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Calcitonin gene-related peptide evokes distinct types of excitatory response in guinea pig coeliac ganglion cells
256
Brain Research, 476 (1989) 256-264 Elsevier
BRE 14134
Calcitonin gene-related peptide evokes distinct types of excitatory response in guinea pig coeliac ganglion cells N.J. Dun and N. Mo* Department of Pharmacology, Loyola UniversityStritch Schoolof Medicine, Maywood, IL 60153 (U.S.A.) (Accepted 28 June 1988) Key words: Calcitonin gene-related peptide; Sympathetic ganglion; Fast excitatory response; Slow excitatory response
Pressure application of calcitonin gene-related peptide (CGRP) evoked in a population of guinea pig coeliac neurons 3 types of response: a fast, a slow and a biphasic depolarization. The responses were not appreciably affected in low Ca/high Mg or tetrodotoxincomaining Krebs solution. The fast depolarization was associated with a fall in membrane resistance; it was made larger on hyperpolarization and the estimated reversal potential was-24 InV. The fast response was reversibly blocked in a Na-free medium as well as by relatively high concentrations of d-tubocurarine (50-100 pM) but not by hexamethonium. The slow, CGRP-induced depolarization resistant to nicotinic and muscarinic antagonists, was associated with either a small increase or decrease of input resistance. Membrane hyperpolarization increased the slow response in the majority of coeliae neurons, with an estimated reversal potential of -44 inV. The biphasic depolarization displayed electrophysiological and pharmacological characteristics resembling the fast and slow responses. These results raise the possibility that CGRP acting via two distinct types of receptor elicits, respectively, a fast, Na-dependent excitatory response and a slow response, the mechanism of which remains to be established.
INTRODUCTION Mammalian sympathetic, particularly the prevertebral (coeliac-superior mesenteric and inferior ruesenteric) ganglia appear to be a repertoire for peptides. For example, immunoreactivities to bombesin, substance P, neurokinin A, cholecystokinin, vasoacrive intestinal polypeptide, enkephalin, dynolphin, gastrin-releasing peptide, and calcitonin gene-related peptide ( C G R P ) have been detected in nerve plexuses of prevertebra! ganglia of the guinea pig and rat 3'9'u'lS'ls'19'22. Electrophysiologica! studies from various laboratories show that the predominant effect of peptides, e.g. substance P, cholecystokinin, neurokinin A, and vasoactive intestinal polypeptide on ganglionic neurons is to cause a slow depolarization and an increased membrane excitability 5,12.13, 19,24
We report here that the action of C G R P , a 37 amino acid peptide widely distributed in the central
and peripheral nervous systems, including the prevertebral ganglia 7,1°,u,15,2°, appears to be more complex. In addition to causing a slow depolarization characteristic of the response evoked by other peptides, C G R P induces a fast, sodium-dependent response that has not been described with other peptides heretofore. MATERIALS AND METHODS Adult male guinea pigs (200-300 kg) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The coeliac-superior mesenteric plexus together with the left greater splanchnic nerves were excised rapidly and transferred to the recording chamber 4. Immediately thereafter the animal was decapitated. The ganglia were superfused continuously with a Krebs solution of the following composition (in raM): NaCI 117, KCI 4.7, CaCI 2 2.5, MgCI2 1.2, N a H C O 3 25; NaH2PO 4 1.2 and glucose 11.5; the solution was
* Present address: Department of Pharmacology, Guangxi Medical College, People's Republic of China. Correspondence: N.J. Dun, Department of Pharmacology, Loyola University Medical Center, Maywood, IL 60] 53, U.S.A.
0006-8993/89/$03.[0 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
257 gassed with 95% 02/5% CO 2 and the temperature of the solution reaching the preparation was maintained at 34 + 1 °C. In experiments in which Na-free medium was used, NaCI, NaHCOa and NaH2PO 4 were replaced with an isomolar amount of Tris-(hydroxymethyl) aminomethane (Sigma), which was converted to Tris-HC! by titration with 1 N HCi t o pH 7.4. In preparing the low Cl solution, NaCl was replaced by Na isothionate. Intracenular recordings were obtained from neurons of the left and right coeliac ganglia by means of fiber-containing glass micro-electrodes filled with 2 M K citrate, with an impedance of 30-60 Mf~. Membrane potentials were amplified via a WPI-707A preamplifier and displayed on a Gould Digital Oscilloscope and a Gould pen recorder, The figures were reproduced from tracings of the pen recorder. CGRP was applied to the ganglion cells by pressure ejection (Picospritzer, General Valve Co.) from a micropipette containing 0.1 mM CGRP. The peptide was ejected onto the ganglion cell from the micropipette using a constant pressure (40 Psi) but variable duration (10-900 ms) of nitrogen gas under visual control. In a few experiments, CGRP was applied to the ganglion cells by superfusion in the concentrations of 0.1-10 ~M. Acetylcholine (ACh) was applied to the ganglion cells by pressure from a micropipette containing 100 mM ACh. Other pharmacological agents were dissolved in Krebs solution and applied by superfusion only. The following compounds were purchased from Sigma: acetylcholine chloride, atropine sulfate, eserine sulfate, hexamethonium bromide, d-tubocurarine chloride, and te-
trodotoxin. The peptide CGRP was obtained from Peninsula Laboratories. RESULTS CGRP was applied by pressure to 86 coeliac ganglion cells of which 31 responded in one of the following 3 ways: a fast depolarization (n = 10, 32%), a slow depolarization (n = 17, 55%) and a biphasic response consisting of the first two responses in succession (n = 4, 13%). Representatives of each type of response a,e shown in Fig. 1. The cells studied had a mean resting membrane potential and input resistance o f - 5 4 + 4.5 mV and 43 + 16 MQ. Generally, CGRP delivered by a short pulse of a few to less than 200 ms in duration was sufficient to elicit a response in sensitive neurons. In neurons insensitive to short pulses of CGRP, increasing the pulse duration to as long as 900 ms induced no detectable response. Furthermore, CGRP (0.1-10/~M) applied by superfusion to 15 randomly selected coeliac neurons that had not responded to pressure application, likewise failed to produce a positive response.
Characteristics of the fast depolarization by CGRP This type of response had a relatively rapid onset (0.4 _+ 0.2 s, mean + S.D., here and subsequently) and a brief duration (4 _+ 1.3 s). The CGRP-induced fast depolarization had a time course comparable to that of ACh-induced depolarization (see Fig. 5). When recorded at the resting membrane potential between -50 and -60 mV, the mean amplitude was 13 -4- 2.7 inV. Spike discharge could often be seen on
__/ t CGRP 8ms
|1
t 15ms
t 20ms 2s
20s
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Fig. 1. Three types of depolarizing responses induced by CGRP in coeliac neurons. Solid triangles indicate duration of ejection pulse in ms. A: fast depolarization accompanied by cell discharge. B: slow depolarization associated with cell discharge. C: hiphasic response consistingof an initial, fast depolarization with spike discharge followedby a slow, smaller depolarization. Note different time scales. The peak amplitude of spike discharge in this and subsequent figures were truncated because of the limited frequencyof the pen recorder. Records A, B and C were taken from 3 different coeliac neurons.
258 the rising phase of the CGRP-induced depolarization (Figs. 1-4). Neither the amplitude nor the duration of CGRP-induced depolarizations were significantly changed in a low Ca (0.25 mM)/high Mg (12 m M ) Krebs solution (Fig. 2B) or tetrodotoxin (TI'X, 0.1 /~M)-containing solution (n = 4). A small hyperpolarization ranging from 2 to 4 mV in amplitude could be detected following the fast depolarizing response in 3 cells (see Figs. 4 and 5). Although this response was not investigated systematically, it was noted that the amplitude was related to the magnitude of the preceding depolarization (for example, Fig. 4). The fast depolarization was associated with a marked fall in membrane input resistance in all 4 neurons investigated (Fig. 3A). This is indicated by a re-
duction of the amplitude of hyperpolarizing electrotonic potentials evoked by hyperpolarizing current pulses ~uring the CGRP-induced depolarization. On the other hand, depolarization of the membrane by current injection to the same potential level as that achieved by C G R P resulted in no apparent decrease of the amplitude of electrotonic potentials, suggesting that the decline was not due to m e m b r a n e depolarization per se (Fig. 3A). The relationship between the amplitude of CGRPinduced depolarization and membrane potential was roughly linear, i.e, membrane depolarization and hyperpolarization de,:reased and increased, respectively, the response (Fig. 3B). The estimated reversal potential w a s - 2 4 + 4 mV (n -- 4).
A
m
A 20ms
A CGRP,5Oms
Low Ca
._k_._ A
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Fig. 2. Lacg ot ettects of low Ca solution on fast and slow depolarizations. A: pressure application of CGRP elicited in this cell a slow depolarization. Downward deflections represent hyperpolarizing electrotonic potentials induced by constant current pulses (not shown). Note there was a small (25%) increase of the amplitude of electrotonic potentials at the peak of CGRP-induced depolarization, indicating increased membrane input resistance. The CGRP-induced slow depolarization evoked in a low Ca (0.25 mM) solution remained the same. B: CGRP evoked a fast depolarization in this cell. Again, low Ca solution had no appreciable effect on the amplitude or time course of the CGRP response. Records A and B were taken from two different coeliac neurons.
259 A
noticeably changed in a low C! solution in 2 cells tested. t
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Fig. 3. Electrophysiological characteristics of fast CGRP-induced depolarizations, Cell A: fast depolarization was accompanied by a marked decrease of membrane input resistance as indicated by a reduction of the amplitude of hyperpolarizing electrotonic potentials. After the response had subsided, depolarization of the membrane to the same potential level seen at the peak of the response did not cause any appreciable change of the electrotonic potential amplitude. Cell B: membrane hyperpolarization increased the amplitude of CGRP-induced fast depolarization. Numerals to the left of each trace denote the membrane potential at which the response was elicited. Graph to the right: amplitude of CGRP response against membrane potential, The intercept of the line fitted by eye indicates the reversal potential, i.e. -24 inV. Records A and B were taken from two different cells. The possible ion species involved in the CGRP-induced fast depolarization were investigated. First, the effects of C G R P on coeliac neurons before and after superfusing the ganglia with a Na-free, TrisHC! Krebs solution were examined. Fig. 4B shows the depolarizing action of CGRP was reversibly abolished in a Na-free medium; a similar result was obtained in 3 other cells. Changing the Krebs solution to a K-free solution by omitting the KCI also reduced the CGRP-induced depolarization by a mean of 26 _+ 7% (n = 3). The response was however not
Effects of cholinergic antagonists and anticholinesterase agent Experiments were initially carried out with d-tubocurarine (d-To) to test whether the fast depolarization caused by CGRP was due secondary to a presynaptic release of ACh. Unexpectedly, d-To at relatively high concentrations suppressed the CGRP-induced depolarization in a dose-dependent manner (Fig. 4A). At the concentrations of 10, 30 and 100 /~M, d-Tc reduced the fast depolarization by 21 + 5% (n = 3), 44 + 6% (n = 4) and 92 + 6% (n = 4), respectively. The depressant effect of d-Tc was reversible upon wash (Fig. 4A). On the other hand, hexamethonium in comparable concentrations caused no appreciable reduction of the CGRP-induced fast depolarization in any of the 4 cells tested. Similarly, atropine (1 #M) was without effect on the fast depolarization in all 3 cells examined. If the CGRP-induced fast depolarization was due secondary to a release of ACh, the response should be enhanced by the anticholinesterase agent eserine 14. In the concentrations of 1 and 5/zM, eserine had no appreciable effect on the fast response induced by CGRP in any of the 3 cells examined (Fig. 5B). In contrast, eserine (1/~M) markedly increased the amplitude and prolonged duration of the depolarization induced by ACh in two cells tested (Fig. 5A).
Characteristics of the slow depolarization by CGRP CGRP elicited a slowly rising arid falling depolarization in slightly over half of the responsive neurons. This type of response had a mean latency, duration and amplitude of 2.6 + 1.6 s, 69 + 7 s and 10 +__3.2 mV (n = 12), respectively. Spike discharges appeared occasionally at the rising or plateau phase of the slow response (e.g. Fig. 1). The slow response was not significantly changed in a low Ca solution or TI'X-containing solution (Fig. 2A). The amplitude before and after low Ca/high Mg solution was 11 + 1.8 mV and 10 + 2.1 mV (n = 3), respectively. Unlike the fast depolarization, the electrophysiological characteristics of the slow CGRP response were somewhat variable depending on the cells studied. When the membrane potential was manually clamped at the resting level, either a small increase
260 A
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Fig. 4. Suppression of fast CGRP-induced depolarizations by d-Tc and Na-free solution. Cell A: d-Tc 10, 30 and 100/~M attenuated the CGRP-induced fast depolarization in a concentration-dependent manner. At the highest concentration used, d-Tc nearly abolished the response. The effect was reversible upon wash. Numerals between the traces indicate the duration of drug application or wash in minutes. Cell B: superfusing the ganglion with a Na-free, Tris-HCI solution blocked the CGRP-induced fast depolarization, and the effect was reversed after wash. Records A and B were obtained from two different coeliac neurons.
A
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Fig. 5. Potentiationof ACh depolarizationbut not CGRP responseby eserine.Cell A: membranedepolarizationwas inducedby pressure application of ACh from a micropipette containing 100 mM ACh. Eserine increased the amplitude as well as the duration of AChinduced depolarization. The response was blocked by d-Tc 10 gM. Cell B: CGRP-induced fast depolarization was not enhanced by eserine, although it was depressed by d-Tc 50/~M. Numerals between traces denote the duration of drug application or wash in minutes. Records A and B were taken from two different coeliac neurons.
261 A
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Fig. 6. Membrane resistance change and effects of membrane potential on CGRP-induced slow depolarization. A: upper trace represents voltage recording and lower trace current recording. Downward deflections are hyperpolarizing electrotonic potentials evoked by constant current pulses (downward deflections of lower trace). CGRP elicited a slow depolarization and at the peak of the response there was a slight increase in membrane resistance as indicated by a small (18%~ increase of the amplitude of electrotonie potenti~!s. When the membrane potential was manually clamped at the resting membrane potential level of-54 mV, the CGRP response was associated with a transient, small (15%) decline followed by a more sustained, also small (12%) increase of input resistance. B: hyperpolarization and depolarization from the initial resting membrane potential of-64 mV increased and decreased the CGRP-induced slow depolarization. Graph of depolarization amplitude against membrane potential is shown to the right. Extrapolation of the fine indicates a reversal potential of-42 inV. Records A and B were taken from two different coeliac neurons.
or decrease ( 1 0 - 3 0 % ) of the input resistance during the slow C G R P - i n d u c e d response was observed in different cells (Fig. 6A). M o r e o v e r , the relationship b e t w e e n the a m p l i t u d e and m e m b r a n e potential was
,o' '
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also n o t uniform. In 4 of the 6 coeliac n e u r o n s , the slow depolarization b e c a m e larger o n m e m b r a n e hyperpolarization; the extrapolated m e a n reversal potential was - 4 4 m V (Fig. 613). M e m b r a n e hyperpo-
. . . . '
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Fig. 7. Biphasic response of a guinea pig coeliac neuron to pressure application of CGRP. CGRP evoked a fast, transient depolarization followed by a more slowly declining response. Superfusing the ganglion with d-Tc blocked the fast, transient phase without noticeably altering the slow phase. The effect was reversible after wash.
262 larization reduced the slow CGRP response in one cell, and in another the response was not substantially changed. With respect to the cholinergic antagonists, d-Tc at the concentration as high as 100 gM caused little or no change of the slow response in all 4 cells tested. Hexamethonium and atropine likewise had no effect on the slow depolarization (n = 3).
Characteristics of the biphasic response A biphasic response to CGRP was noted in 4 coeliac neurons (Figs. 1C and 7). The biphasic response had the appearance of the fast and slow depolarizations mentioned above. The initial fast rising depolarization was followed by a smaller, more sustained depolarization. The fast component was reversibly abolished by d-Tc, leaving the slow component intact (Fig. 7). Thus, the biphasic response appeared to be a composite of the fast and slow depolarization. DISCUSSION The peptide CGRP evoked 3 types of excitatory responses with distinct electrophysiological and pharmacological characteristics in a population of coeliac neurons. Unlike the finding in guinea pig myenteric ganglia where Type 2 neurons appeared to be uniformly sensitive to the peptide roT,less than half of the coeliac ganglion cells responded to CGRP. This may imply a selective distribution of CGRP receptors to a subpopulation of coe!iac neurons. Moreover, CGRP elicited in myenteric neurons only a slow depolarization similar in appearance to toe slow response observed in our study 17. On the other hand, CGRP caused no apparent change of the membrane vo,e,,,,a, u u t p,o,on~ed the duration of the calciumdependent action potential of the cat parasympathetic ganglion cells 16. As a corollary, CGRP may subserve a diverse function in various peripheral autonomic ganglia. Differences in their latency and duration notwithstanding, the fast and slow response displayed distinct electrophysiological and pharmacological characteristics. The fast response was always associated with a marked reduction in membrane resistance, it was increased by hyperpolarization and the estimated reversal potential was about -20 mV. These findings together with the observations that the re-
sponse was redu~:ed in a Na- and K-free, but not low Cl-solution saggest that the peptide increased membrane pe!meability to Na and K ions. This aspect of CGRP action closely resembles the nicotinic action of ACh on autonomic neurons. The observation that d-Tc, a compound frequently regarded as a nicotinic blocker, antagonized the CGRP-induced fast depolarization was somewhat unexpected. The action of CGRP however is not fikely to be mediated by ACh acting on nicotinic receptors for the reasons that the response was not antagonized by another nicotinic blocker hexamethonium nor potentiated by the anticholinesterase agent eserine, whereas the latter augmented the ACh-induced depolarization. It should be noted that d-Tc has been shown to be a non-selective blocker; it antagonizes the action of a variety of putative transmitters including histamine, ACh, and 5-HT in the vertebrate and invertebrate neurons m'23. In Aplysia neurons d-Tc antagonizes the action of a variety of putative transmitters probably by blocking the receptor-activated Na or Cl channels, rather than blocking the different receptors m. Our finding that the CGRP-induced fast depolarization was eliminated in a Na-free solution is consistent with this interpretation. The slow depolarizations induced by CGRP display characteristics that are similar to the slow responses evoked by a number of peptides on prevertebral ganglion cellss'12'13'17'19 and on myenteric neu.tons 17'24. The mechanism underlying the slow CGRP-induced response appears to be less straightforward. The input resistance change irrespective an increase or decrease during the CGRP-induced slow depolarization was modest. Furthermore, membrane hyperpolarization produced variable effects on the slow, CGRP-induced response in different cells; while an increase of the slow response was observed in the majority of cells tested, a decrease or no apparent change was also noted in a few cells. Interestingly, similar findings have been reported with respect to the substance P-induced slow depolarization 5. The mechanism(s) underlying the CGRP-induced slow depolarization in prevertebral ganglion cells is not clear. In the case of substance P response, an increase of Na and a decrease of K membrane permeability has been suggested 5. On the other hand, the primary mechanism underlying the CGRP-induced
263 slow depolarization in the myenteric neurons appears to be an inactivation of Ca-activated K conductance 17. A s the fast and slow depolarization differed with respect to their electrophysiological and pharmacological characteristics, two subpopulations of C G R P receptors may be involved. The biphasic response observed in a small number of neurons would indicate the co-existence of two subsets o f C G R P receptors in a single neuron. Alternatively, one class of C G R P r e c e p t o r may couple to different sets of channels and/or intracellular transducing mechanisms, thereby giving rise to different responses. The question whether C G R P is released from immunoreactive fibers in the coeliac ganglion by physiological stimuli remains to be answered. Also not known is whether synaptically released C G R P may elicit two types of response as shown here by exogenous application. Interestingly, denervation studies show that CGRP-immunoreactive fibers in the coeliac ganglia may arise as collateral branches of afferent fibers from dorsal root ganglion cells some of which contain substance P-immunoreactivities as
REFERENCES 1 Ascher, P., Marty, A. and Neild, T.O., The mode of action of antagonists on the excitatory response to acetylcholine in Aplysia neurones, J. Physiol. (Lond. L 278 (1978) 207-235. 2 Carpenter, D.O., Swann, J.W. and Yarowsky, P.J., Effects of curare on responses to different putative transmitters in Aplysia neurons, J. Neurobiol., 8 (1977) 119-132. 3 Dalsggard, C.J., H6kfelt, T., Schultzberg, M., Lundberg, J.M., Tercnius, L., Dockray, G.J. and Goldstein, M., Origin of peptide-containing fibccs in the inferior mesenteric ganglion of the guinea pig: immunohistochemical studies with antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin, Neuroscience, 9 (1983) 191-211. 4 Dun, N.J. and Ma, R.C., Slow non-cholinergic excitatory potentials in neurones of the guinea pig coeliac ganglia, J. Physiol. (Lond.), 351 (1984) 47-60. 5 Dun, N.J. and Minota, S., Effects of substance P on neurones of the inferior mesenteric ganglia of the guinea pig, J. Physiol. (Lond.), 231 (1981)259-271. 6 Gibbins, I.L., Furness, J.B., Costa, M., Maclntyre, I., Hillyard, C.J. and Girgis, S., Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pig, Neurosci. Leu., 57 (1985) 125-130. 7 Gibson, S.J., Polark, J.M., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101-3111.
well6'It. In this respect, if the action of C G R P in the peripheral ganglion cells is an indication of its central action at the dorsal horn neurons, the possibility that the peptide may subserve a fast and slow excitatory mode of sensory transmission is intriguing. Finally, it is pertinent to mention that C G R P is one of the few peptides known to date to cause a fast, Nadependent excitatory response. Substance P has been shown to cause a fast and slow depolarization in spinal and myenteric neurons in culture 8"21. Generally, peptides are thought to mediate neuronal events of slow time course. O u r finding in conjunction with the observations made in the cultured central and autonomic neurons with respect to the action of substance p8.21 raise the possibility that neuronal events mediated by certain peptides may be swift, analogous to the action of 'classical' transmitters such as nicotinic A C h on motor endplates and autonomic neurons. ACKNOWLEDGEMENT This study was supported by Grant NS18710 from the Department of Health and Human Services.
8 Hanani, M. and Burnstock, G., Substance P evokes slow and fast responses ~n cultured myenteric neurons of the guinea pig, NeuroscL Lett., 48 (1984) 19-23. 9 Kondo, H., lwanaga, T. and Yanaihara, N., On the occurrence of gastrin releasing peptide (GRP)-Iike immunoreactive nerve fibers in the celiac ganglion of rats, Brain Research, 289 (1983) 326-329. 10 Lee, Y., Hayashi, N., H~llyard, C.J., Girgis, S.I., Maclntyre, I., Emson, P.C. and Tohyama, M., Calcitonin gonerelated peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat coeliac ganglion, Brain Research, 407 (1987) 149-151. 11 Lee, Y., Takami, K., Kawai, Y., Girgis, S.I., Hillyard, C.J., Maclntyre, I., Emson, P.C. and Tohyama, M., Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P, Neuroscience, 15 (1985) 1227-1237. 12 Love, J.A. and Szurszewski, J.H., The electrophysiological effects of vasoactive intestinal polypeptide in the guinea pig inferior mesenteric ganglion, J. Physiol. (Lond.), 394 (1987) 67-84. 13 Mo, N. and Dun, N.J., Cholecystokinin octapeptide depolarizes guinea pig inferior mesenteric ganglion cells and facilitates nicotinic transmission, Neurosci. Left.. 64 (1986) 263-268. 14 Mo, N., Dun, N.J. and Karczmar, A.G., Facilitation and inhibition of nicotinic transmission by eserine in the sympathetic ganglia of the rabbit, Neuropharmacology, 24 (1985) 1093-1101. 15 Muldberry, P.K., Ghatei, M.A., Bishop, A.E., Allen, Y.S., Polak, J.M. and Bloom, S.R., Distribution and chro-
264 matographic characterization of CGRP-Iike immunoreactivity in the brain and gut of the rat, Regul. Peptides, 12 (1985) 133-144. 16 Nohmi, M., Shiunick-Gallagher, P., Gean, P.W., Gailagher, J.P. and Cooper, C.W., Calcitonin and calcitonin generelated peptide enhance calcium-dependent potentials, Brain Research, 367 (1986) 346-350. 17 Palmer, J.M., Schemann, M., Tamnra, K. and Wood, J.D., Calcitonin gene-related peptide excites myenteric neurons, Eur. J. Pharmaeol., 132 (1986) 163-170. 18 Reinecki, M., Forssmann, W.G., Thiekotter, G. and Triepel, J., Localiza~on of neurotensin-immunoreactivity in the spinal cord and peripheral nervous system of the guinea pig, Neurosei. Lett., 37 (1983) 37-42. 19 Saria, A., Ma, R.C., Dun, N.J., Theodorssca-Norheim, E. and Lundberg, J.M., Neurokinin A in capsaicin-sensitive neurons of the guinea pig inferior mesenteric ganglia: an additional putative mediator for the non-cholinergicexcita-
tory postsynaptic potential, Neuroscience, 21 (1987) 951-958. 20 Skofitsch, G. and Jacobowitz, D.M., Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system, Peptides, 6 (1985) 1069-1973. 21 Vincent, J.D. and Barker, J.L., Substance P: evidence for diverse roles in neuronal function from cultured mouse spinal neurons, Science, 205 (1979) 1409-1412. 22 Vincent, S.R., Dalsggard, C.J., Schultzberg, M., H6kfelt, T., Christesson, I. and Terenius, L., Dynorphin-immunoreactive neurons in the autonomic nervous system, Neuroscience, 11 (1984) 973-987. 23 Wallis, D.I. and Dun, N.J., A comparison of fast and slow depolarizations evoked by 5-HT in guinea pig coeliac ganglion cells in vitro, Br. J. Pharmacol., 93 (1988) 110-120. 24 Wood, J.D., Enteric neurophysiology,Am. J. PhysioL, 247 (1984) (3585-(3598.
Brain Research, 476 (1989) 256-264 Elsevier
BRE 14134
Calcitonin gene-related peptide evokes distinct types of excitatory response in guinea pig coeliac ganglion cells N.J. Dun and N. Mo* Department of Pharmacology, Loyola UniversityStritch Schoolof Medicine, Maywood, IL 60153 (U.S.A.) (Accepted 28 June 1988) Key words: Calcitonin gene-related peptide; Sympathetic ganglion; Fast excitatory response; Slow excitatory response
Pressure application of calcitonin gene-related peptide (CGRP) evoked in a population of guinea pig coeliac neurons 3 types of response: a fast, a slow and a biphasic depolarization. The responses were not appreciably affected in low Ca/high Mg or tetrodotoxincomaining Krebs solution. The fast depolarization was associated with a fall in membrane resistance; it was made larger on hyperpolarization and the estimated reversal potential was-24 InV. The fast response was reversibly blocked in a Na-free medium as well as by relatively high concentrations of d-tubocurarine (50-100 pM) but not by hexamethonium. The slow, CGRP-induced depolarization resistant to nicotinic and muscarinic antagonists, was associated with either a small increase or decrease of input resistance. Membrane hyperpolarization increased the slow response in the majority of coeliae neurons, with an estimated reversal potential of -44 inV. The biphasic depolarization displayed electrophysiological and pharmacological characteristics resembling the fast and slow responses. These results raise the possibility that CGRP acting via two distinct types of receptor elicits, respectively, a fast, Na-dependent excitatory response and a slow response, the mechanism of which remains to be established.
INTRODUCTION Mammalian sympathetic, particularly the prevertebral (coeliac-superior mesenteric and inferior ruesenteric) ganglia appear to be a repertoire for peptides. For example, immunoreactivities to bombesin, substance P, neurokinin A, cholecystokinin, vasoacrive intestinal polypeptide, enkephalin, dynolphin, gastrin-releasing peptide, and calcitonin gene-related peptide ( C G R P ) have been detected in nerve plexuses of prevertebra! ganglia of the guinea pig and rat 3'9'u'lS'ls'19'22. Electrophysiologica! studies from various laboratories show that the predominant effect of peptides, e.g. substance P, cholecystokinin, neurokinin A, and vasoactive intestinal polypeptide on ganglionic neurons is to cause a slow depolarization and an increased membrane excitability 5,12.13, 19,24
We report here that the action of C G R P , a 37 amino acid peptide widely distributed in the central
and peripheral nervous systems, including the prevertebral ganglia 7,1°,u,15,2°, appears to be more complex. In addition to causing a slow depolarization characteristic of the response evoked by other peptides, C G R P induces a fast, sodium-dependent response that has not been described with other peptides heretofore. MATERIALS AND METHODS Adult male guinea pigs (200-300 kg) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The coeliac-superior mesenteric plexus together with the left greater splanchnic nerves were excised rapidly and transferred to the recording chamber 4. Immediately thereafter the animal was decapitated. The ganglia were superfused continuously with a Krebs solution of the following composition (in raM): NaCI 117, KCI 4.7, CaCI 2 2.5, MgCI2 1.2, N a H C O 3 25; NaH2PO 4 1.2 and glucose 11.5; the solution was
* Present address: Department of Pharmacology, Guangxi Medical College, People's Republic of China. Correspondence: N.J. Dun, Department of Pharmacology, Loyola University Medical Center, Maywood, IL 60] 53, U.S.A.
0006-8993/89/$03.[0 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
257 gassed with 95% 02/5% CO 2 and the temperature of the solution reaching the preparation was maintained at 34 + 1 °C. In experiments in which Na-free medium was used, NaCI, NaHCOa and NaH2PO 4 were replaced with an isomolar amount of Tris-(hydroxymethyl) aminomethane (Sigma), which was converted to Tris-HC! by titration with 1 N HCi t o pH 7.4. In preparing the low Cl solution, NaCl was replaced by Na isothionate. Intracenular recordings were obtained from neurons of the left and right coeliac ganglia by means of fiber-containing glass micro-electrodes filled with 2 M K citrate, with an impedance of 30-60 Mf~. Membrane potentials were amplified via a WPI-707A preamplifier and displayed on a Gould Digital Oscilloscope and a Gould pen recorder, The figures were reproduced from tracings of the pen recorder. CGRP was applied to the ganglion cells by pressure ejection (Picospritzer, General Valve Co.) from a micropipette containing 0.1 mM CGRP. The peptide was ejected onto the ganglion cell from the micropipette using a constant pressure (40 Psi) but variable duration (10-900 ms) of nitrogen gas under visual control. In a few experiments, CGRP was applied to the ganglion cells by superfusion in the concentrations of 0.1-10 ~M. Acetylcholine (ACh) was applied to the ganglion cells by pressure from a micropipette containing 100 mM ACh. Other pharmacological agents were dissolved in Krebs solution and applied by superfusion only. The following compounds were purchased from Sigma: acetylcholine chloride, atropine sulfate, eserine sulfate, hexamethonium bromide, d-tubocurarine chloride, and te-
trodotoxin. The peptide CGRP was obtained from Peninsula Laboratories. RESULTS CGRP was applied by pressure to 86 coeliac ganglion cells of which 31 responded in one of the following 3 ways: a fast depolarization (n = 10, 32%), a slow depolarization (n = 17, 55%) and a biphasic response consisting of the first two responses in succession (n = 4, 13%). Representatives of each type of response a,e shown in Fig. 1. The cells studied had a mean resting membrane potential and input resistance o f - 5 4 + 4.5 mV and 43 + 16 MQ. Generally, CGRP delivered by a short pulse of a few to less than 200 ms in duration was sufficient to elicit a response in sensitive neurons. In neurons insensitive to short pulses of CGRP, increasing the pulse duration to as long as 900 ms induced no detectable response. Furthermore, CGRP (0.1-10/~M) applied by superfusion to 15 randomly selected coeliac neurons that had not responded to pressure application, likewise failed to produce a positive response.
Characteristics of the fast depolarization by CGRP This type of response had a relatively rapid onset (0.4 _+ 0.2 s, mean + S.D., here and subsequently) and a brief duration (4 _+ 1.3 s). The CGRP-induced fast depolarization had a time course comparable to that of ACh-induced depolarization (see Fig. 5). When recorded at the resting membrane potential between -50 and -60 mV, the mean amplitude was 13 -4- 2.7 inV. Spike discharge could often be seen on
__/ t CGRP 8ms
|1
t 15ms
t 20ms 2s
20s
lOmV 2s
Fig. 1. Three types of depolarizing responses induced by CGRP in coeliac neurons. Solid triangles indicate duration of ejection pulse in ms. A: fast depolarization accompanied by cell discharge. B: slow depolarization associated with cell discharge. C: hiphasic response consistingof an initial, fast depolarization with spike discharge followedby a slow, smaller depolarization. Note different time scales. The peak amplitude of spike discharge in this and subsequent figures were truncated because of the limited frequencyof the pen recorder. Records A, B and C were taken from 3 different coeliac neurons.
258 the rising phase of the CGRP-induced depolarization (Figs. 1-4). Neither the amplitude nor the duration of CGRP-induced depolarizations were significantly changed in a low Ca (0.25 mM)/high Mg (12 m M ) Krebs solution (Fig. 2B) or tetrodotoxin (TI'X, 0.1 /~M)-containing solution (n = 4). A small hyperpolarization ranging from 2 to 4 mV in amplitude could be detected following the fast depolarizing response in 3 cells (see Figs. 4 and 5). Although this response was not investigated systematically, it was noted that the amplitude was related to the magnitude of the preceding depolarization (for example, Fig. 4). The fast depolarization was associated with a marked fall in membrane input resistance in all 4 neurons investigated (Fig. 3A). This is indicated by a re-
duction of the amplitude of hyperpolarizing electrotonic potentials evoked by hyperpolarizing current pulses ~uring the CGRP-induced depolarization. On the other hand, depolarization of the membrane by current injection to the same potential level as that achieved by C G R P resulted in no apparent decrease of the amplitude of electrotonic potentials, suggesting that the decline was not due to m e m b r a n e depolarization per se (Fig. 3A). The relationship between the amplitude of CGRPinduced depolarization and membrane potential was roughly linear, i.e, membrane depolarization and hyperpolarization de,:reased and increased, respectively, the response (Fig. 3B). The estimated reversal potential w a s - 2 4 + 4 mV (n -- 4).
A
m
A 20ms
A CGRP,5Oms
Low Ca
._k_._ A
Wash
I lOmV
A 20s
A
m
2s
Fig. 2. Lacg ot ettects of low Ca solution on fast and slow depolarizations. A: pressure application of CGRP elicited in this cell a slow depolarization. Downward deflections represent hyperpolarizing electrotonic potentials induced by constant current pulses (not shown). Note there was a small (25%) increase of the amplitude of electrotonic potentials at the peak of CGRP-induced depolarization, indicating increased membrane input resistance. The CGRP-induced slow depolarization evoked in a low Ca (0.25 mM) solution remained the same. B: CGRP evoked a fast depolarization in this cell. Again, low Ca solution had no appreciable effect on the amplitude or time course of the CGRP response. Records A and B were taken from two different coeliac neurons.
259 A
noticeably changed in a low C! solution in 2 cells tested. t
CGRP,50mt [IOmV
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mV i CGRP.3Oml
21:
-70
~-
--
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.
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-90 mV
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Fig. 3. Electrophysiological characteristics of fast CGRP-induced depolarizations, Cell A: fast depolarization was accompanied by a marked decrease of membrane input resistance as indicated by a reduction of the amplitude of hyperpolarizing electrotonic potentials. After the response had subsided, depolarization of the membrane to the same potential level seen at the peak of the response did not cause any appreciable change of the electrotonic potential amplitude. Cell B: membrane hyperpolarization increased the amplitude of CGRP-induced fast depolarization. Numerals to the left of each trace denote the membrane potential at which the response was elicited. Graph to the right: amplitude of CGRP response against membrane potential, The intercept of the line fitted by eye indicates the reversal potential, i.e. -24 inV. Records A and B were taken from two different cells. The possible ion species involved in the CGRP-induced fast depolarization were investigated. First, the effects of C G R P on coeliac neurons before and after superfusing the ganglia with a Na-free, TrisHC! Krebs solution were examined. Fig. 4B shows the depolarizing action of CGRP was reversibly abolished in a Na-free medium; a similar result was obtained in 3 other cells. Changing the Krebs solution to a K-free solution by omitting the KCI also reduced the CGRP-induced depolarization by a mean of 26 _+ 7% (n = 3). The response was however not
Effects of cholinergic antagonists and anticholinesterase agent Experiments were initially carried out with d-tubocurarine (d-To) to test whether the fast depolarization caused by CGRP was due secondary to a presynaptic release of ACh. Unexpectedly, d-To at relatively high concentrations suppressed the CGRP-induced depolarization in a dose-dependent manner (Fig. 4A). At the concentrations of 10, 30 and 100 /~M, d-Tc reduced the fast depolarization by 21 + 5% (n = 3), 44 + 6% (n = 4) and 92 + 6% (n = 4), respectively. The depressant effect of d-Tc was reversible upon wash (Fig. 4A). On the other hand, hexamethonium in comparable concentrations caused no appreciable reduction of the CGRP-induced fast depolarization in any of the 4 cells tested. Similarly, atropine (1 #M) was without effect on the fast depolarization in all 3 cells examined. If the CGRP-induced fast depolarization was due secondary to a release of ACh, the response should be enhanced by the anticholinesterase agent eserine 14. In the concentrations of 1 and 5/zM, eserine had no appreciable effect on the fast response induced by CGRP in any of the 3 cells examined (Fig. 5B). In contrast, eserine (1/~M) markedly increased the amplitude and prolonged duration of the depolarization induced by ACh in two cells tested (Fig. 5A).
Characteristics of the slow depolarization by CGRP CGRP elicited a slowly rising arid falling depolarization in slightly over half of the responsive neurons. This type of response had a mean latency, duration and amplitude of 2.6 + 1.6 s, 69 + 7 s and 10 +__3.2 mV (n = 12), respectively. Spike discharges appeared occasionally at the rising or plateau phase of the slow response (e.g. Fig. 1). The slow response was not significantly changed in a low Ca solution or TI'X-containing solution (Fig. 2A). The amplitude before and after low Ca/high Mg solution was 11 + 1.8 mV and 10 + 2.1 mV (n = 3), respectively. Unlike the fast depolarization, the electrophysiological characteristics of the slow CGRP response were somewhat variable depending on the cells studied. When the membrane potential was manually clamped at the resting level, either a small increase
260 A
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_
t'
A CGRP,50ms
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t'
Wash
d-Tc,lO.uM
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Na-free
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Fig. 4. Suppression of fast CGRP-induced depolarizations by d-Tc and Na-free solution. Cell A: d-Tc 10, 30 and 100/~M attenuated the CGRP-induced fast depolarization in a concentration-dependent manner. At the highest concentration used, d-Tc nearly abolished the response. The effect was reversible upon wash. Numerals between the traces indicate the duration of drug application or wash in minutes. Cell B: superfusing the ganglion with a Na-free, Tris-HCI solution blocked the CGRP-induced fast depolarization, and the effect was reversed after wash. Records A and B were obtained from two different coeliac neurons.
A
A
A~,,.~.,.
t
A
A
A
t
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t
d-Tc,10 I~M
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A Wash
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Fig. 5. Potentiationof ACh depolarizationbut not CGRP responseby eserine.Cell A: membranedepolarizationwas inducedby pressure application of ACh from a micropipette containing 100 mM ACh. Eserine increased the amplitude as well as the duration of AChinduced depolarization. The response was blocked by d-Tc 10 gM. Cell B: CGRP-induced fast depolarization was not enhanced by eserine, although it was depressed by d-Tc 50/~M. Numerals between traces denote the duration of drug application or wash in minutes. Records A and B were taken from two different coeliac neurons.
261 A
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Fig. 6. Membrane resistance change and effects of membrane potential on CGRP-induced slow depolarization. A: upper trace represents voltage recording and lower trace current recording. Downward deflections are hyperpolarizing electrotonic potentials evoked by constant current pulses (downward deflections of lower trace). CGRP elicited a slow depolarization and at the peak of the response there was a slight increase in membrane resistance as indicated by a small (18%~ increase of the amplitude of electrotonie potenti~!s. When the membrane potential was manually clamped at the resting membrane potential level of-54 mV, the CGRP response was associated with a transient, small (15%) decline followed by a more sustained, also small (12%) increase of input resistance. B: hyperpolarization and depolarization from the initial resting membrane potential of-64 mV increased and decreased the CGRP-induced slow depolarization. Graph of depolarization amplitude against membrane potential is shown to the right. Extrapolation of the fine indicates a reversal potential of-42 inV. Records A and B were taken from two different coeliac neurons.
or decrease ( 1 0 - 3 0 % ) of the input resistance during the slow C G R P - i n d u c e d response was observed in different cells (Fig. 6A). M o r e o v e r , the relationship b e t w e e n the a m p l i t u d e and m e m b r a n e potential was
,o' '
CGRP.8Oms
!
d-Tc.50~M
also n o t uniform. In 4 of the 6 coeliac n e u r o n s , the slow depolarization b e c a m e larger o n m e m b r a n e hyperpolarization; the extrapolated m e a n reversal potential was - 4 4 m V (Fig. 613). M e m b r a n e hyperpo-
. . . . '
2o' t
Wash
,
40 s
llOmV
Fig. 7. Biphasic response of a guinea pig coeliac neuron to pressure application of CGRP. CGRP evoked a fast, transient depolarization followed by a more slowly declining response. Superfusing the ganglion with d-Tc blocked the fast, transient phase without noticeably altering the slow phase. The effect was reversible after wash.
262 larization reduced the slow CGRP response in one cell, and in another the response was not substantially changed. With respect to the cholinergic antagonists, d-Tc at the concentration as high as 100 gM caused little or no change of the slow response in all 4 cells tested. Hexamethonium and atropine likewise had no effect on the slow depolarization (n = 3).
Characteristics of the biphasic response A biphasic response to CGRP was noted in 4 coeliac neurons (Figs. 1C and 7). The biphasic response had the appearance of the fast and slow depolarizations mentioned above. The initial fast rising depolarization was followed by a smaller, more sustained depolarization. The fast component was reversibly abolished by d-Tc, leaving the slow component intact (Fig. 7). Thus, the biphasic response appeared to be a composite of the fast and slow depolarization. DISCUSSION The peptide CGRP evoked 3 types of excitatory responses with distinct electrophysiological and pharmacological characteristics in a population of coeliac neurons. Unlike the finding in guinea pig myenteric ganglia where Type 2 neurons appeared to be uniformly sensitive to the peptide roT,less than half of the coeliac ganglion cells responded to CGRP. This may imply a selective distribution of CGRP receptors to a subpopulation of coe!iac neurons. Moreover, CGRP elicited in myenteric neurons only a slow depolarization similar in appearance to toe slow response observed in our study 17. On the other hand, CGRP caused no apparent change of the membrane vo,e,,,,a, u u t p,o,on~ed the duration of the calciumdependent action potential of the cat parasympathetic ganglion cells 16. As a corollary, CGRP may subserve a diverse function in various peripheral autonomic ganglia. Differences in their latency and duration notwithstanding, the fast and slow response displayed distinct electrophysiological and pharmacological characteristics. The fast response was always associated with a marked reduction in membrane resistance, it was increased by hyperpolarization and the estimated reversal potential was about -20 mV. These findings together with the observations that the re-
sponse was redu~:ed in a Na- and K-free, but not low Cl-solution saggest that the peptide increased membrane pe!meability to Na and K ions. This aspect of CGRP action closely resembles the nicotinic action of ACh on autonomic neurons. The observation that d-Tc, a compound frequently regarded as a nicotinic blocker, antagonized the CGRP-induced fast depolarization was somewhat unexpected. The action of CGRP however is not fikely to be mediated by ACh acting on nicotinic receptors for the reasons that the response was not antagonized by another nicotinic blocker hexamethonium nor potentiated by the anticholinesterase agent eserine, whereas the latter augmented the ACh-induced depolarization. It should be noted that d-Tc has been shown to be a non-selective blocker; it antagonizes the action of a variety of putative transmitters including histamine, ACh, and 5-HT in the vertebrate and invertebrate neurons m'23. In Aplysia neurons d-Tc antagonizes the action of a variety of putative transmitters probably by blocking the receptor-activated Na or Cl channels, rather than blocking the different receptors m. Our finding that the CGRP-induced fast depolarization was eliminated in a Na-free solution is consistent with this interpretation. The slow depolarizations induced by CGRP display characteristics that are similar to the slow responses evoked by a number of peptides on prevertebral ganglion cellss'12'13'17'19 and on myenteric neu.tons 17'24. The mechanism underlying the slow CGRP-induced response appears to be less straightforward. The input resistance change irrespective an increase or decrease during the CGRP-induced slow depolarization was modest. Furthermore, membrane hyperpolarization produced variable effects on the slow, CGRP-induced response in different cells; while an increase of the slow response was observed in the majority of cells tested, a decrease or no apparent change was also noted in a few cells. Interestingly, similar findings have been reported with respect to the substance P-induced slow depolarization 5. The mechanism(s) underlying the CGRP-induced slow depolarization in prevertebral ganglion cells is not clear. In the case of substance P response, an increase of Na and a decrease of K membrane permeability has been suggested 5. On the other hand, the primary mechanism underlying the CGRP-induced
263 slow depolarization in the myenteric neurons appears to be an inactivation of Ca-activated K conductance 17. A s the fast and slow depolarization differed with respect to their electrophysiological and pharmacological characteristics, two subpopulations of C G R P receptors may be involved. The biphasic response observed in a small number of neurons would indicate the co-existence of two subsets o f C G R P receptors in a single neuron. Alternatively, one class of C G R P r e c e p t o r may couple to different sets of channels and/or intracellular transducing mechanisms, thereby giving rise to different responses. The question whether C G R P is released from immunoreactive fibers in the coeliac ganglion by physiological stimuli remains to be answered. Also not known is whether synaptically released C G R P may elicit two types of response as shown here by exogenous application. Interestingly, denervation studies show that CGRP-immunoreactive fibers in the coeliac ganglia may arise as collateral branches of afferent fibers from dorsal root ganglion cells some of which contain substance P-immunoreactivities as
REFERENCES 1 Ascher, P., Marty, A. and Neild, T.O., The mode of action of antagonists on the excitatory response to acetylcholine in Aplysia neurones, J. Physiol. (Lond. L 278 (1978) 207-235. 2 Carpenter, D.O., Swann, J.W. and Yarowsky, P.J., Effects of curare on responses to different putative transmitters in Aplysia neurons, J. Neurobiol., 8 (1977) 119-132. 3 Dalsggard, C.J., H6kfelt, T., Schultzberg, M., Lundberg, J.M., Tercnius, L., Dockray, G.J. and Goldstein, M., Origin of peptide-containing fibccs in the inferior mesenteric ganglion of the guinea pig: immunohistochemical studies with antisera to substance P, enkephalin, vasoactive intestinal polypeptide, cholecystokinin and bombesin, Neuroscience, 9 (1983) 191-211. 4 Dun, N.J. and Ma, R.C., Slow non-cholinergic excitatory potentials in neurones of the guinea pig coeliac ganglia, J. Physiol. (Lond.), 351 (1984) 47-60. 5 Dun, N.J. and Minota, S., Effects of substance P on neurones of the inferior mesenteric ganglia of the guinea pig, J. Physiol. (Lond.), 231 (1981)259-271. 6 Gibbins, I.L., Furness, J.B., Costa, M., Maclntyre, I., Hillyard, C.J. and Girgis, S., Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pig, Neurosci. Leu., 57 (1985) 125-130. 7 Gibson, S.J., Polark, J.M., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101-3111.
well6'It. In this respect, if the action of C G R P in the peripheral ganglion cells is an indication of its central action at the dorsal horn neurons, the possibility that the peptide may subserve a fast and slow excitatory mode of sensory transmission is intriguing. Finally, it is pertinent to mention that C G R P is one of the few peptides known to date to cause a fast, Nadependent excitatory response. Substance P has been shown to cause a fast and slow depolarization in spinal and myenteric neurons in culture 8"21. Generally, peptides are thought to mediate neuronal events of slow time course. O u r finding in conjunction with the observations made in the cultured central and autonomic neurons with respect to the action of substance p8.21 raise the possibility that neuronal events mediated by certain peptides may be swift, analogous to the action of 'classical' transmitters such as nicotinic A C h on motor endplates and autonomic neurons. ACKNOWLEDGEMENT This study was supported by Grant NS18710 from the Department of Health and Human Services.
8 Hanani, M. and Burnstock, G., Substance P evokes slow and fast responses ~n cultured myenteric neurons of the guinea pig, NeuroscL Lett., 48 (1984) 19-23. 9 Kondo, H., lwanaga, T. and Yanaihara, N., On the occurrence of gastrin releasing peptide (GRP)-Iike immunoreactive nerve fibers in the celiac ganglion of rats, Brain Research, 289 (1983) 326-329. 10 Lee, Y., Hayashi, N., H~llyard, C.J., Girgis, S.I., Maclntyre, I., Emson, P.C. and Tohyama, M., Calcitonin gonerelated peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat coeliac ganglion, Brain Research, 407 (1987) 149-151. 11 Lee, Y., Takami, K., Kawai, Y., Girgis, S.I., Hillyard, C.J., Maclntyre, I., Emson, P.C. and Tohyama, M., Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P, Neuroscience, 15 (1985) 1227-1237. 12 Love, J.A. and Szurszewski, J.H., The electrophysiological effects of vasoactive intestinal polypeptide in the guinea pig inferior mesenteric ganglion, J. Physiol. (Lond.), 394 (1987) 67-84. 13 Mo, N. and Dun, N.J., Cholecystokinin octapeptide depolarizes guinea pig inferior mesenteric ganglion cells and facilitates nicotinic transmission, Neurosci. Left.. 64 (1986) 263-268. 14 Mo, N., Dun, N.J. and Karczmar, A.G., Facilitation and inhibition of nicotinic transmission by eserine in the sympathetic ganglia of the rabbit, Neuropharmacology, 24 (1985) 1093-1101. 15 Muldberry, P.K., Ghatei, M.A., Bishop, A.E., Allen, Y.S., Polak, J.M. and Bloom, S.R., Distribution and chro-
264 matographic characterization of CGRP-Iike immunoreactivity in the brain and gut of the rat, Regul. Peptides, 12 (1985) 133-144. 16 Nohmi, M., Shiunick-Gallagher, P., Gean, P.W., Gailagher, J.P. and Cooper, C.W., Calcitonin and calcitonin generelated peptide enhance calcium-dependent potentials, Brain Research, 367 (1986) 346-350. 17 Palmer, J.M., Schemann, M., Tamnra, K. and Wood, J.D., Calcitonin gene-related peptide excites myenteric neurons, Eur. J. Pharmaeol., 132 (1986) 163-170. 18 Reinecki, M., Forssmann, W.G., Thiekotter, G. and Triepel, J., Localiza~on of neurotensin-immunoreactivity in the spinal cord and peripheral nervous system of the guinea pig, Neurosei. Lett., 37 (1983) 37-42. 19 Saria, A., Ma, R.C., Dun, N.J., Theodorssca-Norheim, E. and Lundberg, J.M., Neurokinin A in capsaicin-sensitive neurons of the guinea pig inferior mesenteric ganglia: an additional putative mediator for the non-cholinergicexcita-
tory postsynaptic potential, Neuroscience, 21 (1987) 951-958. 20 Skofitsch, G. and Jacobowitz, D.M., Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system, Peptides, 6 (1985) 1069-1973. 21 Vincent, J.D. and Barker, J.L., Substance P: evidence for diverse roles in neuronal function from cultured mouse spinal neurons, Science, 205 (1979) 1409-1412. 22 Vincent, S.R., Dalsggard, C.J., Schultzberg, M., H6kfelt, T., Christesson, I. and Terenius, L., Dynorphin-immunoreactive neurons in the autonomic nervous system, Neuroscience, 11 (1984) 973-987. 23 Wallis, D.I. and Dun, N.J., A comparison of fast and slow depolarizations evoked by 5-HT in guinea pig coeliac ganglion cells in vitro, Br. J. Pharmacol., 93 (1988) 110-120. 24 Wood, J.D., Enteric neurophysiology,Am. J. PhysioL, 247 (1984) (3585-(3598.