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Actions of substance P on sympathetic neurons
Veuropharrr~colo~
@Pergamon
0028-3908/79/0201-0215$02.00/o
Vol. 78, pp. 215-218 Press Ltd 1979. Printed in Great Britain PRELIMINARY
NOTES
ACTiONS OF SUBSTANCE P ON SYMPATHETIC NEURONS
N. J.
Dun and A.
Department of Stritch School
G. Karczmar
Pharmacology, of Medicine,
Loyola University Maywood, Ill. 60153
When applied to the neurons of isolated inferior mesenteric ganglia SUMMARY of guinea pig, substance P consistently caused a membrane depolarization accompan ied, in many instances, by intense neuronal discharges. The substance Pinduced depolarization or spikings were not abolished by d-tubocurarine and/or atropine or by superfusing the ganglia with a low Ca/high Hg solution. Repetitive stimulation of hypogastric nerves elicited a slow non-cholinergic depolarization which exhibited electrophysiological and pharmacological properties similar to those of substance P-induced &polarization. When ganglion cells were desensitized by continuous application of substance P, preganglionic stimulation failed to induce the slow non-cholinergic depolarization, These results suggest the poss i bility that the slow non-cholinergic depolarization may be due to the synaptic release of substance P.
There has been a growing interest in the possible synaptic function of subThis peptide is distributed widely in the central as well as peripheral stance P. nervous system; while several proposals were made as to the synaptic function of this peptide in the CNS (Krnjevic, 1977; Otsuka and Konishi, 1977). recent findings suggest that it may play as well a role in autonomic transmission. Substance P produced a membrane depolarization of the neurons of guinea pig myenteric plexus, mimicking the slow depolarization elicited by focal stimulation of the ganglion (Katayama and North, 1978). Furthermore, in the case of the inferior masenteric ganglion of the guinea pig where dense, substance P-positive fibers encircle and terminate on, the principal noradrenergic neurons (Hokfelt et al., 1977), preganglionic stimulation elicited a slowly developing depolarization which was resistant to d-tubocurarine and atropine (Neild, 1978). Accordingly, the present study was undertaken to investigate the electrophysiological effects of substance P on the inferior mesenteric ganglion of the guinea pig and to evaluate the possibility that substance P may be the transmitter mediating the synaptic slow non-cholinergic depolarization. intracellular recordings were made from the neurons of isolated inferior mesenteric ganglia of guinea pigs. The recording techniques have been described previously (Dun and Nishi, 1974); substance P (Sigma Co.) and other drugs were applied to the ganglia by superfusion. Both hypogastric nerves were drawn into a suction electrode for presynaptic stimulation (Dun and Nishi, 1974). In effective concentrations, substance P (lo-100 nM) caused a dose-dependent membrane depolarization reaching peak amplitude in less than 1 min; the amplitude of depolarization varied and ranged from 2 to 23 mV. The duration of substance Pinduced depolarization also varied considerably as it lasted for 1 to 3 min. Intense neuronal discharges were frequently encountered during the rising phase of the substance P-induced depolarization, particularly when higher concentrations (So-100 nM) were used (Fig. 1 & 28). The effect of substance P was not antagonized by cholinergic blocking agents; pretreating the ganglia with d-tubocurarine (SO-100 p/l) and/or atropine (5 pM) for 10 min did not affect the depolarization or spikings Similarly, elicited by substance P. superfusing the ganglia with a low Ca (0.25 mH)/high Mg (12mM) solution which effectively abolished synaptic transmission had no detectable effect on substance P-evoked depolarization or subsequent repetitive neuronal discharges, whereas the firings were prevented by repolarizing the me&rane potential to the resting level.
N.P
18 2--H
215
216
Preliminary Notes
Legend
to
Figure
1
The effect of substance P (100 nM) on medrane potential and membrane resistance changes of a single inferior mesenteric ganglion cell of guinea pig. The upper tracing depicts the current pulses at 0.3 Hz used to elicit the hyperpolarizing electrotonic potentials (vertical deflections of lower tracing); the membrane potential is shcwn in the lower tracing. Irregular spontaneous synaptic potentials can be observed in the lower tracing. Intense neuronal spikings occurred in the plateau phase of the me&rane depolarization; the full amplitude of action potentials cannot be photographically reproduced. Arrmheads mark the start and withdrawal of substance P superfusion. Calibration: 20 mV and 10 sec. The depolarization induced by substance P was accompanied, in the majority of neurons (55 out of 62), by moderate to marked reduction in membrane resistance as shown by the change in the amplitude of hyperpolarizing electrotonic potentials induced by constant current pulses (Fig. 1); it ranged from 8 to 37%. The reduction in membrane resistance was not due to rectification as it still occurred when the membrane potential was manually clamped at the resting level by passing a steady anodal current through the recording electrode. Furthermore, in som neurons the amplitude of electrotonic potentials was substantially reduced prior to any detectable change in the membrane potential (Fig. l), indicating again, that the fall in membrane resistance was not directly related to depolarization. In 7 the substance P-induced mehrane depolarization was associated with a cells, small but definite increase in mmbrane resistance; the increase never amounted to more than 20% of the control value. Repetitive
stimulation
of
both
hypogastric
nerves
elicited,
in
the
majority
of ganglion cells treated with d-tubocurarine (SO PM) and atropine (5 PM), a 2) similar to the response reporslowly developing membrane depolarization (Fig. In 30 of the 34 cells tested, the non-cholinergic depolarited by Nei Id (1578). zation was accompanied by a fall in the mesbiane resistance which varied from 6 the membrane resistance was not appreciablly to 41 percent of control values; Following experiments were carried out to examine changed in other 4 cells. whether the receptors mediating depolarization evoked by substance P and by presynaptic stimulation exhibited cross-reactivity. Synaptically induced slow noncholinergic depolarization was first obtained by stimulating repetitively the hypogastric nerves (Fig. 2 A); following a 10 min wash with Krebs solution, the ganglion was superfused with a Krebs solution containing substance P (100 nil), which caused rapid merrbrane depolarization (Fig. 2 B). In the continuous presence of substance P, the me&rane potential repolarized to the resting level; at this point, repetitive stimulation of hypogastric nerves failed to elicit the slow non-cholinergic depolarization (Fig. 2 C). Following a 30 min wash interval with Krebs solution, the slow non-cholinergic depolarization could again be elicited by presynaptic stimulatirn (Fig. 2 0). This effect was consistently observed in all of the 5 neurons tested.
Preliminary
Notes
A’
217
Bm J
D-
cLegend
for
Figure
2
The effect of continuous application of substance P (100 nH) on the synaptically induced trerrt,rane depolarization. The ganglion was continuously superfused with a Krebs solution containing d-tubocurarine (50 uM) and atropine (5 NM). A: SIW merrbrane &polarization elicited by repetitive supramaximal stimulation of both hypogastric nerves at 30 Hz for 2 sec. B: membrane depolarization and neuronal firings elicited by application of substance P (100 nM) commencing at the superfusion of substance P was maintained the point marked by an arrwhead; for 5 min. C: 3 min after superfusing the ganglion with substance P, repetitive stimulation elicited no detectable membrane potential change. D: merrbrane depolarization induced by preganglicnic stimulation following a 30 min wash with Recordings A-D were taken from the same gang1 ion cell. Vertical Krebs sol ut ion. calibration mark: 20 mV for A, C, & D; 40 mV for B; horizontal calibration mark: 10 sec. The present report demonstrates clearly that substance P exerts a potent depolarizing effect on sympathetic neurons. This response is not cholinergic in nature as it was not abolished by anticholinergic agents. Furthermore, as low Ca/high Hg solution failed to diminish the depolarizing action of substance P, this effect must be exerted directly on ganglionic neurons. The most interesting aspect of this study relates to the finding of the similarity between the depolarization induced by substance P on the one hand and by stimulation of the hypogastric nerves on the other; both responses were insensitive to anticholinergic agents, and thei r ionic mechanisms appear to be similar, as in both cases membrane depolarization seems to be associated, in the majority of neurons, with a fall in medrane resistance (Neild, 1978). Similar significance must be attached to the finding that the desensitization of the ganglion cells caused by prolonged application of substance P renders the neurons incapable of generating the slew non-cholinergic depolarization upon repetitive stimulation of preganglionic fibers; this result suggests that substance P and the transmitter responsible for the slow membrane depolarization may be acting upon the same postsynaptic receptor. It must be emphasized that a rise in membrane resistance occurred during substance P-induced depolarization in a small nur&er of neurons. Similarly, Neild (1978) observed an increase in membrane resistance during the s.1~ mmbrane depolarization in 4 of 94 neurons studied. The significance of two types of conductance changes observed in the present study and that of Neild is at present not clear. Substance P caused membrane depolarization associated with a fall in membrane resistance in the frog (Nicoll, 1978) and rat (Otsuka and 1577) motoneurons, and with a rise in membrane resistance in the cat Konishi, motoneurons (Krnjevir!, 1977) and guinea pig myenteric neurons (Katayama and North, 1978). The transmitter responsible for the slow non-cholinergic depolarization As subobserved in inferior mesenteric ganglion of guinea pig is not known. stance P is densely localized in the varicosites of fibers surrounding the 1977), as the metrbrane depolarization sympathetic neurons (Hbkfelt et al., induced by substance P is indistinguishable from the synaptically induced and as the receptors mediating these two responses show cross response, P may be the endogenous transmitter mediating the reactivity , substance slow non-cholinergic depolarization in the inferior mesenteric ganglion of It has been suggested that substance P-containing fibers which guinea pig. encircle the sympathetic neurons may originate from primary sensory neurons to speculate then that sensory inner(Hokfelt et al., 1977); it is tempting vation affects the activity of autonomic ganglia via the release of substance P.
218
Preliminary Yates
It is interesting to point out that presynaptic stimulation of amphibian besides the well known fast and slow cholinergic paravertebral ganglia evokes, also a non-cholinergic slow depolarization (Nishi and excitatory potentials, Koke ts u, 1968); the metirane characteristics of the latter seem to resemble those of the response to substance P as observed in the present study.
ACKNOWLEDGEMENT: this study was supported Loyola University.
by grants
from NIH NS 06455
and
REFERENCES Dun, N. and Nishi, 5. gang1 ion of the rabbit. Hokfelt, (1977). ganglia:
Effects of dopamine on the (1974). J. Physiol. (Land), -239: 155-164.
L. G., Schultzberg, T., Elfvin, On the occurrence of substance Immunohistochemical evidence.
Katayama, Y. and North, R. A. synaptic excitation within the
superior
cervicai
M., Goldstein, M. and Nilsson, G. P-containing fibers in sympathetic Brain Res. -132: 29-41.
(1978). myenteric
Does substance P mediate slow plexus? Nature 274: 387-388. --
Effects of substance P on central neurons in cats. In: Krnjevi 4, K. Substance P, U. S. von Euler, and B. Pernow, Eds., pp. 217-230, Raven Press, New York, 1977. Slmly-developing depolarization of neruons in the Neild, T. 0. (1978). guinea-pig inferior mesenteric ganglion following repetitive stimulation of Brain Res. -140: 231-239. the preganglionic nerves. Nicoll, R. A. P and related in press,
(1978). peptides
The action of on frog spinal
Nishi, S, and Koketsu, K. sympathetic ganglion cells.
thyrotropin motoneurons.
releasing hot-none, substance J. Pharmacol. Exp. Ther.
Early and late afterdischarges (1968). J. Neurophysiol. -31: 109-121.
of
amphibian
Electrophysiological and neurochemical evidence for Otsuka, N. and Konishi, K. S&stance P, substance P as a transmitter of primary sensory neurons. In: U.S. von Euler and 8. PernaJ, Eds., Raven Press, New York, 1977, pp. 207-214.
@Pergamon
0028-3908/79/0201-0215$02.00/o
Vol. 78, pp. 215-218 Press Ltd 1979. Printed in Great Britain PRELIMINARY
NOTES
ACTiONS OF SUBSTANCE P ON SYMPATHETIC NEURONS
N. J.
Dun and A.
Department of Stritch School
G. Karczmar
Pharmacology, of Medicine,
Loyola University Maywood, Ill. 60153
When applied to the neurons of isolated inferior mesenteric ganglia SUMMARY of guinea pig, substance P consistently caused a membrane depolarization accompan ied, in many instances, by intense neuronal discharges. The substance Pinduced depolarization or spikings were not abolished by d-tubocurarine and/or atropine or by superfusing the ganglia with a low Ca/high Hg solution. Repetitive stimulation of hypogastric nerves elicited a slow non-cholinergic depolarization which exhibited electrophysiological and pharmacological properties similar to those of substance P-induced &polarization. When ganglion cells were desensitized by continuous application of substance P, preganglionic stimulation failed to induce the slow non-cholinergic depolarization, These results suggest the poss i bility that the slow non-cholinergic depolarization may be due to the synaptic release of substance P.
There has been a growing interest in the possible synaptic function of subThis peptide is distributed widely in the central as well as peripheral stance P. nervous system; while several proposals were made as to the synaptic function of this peptide in the CNS (Krnjevic, 1977; Otsuka and Konishi, 1977). recent findings suggest that it may play as well a role in autonomic transmission. Substance P produced a membrane depolarization of the neurons of guinea pig myenteric plexus, mimicking the slow depolarization elicited by focal stimulation of the ganglion (Katayama and North, 1978). Furthermore, in the case of the inferior masenteric ganglion of the guinea pig where dense, substance P-positive fibers encircle and terminate on, the principal noradrenergic neurons (Hokfelt et al., 1977), preganglionic stimulation elicited a slowly developing depolarization which was resistant to d-tubocurarine and atropine (Neild, 1978). Accordingly, the present study was undertaken to investigate the electrophysiological effects of substance P on the inferior mesenteric ganglion of the guinea pig and to evaluate the possibility that substance P may be the transmitter mediating the synaptic slow non-cholinergic depolarization. intracellular recordings were made from the neurons of isolated inferior mesenteric ganglia of guinea pigs. The recording techniques have been described previously (Dun and Nishi, 1974); substance P (Sigma Co.) and other drugs were applied to the ganglia by superfusion. Both hypogastric nerves were drawn into a suction electrode for presynaptic stimulation (Dun and Nishi, 1974). In effective concentrations, substance P (lo-100 nM) caused a dose-dependent membrane depolarization reaching peak amplitude in less than 1 min; the amplitude of depolarization varied and ranged from 2 to 23 mV. The duration of substance Pinduced depolarization also varied considerably as it lasted for 1 to 3 min. Intense neuronal discharges were frequently encountered during the rising phase of the substance P-induced depolarization, particularly when higher concentrations (So-100 nM) were used (Fig. 1 & 28). The effect of substance P was not antagonized by cholinergic blocking agents; pretreating the ganglia with d-tubocurarine (SO-100 p/l) and/or atropine (5 pM) for 10 min did not affect the depolarization or spikings Similarly, elicited by substance P. superfusing the ganglia with a low Ca (0.25 mH)/high Mg (12mM) solution which effectively abolished synaptic transmission had no detectable effect on substance P-evoked depolarization or subsequent repetitive neuronal discharges, whereas the firings were prevented by repolarizing the me&rane potential to the resting level.
N.P
18 2--H
215
216
Preliminary Notes
Legend
to
Figure
1
The effect of substance P (100 nM) on medrane potential and membrane resistance changes of a single inferior mesenteric ganglion cell of guinea pig. The upper tracing depicts the current pulses at 0.3 Hz used to elicit the hyperpolarizing electrotonic potentials (vertical deflections of lower tracing); the membrane potential is shcwn in the lower tracing. Irregular spontaneous synaptic potentials can be observed in the lower tracing. Intense neuronal spikings occurred in the plateau phase of the me&rane depolarization; the full amplitude of action potentials cannot be photographically reproduced. Arrmheads mark the start and withdrawal of substance P superfusion. Calibration: 20 mV and 10 sec. The depolarization induced by substance P was accompanied, in the majority of neurons (55 out of 62), by moderate to marked reduction in membrane resistance as shown by the change in the amplitude of hyperpolarizing electrotonic potentials induced by constant current pulses (Fig. 1); it ranged from 8 to 37%. The reduction in membrane resistance was not due to rectification as it still occurred when the membrane potential was manually clamped at the resting level by passing a steady anodal current through the recording electrode. Furthermore, in som neurons the amplitude of electrotonic potentials was substantially reduced prior to any detectable change in the membrane potential (Fig. l), indicating again, that the fall in membrane resistance was not directly related to depolarization. In 7 the substance P-induced mehrane depolarization was associated with a cells, small but definite increase in mmbrane resistance; the increase never amounted to more than 20% of the control value. Repetitive
stimulation
of
both
hypogastric
nerves
elicited,
in
the
majority
of ganglion cells treated with d-tubocurarine (SO PM) and atropine (5 PM), a 2) similar to the response reporslowly developing membrane depolarization (Fig. In 30 of the 34 cells tested, the non-cholinergic depolarited by Nei Id (1578). zation was accompanied by a fall in the mesbiane resistance which varied from 6 the membrane resistance was not appreciablly to 41 percent of control values; Following experiments were carried out to examine changed in other 4 cells. whether the receptors mediating depolarization evoked by substance P and by presynaptic stimulation exhibited cross-reactivity. Synaptically induced slow noncholinergic depolarization was first obtained by stimulating repetitively the hypogastric nerves (Fig. 2 A); following a 10 min wash with Krebs solution, the ganglion was superfused with a Krebs solution containing substance P (100 nil), which caused rapid merrbrane depolarization (Fig. 2 B). In the continuous presence of substance P, the me&rane potential repolarized to the resting level; at this point, repetitive stimulation of hypogastric nerves failed to elicit the slow non-cholinergic depolarization (Fig. 2 C). Following a 30 min wash interval with Krebs solution, the slow non-cholinergic depolarization could again be elicited by presynaptic stimulatirn (Fig. 2 0). This effect was consistently observed in all of the 5 neurons tested.
Preliminary
Notes
A’
217
Bm J
D-
cLegend
for
Figure
2
The effect of continuous application of substance P (100 nH) on the synaptically induced trerrt,rane depolarization. The ganglion was continuously superfused with a Krebs solution containing d-tubocurarine (50 uM) and atropine (5 NM). A: SIW merrbrane &polarization elicited by repetitive supramaximal stimulation of both hypogastric nerves at 30 Hz for 2 sec. B: membrane depolarization and neuronal firings elicited by application of substance P (100 nM) commencing at the superfusion of substance P was maintained the point marked by an arrwhead; for 5 min. C: 3 min after superfusing the ganglion with substance P, repetitive stimulation elicited no detectable membrane potential change. D: merrbrane depolarization induced by preganglicnic stimulation following a 30 min wash with Recordings A-D were taken from the same gang1 ion cell. Vertical Krebs sol ut ion. calibration mark: 20 mV for A, C, & D; 40 mV for B; horizontal calibration mark: 10 sec. The present report demonstrates clearly that substance P exerts a potent depolarizing effect on sympathetic neurons. This response is not cholinergic in nature as it was not abolished by anticholinergic agents. Furthermore, as low Ca/high Hg solution failed to diminish the depolarizing action of substance P, this effect must be exerted directly on ganglionic neurons. The most interesting aspect of this study relates to the finding of the similarity between the depolarization induced by substance P on the one hand and by stimulation of the hypogastric nerves on the other; both responses were insensitive to anticholinergic agents, and thei r ionic mechanisms appear to be similar, as in both cases membrane depolarization seems to be associated, in the majority of neurons, with a fall in medrane resistance (Neild, 1978). Similar significance must be attached to the finding that the desensitization of the ganglion cells caused by prolonged application of substance P renders the neurons incapable of generating the slew non-cholinergic depolarization upon repetitive stimulation of preganglionic fibers; this result suggests that substance P and the transmitter responsible for the slow membrane depolarization may be acting upon the same postsynaptic receptor. It must be emphasized that a rise in membrane resistance occurred during substance P-induced depolarization in a small nur&er of neurons. Similarly, Neild (1978) observed an increase in membrane resistance during the s.1~ mmbrane depolarization in 4 of 94 neurons studied. The significance of two types of conductance changes observed in the present study and that of Neild is at present not clear. Substance P caused membrane depolarization associated with a fall in membrane resistance in the frog (Nicoll, 1978) and rat (Otsuka and 1577) motoneurons, and with a rise in membrane resistance in the cat Konishi, motoneurons (Krnjevir!, 1977) and guinea pig myenteric neurons (Katayama and North, 1978). The transmitter responsible for the slow non-cholinergic depolarization As subobserved in inferior mesenteric ganglion of guinea pig is not known. stance P is densely localized in the varicosites of fibers surrounding the 1977), as the metrbrane depolarization sympathetic neurons (Hbkfelt et al., induced by substance P is indistinguishable from the synaptically induced and as the receptors mediating these two responses show cross response, P may be the endogenous transmitter mediating the reactivity , substance slow non-cholinergic depolarization in the inferior mesenteric ganglion of It has been suggested that substance P-containing fibers which guinea pig. encircle the sympathetic neurons may originate from primary sensory neurons to speculate then that sensory inner(Hokfelt et al., 1977); it is tempting vation affects the activity of autonomic ganglia via the release of substance P.
218
Preliminary Yates
It is interesting to point out that presynaptic stimulation of amphibian besides the well known fast and slow cholinergic paravertebral ganglia evokes, also a non-cholinergic slow depolarization (Nishi and excitatory potentials, Koke ts u, 1968); the metirane characteristics of the latter seem to resemble those of the response to substance P as observed in the present study.
ACKNOWLEDGEMENT: this study was supported Loyola University.
by grants
from NIH NS 06455
and
REFERENCES Dun, N. and Nishi, 5. gang1 ion of the rabbit. Hokfelt, (1977). ganglia:
Effects of dopamine on the (1974). J. Physiol. (Land), -239: 155-164.
L. G., Schultzberg, T., Elfvin, On the occurrence of substance Immunohistochemical evidence.
Katayama, Y. and North, R. A. synaptic excitation within the
superior
cervicai
M., Goldstein, M. and Nilsson, G. P-containing fibers in sympathetic Brain Res. -132: 29-41.
(1978). myenteric
Does substance P mediate slow plexus? Nature 274: 387-388. --
Effects of substance P on central neurons in cats. In: Krnjevi 4, K. Substance P, U. S. von Euler, and B. Pernow, Eds., pp. 217-230, Raven Press, New York, 1977. Slmly-developing depolarization of neruons in the Neild, T. 0. (1978). guinea-pig inferior mesenteric ganglion following repetitive stimulation of Brain Res. -140: 231-239. the preganglionic nerves. Nicoll, R. A. P and related in press,
(1978). peptides
The action of on frog spinal
Nishi, S, and Koketsu, K. sympathetic ganglion cells.
thyrotropin motoneurons.
releasing hot-none, substance J. Pharmacol. Exp. Ther.
Early and late afterdischarges (1968). J. Neurophysiol. -31: 109-121.
of
amphibian
Electrophysiological and neurochemical evidence for Otsuka, N. and Konishi, K. S&stance P, substance P as a transmitter of primary sensory neurons. In: U.S. von Euler and 8. PernaJ, Eds., Raven Press, New York, 1977, pp. 207-214.