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Galanin as an inhibitory transmitter in the mudpuppy cardiac ganglion
119
Effect of Ieu-ENK on release of CGRP in IMGs Normal Krebs solution
Leu-ENK (10 ~M)
Basal
Distension
Basal
Distension
Naloxone (I#M) + Leu-ENK (10 ~tM) Basal
Distension
60.1 - 10.0
117.2 _+ 24.0 *
55.4 + 10.9
72.0 - 17.1
56.7 + 7.9
79.4 + 8.7 *
* P < 0.05
Although SP in the IMGs was almost totally depleted by capsaicin treatment in vivo, C G R P in the IMGs was reduced by only 36.6%. In both groups of animals, colonic distension to an internal pressure of 15 cm HzO maintained for 2 min or exposure to 80 mM K + for 2 min released CGRP; however, the release in capsaicin-treated animals was significantly reduced compared to control animals. The effect of leu-ENK on release of C G R P (pg/ml) during colonic distension was tested in preparations removed from untreated animals (n = 20). The results were as shown in table above. These studies suggest that: (1) approximately one third of the CGRP-containing dorsal root ganglion neurones contained SP as a co-trans-
mitter and were capsaicin sensitive; (2) C G R P was released during colonic distension at moderate pressure; (3) approximately two-thirds of the CGRP-containing neurones were insensitive to capsaicin; and (4) central efferent nerves containing ENK modulate release of C G R P during colonic distension. Supported by D K 17632. Reference 1 Ma, R.C. and Szurszewski, J.H., Effect of the enkephalins and neurotensin on release of substance P and vasoactive intestinal polypeptide in guinea pig inferior mesenteric ganglion during colonic distension, Supplement to Gastroenterology, 98 (1990) (5): A372.
Galanin as an inhibitory transmitter in the mudpuppy cardiac ganglion T.W. McKeon
1, L . M . K o n o p k a
and R.L. Parsons
Department of Anatomy and Neurobiology, University of Vermont, Burlington, VT 05405, and i Department of Neuroscience, Case Western Reserve University, Cleveland, OH 44106, U.S.A.
Galanin, a 29-amino acid peptide originally isolated from pig intestine, is now known to be present in many regions of both the peripheral and central nervous system of numerous species [1]. We have demonstrated that a galanin-like peptide is present in parasympathetic postganglionic neurones and in the small intraganglionic neurones of the mudpuppy cardiac ganglion [2]. None of the galanin immunoreactive fibres in the mudpuppy cardiac ganglion are preganglionic axons, afferent fibre processes or sympathetic postganglionic axons [3]. Further, at least 40% of the small intraganglionic neurones exhibited galanin immunoreactivity and processes from approximately 50% of these cells make close association with adjacent parasympathetic postganglionic
neurones [3]. The most prevalent response following brief galanin application to individual parasympathetic postganglionic neurones is membrane hyperpolarization and decreased excitability [2,4]. The decrease in excitability remains even when the hyperpolarization is negated electrotonically. The hyperpolarization is due to an increase in potassium conductance, and the decrease in excitability is evident as a reduction in the number of spikes generated by long suprathreshold depolarizing current pulses or a decrease in spontaneous action potential generation [4]. The extent of the decrease in excitability is not dependent on the amplitude of the peak galanin-induced hyperpolarization, measured just prior to negating the potential change. Further, the galanin-induced decrease in
120
excitability was observed in cells in which there was either a very small (1-2 mV) or no measurable hyperpolarization. A small decrease in input resistance (less than 15% in most cells) was measured at the peak of a 10-20 mV galanin-induced hyperpolarization and the decrease was even less (approximately 2%) when the hyperpolarization was negated electrotonically. These results suggested that the galanin-induced conductance change might be voltage dependent. Evidence in support of this conclusion was obtained in three voltage-clamped cells. The galanin-induced membrane current was measured and the conductance change, calculated using the expression: GGa~= I~al(E~, I - Eclamp) , increased with hyperpolarization. Galanin decreased excitability in preparations exposed to either 100-200 /LM cadmium or 5 - 1 0 mM tetraethylammonium (TEA). The galanin-induced decrease in excitability resulted from a shift in the threshold for spike generation to more positive values of membrane potential both in control preparations and in preparations treated with tetrodotoxin (TTX) and TEA. The present study provided further evidence that the galanin-induced decrease in excitability cannot be simply attributed to the agonist-induced increase in potassium conductance. Rather, the decrease in excitability, particularly when hyperpolarization is
negated, results from a direct action of galanm on the ionic conductance mechanisms involved in action potential generation. We suggest that the decrease in excitability is due not to the potentiation of an outward current such as I, but to an alteration in the voltage dependence of the sodium/calcium conductances associated with action potential generation in these cells. Lastly, because the major action of galanin is inhibition and most of the innervation by galanin-positive fibres is derived from small intraganglionic neurones, we propose that some of the small intraganglionic neurones function as inhibitory interneurones utilizing a galanin-like peptide as an inhibitory transmitter. Supported by PHS grants NS 23978 and NS 25973. References 1 ROkaeus, A., TINS, 10 (1987) 158-t64. 2 Parsons, R.L., Neel; D.S., Konopka, L.M. and McKeon, T.W., Neuroscience, 29 (1989) 749-759. 3 McKeon, T.W. and Parsons, R.L., J. Auton. Nerv. Sys., 31 (1990) 135-140. 4 Konopka, L.M., McKeon, T.W. and Parsons, R.L., J. Physiol. 410 (1989) 107-122.
of catechotM me biosy by and
in
R . E . Z i g r n o n d , C. B a l d w i n , H . H y a t t - S a c h s , A . R i t t e n h o u s e , C. S a s e k and M.A. Schwarzschild Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, OH. U.S.A.
Previous studies in our laboratory have established that the acute effect of preganglionic nerve stimulation in vitro on tyrosine hydroxylase (TH) activity in the rat superior cervical ganglion (SCG) is mediated partly by a nicotinic and partly by a non-cholinergic mechanism. A subgroup of peptides of the secretin-glucagon family - namely, secretin, vasoactive intestinal peptide (VIP), pep-
tide histidine isoleucine amide (PHI), rat growth hormone-releasing factor and hetodermin H38 were subsequently found to stimulate T H activity in the ganglion. Other members of this peptide family, such as glucagon or human growth hormone-releasing hormone, had no effect. The increases in T H activity produced by these peptides correlated with their ability to increase
Effect of Ieu-ENK on release of CGRP in IMGs Normal Krebs solution
Leu-ENK (10 ~M)
Basal
Distension
Basal
Distension
Naloxone (I#M) + Leu-ENK (10 ~tM) Basal
Distension
60.1 - 10.0
117.2 _+ 24.0 *
55.4 + 10.9
72.0 - 17.1
56.7 + 7.9
79.4 + 8.7 *
* P < 0.05
Although SP in the IMGs was almost totally depleted by capsaicin treatment in vivo, C G R P in the IMGs was reduced by only 36.6%. In both groups of animals, colonic distension to an internal pressure of 15 cm HzO maintained for 2 min or exposure to 80 mM K + for 2 min released CGRP; however, the release in capsaicin-treated animals was significantly reduced compared to control animals. The effect of leu-ENK on release of C G R P (pg/ml) during colonic distension was tested in preparations removed from untreated animals (n = 20). The results were as shown in table above. These studies suggest that: (1) approximately one third of the CGRP-containing dorsal root ganglion neurones contained SP as a co-trans-
mitter and were capsaicin sensitive; (2) C G R P was released during colonic distension at moderate pressure; (3) approximately two-thirds of the CGRP-containing neurones were insensitive to capsaicin; and (4) central efferent nerves containing ENK modulate release of C G R P during colonic distension. Supported by D K 17632. Reference 1 Ma, R.C. and Szurszewski, J.H., Effect of the enkephalins and neurotensin on release of substance P and vasoactive intestinal polypeptide in guinea pig inferior mesenteric ganglion during colonic distension, Supplement to Gastroenterology, 98 (1990) (5): A372.
Galanin as an inhibitory transmitter in the mudpuppy cardiac ganglion T.W. McKeon
1, L . M . K o n o p k a
and R.L. Parsons
Department of Anatomy and Neurobiology, University of Vermont, Burlington, VT 05405, and i Department of Neuroscience, Case Western Reserve University, Cleveland, OH 44106, U.S.A.
Galanin, a 29-amino acid peptide originally isolated from pig intestine, is now known to be present in many regions of both the peripheral and central nervous system of numerous species [1]. We have demonstrated that a galanin-like peptide is present in parasympathetic postganglionic neurones and in the small intraganglionic neurones of the mudpuppy cardiac ganglion [2]. None of the galanin immunoreactive fibres in the mudpuppy cardiac ganglion are preganglionic axons, afferent fibre processes or sympathetic postganglionic axons [3]. Further, at least 40% of the small intraganglionic neurones exhibited galanin immunoreactivity and processes from approximately 50% of these cells make close association with adjacent parasympathetic postganglionic
neurones [3]. The most prevalent response following brief galanin application to individual parasympathetic postganglionic neurones is membrane hyperpolarization and decreased excitability [2,4]. The decrease in excitability remains even when the hyperpolarization is negated electrotonically. The hyperpolarization is due to an increase in potassium conductance, and the decrease in excitability is evident as a reduction in the number of spikes generated by long suprathreshold depolarizing current pulses or a decrease in spontaneous action potential generation [4]. The extent of the decrease in excitability is not dependent on the amplitude of the peak galanin-induced hyperpolarization, measured just prior to negating the potential change. Further, the galanin-induced decrease in
120
excitability was observed in cells in which there was either a very small (1-2 mV) or no measurable hyperpolarization. A small decrease in input resistance (less than 15% in most cells) was measured at the peak of a 10-20 mV galanin-induced hyperpolarization and the decrease was even less (approximately 2%) when the hyperpolarization was negated electrotonically. These results suggested that the galanin-induced conductance change might be voltage dependent. Evidence in support of this conclusion was obtained in three voltage-clamped cells. The galanin-induced membrane current was measured and the conductance change, calculated using the expression: GGa~= I~al(E~, I - Eclamp) , increased with hyperpolarization. Galanin decreased excitability in preparations exposed to either 100-200 /LM cadmium or 5 - 1 0 mM tetraethylammonium (TEA). The galanin-induced decrease in excitability resulted from a shift in the threshold for spike generation to more positive values of membrane potential both in control preparations and in preparations treated with tetrodotoxin (TTX) and TEA. The present study provided further evidence that the galanin-induced decrease in excitability cannot be simply attributed to the agonist-induced increase in potassium conductance. Rather, the decrease in excitability, particularly when hyperpolarization is
negated, results from a direct action of galanm on the ionic conductance mechanisms involved in action potential generation. We suggest that the decrease in excitability is due not to the potentiation of an outward current such as I, but to an alteration in the voltage dependence of the sodium/calcium conductances associated with action potential generation in these cells. Lastly, because the major action of galanin is inhibition and most of the innervation by galanin-positive fibres is derived from small intraganglionic neurones, we propose that some of the small intraganglionic neurones function as inhibitory interneurones utilizing a galanin-like peptide as an inhibitory transmitter. Supported by PHS grants NS 23978 and NS 25973. References 1 ROkaeus, A., TINS, 10 (1987) 158-t64. 2 Parsons, R.L., Neel; D.S., Konopka, L.M. and McKeon, T.W., Neuroscience, 29 (1989) 749-759. 3 McKeon, T.W. and Parsons, R.L., J. Auton. Nerv. Sys., 31 (1990) 135-140. 4 Konopka, L.M., McKeon, T.W. and Parsons, R.L., J. Physiol. 410 (1989) 107-122.
of catechotM me biosy by and
in
R . E . Z i g r n o n d , C. B a l d w i n , H . H y a t t - S a c h s , A . R i t t e n h o u s e , C. S a s e k and M.A. Schwarzschild Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, OH. U.S.A.
Previous studies in our laboratory have established that the acute effect of preganglionic nerve stimulation in vitro on tyrosine hydroxylase (TH) activity in the rat superior cervical ganglion (SCG) is mediated partly by a nicotinic and partly by a non-cholinergic mechanism. A subgroup of peptides of the secretin-glucagon family - namely, secretin, vasoactive intestinal peptide (VIP), pep-
tide histidine isoleucine amide (PHI), rat growth hormone-releasing factor and hetodermin H38 were subsequently found to stimulate T H activity in the ganglion. Other members of this peptide family, such as glucagon or human growth hormone-releasing hormone, had no effect. The increases in T H activity produced by these peptides correlated with their ability to increase