a local anaesthetic action of these agents is unlikely, however, the possibility of a specific blockade of action potential propagation from terminal axons to varicosities must be taken into consideration (see Ref. 10). Whereas nerve stimulation-induced transmitter release is directly proportional to the concentration of C#+-ions in the
338
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noradrenaline at the sub-junctional membrane is certainly a most reliable indicator for transmitter effects. The registration of e.j.ps supplies us with information about the amount of transmitter released per impulse and the s.e.j.p, frequency is the equivalent of spontaneous quantal transmitter release. Thus, in a number of cases electrophysiology is the method of choice for studying transmitter release in smoothmuscle organs.
Reading list
Fig 3. An action potential invades the varicostty and causes an increase in the axoplasmic concentration o f free calctum ([Ca~+]O wa a temporary permeability increase o f the varicosity membrane, resulting in an inward flux o f extracellular calcium ([Ca ~+]o). The mcreased axoplasmic calctum level trtggers the release o f vestcular noradrenahne (NA). Noradrenaline crosses the juncttonal cleft, occupies post-juncttonal adrenoceptors, andevokesane.j.p Possiblemechanismsbywhichtheactivationofpre-tunctionalreceptors leads to a depression o f noradrenaline release. 1: Specific blockade o f action potential propagation to the varicostty. 2: Interference wtth the mflux o f Ca 2+through voltage-dependent calctum channels. 3: Interference with the sequestratton o f Ca 2+into an mtraceUular pool other than the mttochondrium.
medium, the depression of e.j.p, amplitudes by prostaglandins and opiates shows an inverse dependence on external Ca z+. In general, manipulations designed to increase intracellular free calcium enhanced the e.j.p, amplitude and prevented the inhibitory action of these agents n. Therefore, transmitter release may be inhibited by a reduction in the supply of Ca2+-ions to the stimuhls-release coupling mechanism. Mg: m~d Ca 2+ were suggested to compete for a pre-junctional site X which is responsible for the influx of Ca ~÷ through voltagedependent channels. By contrast, normorphine and PGE~ inhibited nerve stimulation-induced transmitter release by a different mechanism, as shown by their non-competitive interference with the effect of Ca 2÷. The s.e.j.p, frequency was reduced by Mg 2., implying that a significant portion of spontaneous noradrenaline release is the consequence of calcium entry into the varicosity. Both normorphine and PGE1 failed to affect the s.e.j.p, frequency, again in favour of a different mode of action from that of Mg 2+. A third alternative is that some of these agents may inhibit the binding of calcium to an intracellular site. Electrophysiological
evidence obtained in enteric ganglia and locus coeruleus neurons favours the existance of such a site, which may be important for buffering increased intracellular free Ca2+-concentrations during neuronal activityTM. Inhibition of the Ca 2÷sequestration would prolong the period of increased intracellular Ca 2+, and in a consequence would reduce the concentration gradient between extra- and intra-cellular Ca ~÷. This would result in a reduced driving force for Ca 2+- influx into the cell and also in a depression of stimulus-release coupling. Moreover, the enhanced free calcium concentration could activate a calcium-dependent potassium conductance, which in turn, would hyperpolarize the resting membrane and lead to a block of action potential propagation.
1 Holman, M. E. (1970) inSmooth Muscle (Biilbrrag, E., Brading, A. F. and Jones, A. W., eds), pp. 244-288, Edward Arnold, London 2 Burnstock, G. and Bell, G. (1974) in The Peripheral Nervous System (Hubbard, J. 1., ed.), pp. 277-327, Plenum Press, New York, London 3 Blakeley, A. G. H and Cunnane, T. C. (1979)J. Physiol (London) 296, 85-96 4 Hirst, G. D. S. and Neild, T. O. (1980)J. Phystol. (London) 303, 43-60 5 Illes, P. and Starke, K. (1983) Br. J. Pharmacol. 78,365-373 6 Holman, M. E. and Surprenant, A. (1980) Br. J. Pharmacol. 71,651-661 7 Hirst, G. D. S and Neild, T. O. (1981)J. Physiol. (London) 313, 343-350 8 Kuriyama, H and Makita, Y. (1982)J. Physiol. (London) 327, 431-448 9 flies, P. (1982) m Regulatory Peptides: From Molecular Biology to Function (Costa, E. and Trabucchi, M,, eds), pp. 347-352, Raven Press, New York 10 files, P. and North, R. A. (1982) Br. J. Pharmacol. 75, 599--604 11 Illes, P., Zieglgansberger, W. and Herz, A. (1980) Brain Res. 191,511-522 12 Williams, J. T., Egan, T. M. and North, R. A. (1982)Nature (London) 299, 74--77
Conclusion The investigation of mechanisms that modulate the release of noradrenaline requires a multi-disciplinary approach. Although biochemical and contraction measurements have yielded a wealth of information, it is important to supplement them with electrophysiological methods. The conductance increase induced by
Peter llles received his M D. and Ph.D degrees from the University o f Budapest under Professor J. Knoll. He worked as an Assistant Professor o f Pharmacology at the Untverstty untd 1978. He subsequently spent a year at the Universtty o f Lund, Sweden, and 2 years at the Max.Planck Institute o f Psychiatry, Munich. In 1981 he moved to the Department o f Pharmacology, University o f Fretburg. His main interest is in neurotransmission.