Chemosensitivity of C-cells in bullfrog dorsal root ganglia to substance P and adenosine 5′-triphosphate

Neuroscience Letters, 163 (1993) 169 172 © 1993 Elsevier Ireland Ltd. All rights reserved 0304-3940/93/5 06.00

169

NSL 09977

Chemosensitivity of C-cells in bullfrog dorsal root ganglia to substance P and adenosine 5'-triphosphate Takayuki Tokimasa*, Masashi Tsurusaki, Takashi Akasu Department of Physiology, Kurume University School of Medicine, 67 Asahi-maehi, Kurume 830. Japan (Received 15 April 1993: Revised version received 27 July 1993; Accepted 20 August 19931

Key words:

Substance P; ATP: G-protein; M-current; ATP channel: Sensory neuron

Dissociated bullfrog dorsal root ganglion cells were voltage clamped in the whole-cell configuration. In small C-cells having 20/am as averaged diameter, substance-P (0.1 1 /aM) inhibited an M-type potassium current while ATP (1 10/aM) activated a sodium-potassium current. In large A-cells (~ 65 ~m in diameter) in which ATP has been shown to inhibit M-current, substance P (0.1 1 yM) also inhibited this potassium current without activating the sodium-potassium current. Results provided evidence for the distinction between A- and C-cells in terms of their chemosensilivity.

Dorsal root ganglia contain cell bodies of myelinated A-fibers and non-myelinated C-fibers [6, 8]. Previous studies on bullfrog dorsal root ganglia demonstrated that C-cells differ significantly from A-cells in terms of their morphological and electrophysiological properties [9, 10, 13, 14, 17]. In the present study, we aimed to demonstrate further evidence for the distinction between these cell types in terms of their sensitivity to substance P (SP) and adenosine 5"-triphosphate (ATP). Experiments were carried out at 22 24°C on dissociated bullfrog dorsal root ganglion cells with methods described previously [16-19]. A standard pipette solution had the following composition: KC1, 100 raM; MgCI2, 4 mM; EGTA, 1 raM; KOH, 6 mM; Na2ATR 5 mM and sodium salt of HEPES, 2.5 mM (pH adjusted with HEPES and HC1 to 7.0). A cesium-rich (96 raM) pipette solution was used to eliminate potassium currents (see legend of Fig. 2 for its composition). Unless otherwise mentioned, the superfusate contained tetrodotoxin (3 /aM, Sankyo). Guanosine 5'-O-(3-thiotriphosphate) (GTP-y-S, tetralithium salt) and other drugs were obtained from Sigma. Statistics are expressed as mean + S.E.M. Consistent with our previous observations [16, 17], C-cells were smaller than A-cells in their diameter (e.g. 25/am in Fig. 1) and showed the action potential lasting for 7-12 ms (Fig. IA). None of these small cells showed a prominent hyperpolarization-acti-

*Corresponding author.

vated inward rectifier (n = 35) and therefore identified as C-cells [15-18]. C-cells were superfused with a Ringer solution (see legend of Fig. 1 for its composition) then subjected to 0.5 s step commands from the holding potential of - 5 5 mV. Upon stepping up to - 3 0 mV, an inward calcium current was activated (Fig. 1B,C; full height not shown here) followed by an outward current during the commands and its tail after the termination of the commands (Fig. 1B,C). Current responses during the step commands from -55 to - 6 5 mV were ohmic (Fig. 1B,C). SP (0.1 /aM) inhibited the outward current at - 3 0 mV while leaving the ohmic current between - 5 5 and -65 mV unaffected (Fig. I B). The calcium current was not affected by SP (monitored on oscilloscope, data not shown). In the same cell, however, the main action of ATP (10/aM) was to produce an inward current at -55 mV which was associated with an increased amplitude of the ohmic current (Fig. 1C). Similar results were obtained from 5 of 6 other cells. Remaining 1 cell was sensitive to SP but not to ATE In 6 cells, an averaged amplitude was 92 + 8 pA (e.g. 80 pA in Fig. 1C) for the ATP-induced inward current at -55 mV. Results illustrated in Fig. 1C were reminiscent of the ATP-induced activation of a sodium-potassium current (IArp) in a subpopulation of dorsal root ganglion cells in frogs [4, 5] and rats [4, 5, 12]. Hence, the actions of ATP were tested on identified C-cells under conditions at which voltage-dependent potassium currents had been eliminated by intracellular cesium ions (Fig. 2A). In l0

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Fig. 1. Actions of substance P (SP) and ATP in C-cells. A: the action potential (rest, -55 mV) evoked by injecting a 23 pA current pulse for 20 ms. After recording the action potential of this cell (25/am in diameter), tetrodotoxin (3/aM) was added to a Ringer solution. The cell was then subjected to step commands (-10 mV or +25 mV) from the holding potential (V0 o f - 5 5 mV. B: from left to right are current responses in control, in the presence of SP (0.1/aM) and after wash out. C: results with ATP (10/aM). A 80 pA inward shift of the base line from a dashed line denotes the ATP-activated current. Composition of Ringer solution (mM): NaCI, 112; KC1, 2; CaCI2, 1.8; HEPES, 4 and Tris, 1 (pH adjusted with HC1 to 7.2).

cells with the concentration of ATP at 3 p M , IAVphad an averaged amplitude of 85 + 9 pA at -55 mV and 378 + 24 pA at -95 mV (Fig. 2B,C). IATphad the reversal potential near zero mV (-3.0 + 2.0 mV, n = 6) and the conductance-voltage relationship showed inward rectification between zero and - 115 mV (Fig. 2B) [4, 5]. Intra-

cellular loading with GTP-?'-S (150 pM) failed to make the actions of ATP irreversible (n = 3); results illustrated in Fig. 2B,C were obtained from one of these 3 cells. With regard to results shown in Fig. 1B, the action of SP (0.1 -1 pM) were tested on a M-type potassium current (IM) in the presence of apamin (30 nM) and tetraethylammonium (TEA, 20 30 mM) [15, 16, 19]. In 30 of 35 A-cells, SP (1 pM) reduced the amplitude of&~ at -35 mV from 1139 _ 38 pA to 244 _ 17 pA (e.g. from 1020 to 390 pA in Fig. 3A). ATP (1 10pM) also inhibited IM in 26 of these 30 cells (Fig. 3A) and 3 of 5 cells which were insensitive to SP. IATpcould not be detected even when it was sought at -95 mV (n = 11). In 10 of 12 C-cells, the amplitude of IM at -35 mV (204 + 19 pA in control) was reduced to 62 __ 10 pA by SP (1 pM) (e.g. from 200 to 90 pA in Fig. 3B). The remaining 2 cells were insensitive to SP (1 pM). Intracellular loading with GTP-?'-S (30--100 pM) made the actions of SP (1 pM) on IM irreversible not only in A-cells (n = 2, not shown) but in C-cells (n = 3~ Fig. 3C), indicating that a class of GTP-binding proteins (G-protein [7]) mediates the receptor--effector coupling. We have provided further evidence suggesting that Aand C-cells of bullfrog dorsal root ganglia can be distinguished from each other on the basis of their sensitivity to ATP but not to SP. Results also show that ATP does not inhibit IM in Ccells in spite of the presence of a class of G-proteins which mediates the receptor-effector coupling for SP. Previously, Adams and others reported that acetylcholine acting at muscarinic receptors fails to inhibit IM in type-C sympathetic neurons even though the current can be inhibited by luteinizing hormone-releasing hormone in the same cell [1-3, 11]. One difference between these two preparations is that muscarinic receptors co-exist with nicotinic receptors (now called acetylcholine-channels) in both B- and C-cells in sympathetic ganglia [11, 20] whereas in sensory ganglia purinoceptors and ATP-channels respectively distribute among A- and C-cells. Further experiments are necessary to

---) Fig. 2. ATP-activated current in C-cells. A: current-voltage curve for the calcium-channel current (barium ions as charge carriers). Ordinate denotes the amplitude of the barium current during 100 ms step commands from -70 mV. e, control, c~, cobalt (0.6 raM, bath application). Inset; sample recordings at +10 mV (I; current. V, voltage. Bars=50 ms, 1 nA and 100 mY). B: results from the second cell. Ordinate denotes the peak amplitude of the ATP-activated current. Sample recordings are shown in C. ATP (3 pM) was added to the superfusate for periods indicated by solid lines. Holding potential is indicated beside each trace. Composition of the cesium-rich pipette solution: CsC1, 96 mM; NaC1, 9 mM; T E A - a , 5 mM; EGTA, 2 mM; Na2ATP, 5 mM and HEPES (sodium salt), 2.5 mM (pH adjusted with CsOH to 6.9). Fig. 3. M-current inhibition by substance P (SP). M-current (IM) was continuously activated by setting a holding potential at -35 mV then briefly deactivated during hyperpolarizing step commands for 0.5 s. A: results obtained from an A-cell. ATP (3/aM) and SP (1 gM) were added to the superfusate for periods indicated by solid lines. B: essentially the same experiments as that in A but in an C-cell. On two occasions, the chart speed was increased by a factor of 10 so that IM can be observed on a faster time scale. C: results from another C-cell with the standard pipette solution containing GTP-y-S (100 gM). Left; control. Middle; SP (1/aM). Right; wash. The amplitude of the step commands was 25 mV in A C. The concentration of TEA in the superfusate was 30 mM in A and 20 mM in B and C.

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clarify physiological significance of such heterogeneity in amphibian primary afferent neurons. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, The Ishibashi Research Fund and a Grant-in-aid for Research by The Ishibashi Foundation. 1 Adams, P.R. and Brown, D.A., Luteinizing hormone-releasing factor and muscarinic agonists act on the same voltage-sensitive K +current in bullfrog sympathetic neurones, Br. J. Pharmacol., 68 (1980) 353- 355. 2 Adams, ER., Brown, D.A. and Constanti, A., M-currents and other potassium currents in bullfrog sympathetic neurones, J. Physiol., 330 (1982) 537-572. 3 Adams, ER., Brown, D.A. and Jones, S.W., Substance P inhibits the M-current in bullfrog sympathetic neurones, Br. J. Pharmacol., 79 (1983) 330.-333. 4 Bean, B.P., ATP-activated channels in rat and bullfrog sensory neurons: concentration dependence and kinetics, J. Neurosci., 10 (1990) 1 10.

5 Bean, B.P., Williams, C.A. and Ceelen, P.W., ATP-activated channels in rat and bullfrog sensory neurons: current-voltage relation and single-channel behavior, J. Neurosci., 10 (1990) 11 19. 6 Gallego, R. and Eyzaguirre, C., Membrane and action potential characteristics of A and C nodose ganglion cells studied in whole ganglia and in tissue slices, J. Neurophysiol., 41 (1978) 1217--1232. 7 Gilman, A.G., G proteins: transducers of receptor-generated signals, Annu. Rev. Biochem., 56 (1987) 615 649. 8 Higashi, H., Pharmacological aspects of visceral sensory receptors, Prog. Brain Res., 67 (1986) 149 162. 9 Holz, G.G., Shefner, S.A. and Anderson, E.G., Serotonin depolarizes type A and C primary afferents: an intracellular study in bullfrog dorsal root ganglion, Brain Res., 327 (1985) 71-79.

10 Holz, G.G., Shefner, S.A. and Anderson. E.G.. Sciotonm decrease~ the duration of action potentials recorded from tetraethylammonium-treated bullfrog dorsal root ganglion cells. J. Neurosci.. *~ (1986) 620 626. 11 Jones, S.W. and Adams, ER., The M-current and other potassium currents of vertebrate neurons. In L.K. Kaczmarek and I.B. Lcvitan (Eds.), Neuromodulation: The Biochemical Control of Neuronal Excitability, Oxford University Press, New York, 1987, pp. 159 186. t2 Krishtal, O.A., Marchenko, S.M. and Obukhov, A.G., Cationic channels activated by extracellular ATP in rat sensory neurons, Neuroscience, 27 (1988) 995-1000. 13 Morita, K. and Katayama, Y., 5-Hydroxytryptamine effects on the somata of bullfrog primary afferent neurons. Neuroscience. 21 (1987) 1007-1018. 14 Morita, K., Katayama, Y., Akasu, T. and Koketsu, K., Chemosensitivities of bullfrog spinal ganglion cells, J. Physiol. Soc. Jpn.. 45 (1983) 423. 15 Tokimasa, T. and Akasu, T., Cyclic AMP regulates an inward rectifying sodium-potassium current in dissociated bull-frog sympathetic neurones, J. Physiol., 420 (1990) 409 429. 16 Tokimasa, T. and Akasu, T., ATP regulates muscarine-sensitive potassium current in dissociated bull-frog primary afferent neutones, J. Physiol., 426 (1990) 241 264. 17 Tokimasa, T., Shiraishi, M. and Akasu, T., Morphological and electrophysiological properties of C-cells in bullfrog dorsal root ganglia, Neurosci. Lett., 116 (1990) 304-308. 18 Tokimasa, T., Sugiyama, K., Akasu, T. and Muteki, T., Volatile anaesthetics inhibit a cyclic AMP-dependent sodium-potassium current in cultured sensory neurones of bullfrog, Br. J. Pharmacol., 101 (1990) 190 192. 19 Tokimasa, T., Tsurusaki, M. and Akasu, T., Slowly inactivating potassium current in cultured bull-frog primary afferent and sympathetic neurones, J. Physiol., 435 (1991) 585--604. 20 Selyanko, A.A., Smith, P.A. and Zidichouski, J.A., Effects of muscarine and adrenaline on neurones from Rana pipiens sympathetic ganglia, J. Physiol., 425 (1990) 471 500.