Neuropeptide Y hyperpolarizes submucosal neurons of the guinea-pig descending colon

Neuroscience Letters 227 (1997) 212–214

Neuropeptide Y hyperpolarizes submucosal neurons of the guinea-pig descending colon Keiji Hirai*, Kirsteen N. Browning1, Gordon M. Lees2 University of Aberdeen, Department of Biomedical Sciences, Marischal College, Aberdeen AB9 1AS, UK Received 25 November 1996; revised version received 28 April 1997; accepted 28 April 1997

Abstract Effects of neuropeptide Y (NPY) on submucosal neurons of the guinea-pig descending colon were investigated electrophysiologically by means of intracellular electrophysiological recordings. NPY (100 nM) induced a marked and prolonged hyperpolarization, accompanied by a decrease in input resistance in most (90%) neurons. This NPY-induced hyperpolarization was diminished and augmented by membrane hyperpolarization and depolarization, respectively. The NPY-hyperpolarization was not affected by exposure to either calcium-free solutions or the a2-adrenoceptor antagonist, idazoxan (1 mM). When more than one peptide was applied to a neuron, NPY, PYY and Pro34-NPY were equipotent, whilst NPY13–36 was less potent. It was concluded that NPY hyperpolarized submucosal neurons of the guinea-pig descending colon, possibly via a direct action on postsynaptic Y1-receptor and increasing potassium conductance.  1997 Elsevier Science Ireland Ltd. Keywords: Neuropeptide Y; Membrane hyperpolarization; Intracellular recordings; Y1-receptors; Submucosal neurone; Descending colon; Guinea-pig; Electrophysiology

Generally, NPY is thought to inhibit neurotransmitter release through activation of presynaptic Y2-receptors without affecting the electrophysiological properties of the postsynaptic membrane. In the guinea-pig enteric nervous system, for example, NPY depresses both fast (cholinergic) and slow (non-cholinergic) excitatory synaptic transmission in myenteric neurons 2] and slow (noradrenergic) inhibitory synaptic transmission in submucosal neurons of guinea-pig caecum 4]. In the present study, however, it was found that NPY hyperpolarized submucosal neurons of the guinea-pig descending colon by a direct postsynaptic action. Eight male albino guinea-pigs (Duncan–Hartley strain; 220–650 g) were killed by stunning and exsanguination and a segment of descending colon was isolated. Prepara* Corresponding author. Department of Autonomic Physiology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan. Tel.: +81 3 52808078; fax: +81 3 52808077; e-mail: [email protected] 1 Department of Physiology, West Virginia University, Morgantown WV 26506-9229, USA. 2 Department of Biomedical Sciences, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK.

tions of submucosa (about 1 cm2), free of both mucosa and external muscle layers, were pinned to the Sylgard (Dow Corning) floor of the small recording chamber; they were continuously superfused with Krebs solution (35–37°C, gassed with 95% O2/5% CO2) at 5–10 ml/min. Ganglia were viewed at a magnification of 250 (Zeiss Nomarski DIC). The composition of Krebs solution was (mM): NaCl 118.2; KCl 4.75; CaCl2 2.54; MgCl2 1.18; KH2PO4 1.35; NaHCO3 25.0 and glucose 11.0. The nominally-zero calcium Krebs solution (Ca2+-free solution) contained 2.36 mM MgCl2 and no added CaCl2. Conventional intracellular electrical recordings were made with glass microelectrodes filled with 2 M KCl (tip resistance of 70–150 MQ) as previously described 4]. Postsynaptic potentials (PSPs) were evoked by interganglionic nerve stimulation with justsupramaximal, negative current pulses (0.3 ms, 1–5 pulses at 20 Hz) from a saline-filled focal electrode (tip diameter 5–15 mm). Values are quoted as means ± SD. The mean resting membrane potential of submucosal neurons of the descending colon was −57 ± 10 mV (n = 38; range −45–−75 mV) and mean input resistance (R in) was 377 ± 134 MQ (n = 32; range 240–500 MQ). Almost all

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0325-X

K. Hirai et al. / Neuroscience Letters 227 (1997) 212–214

(.96%) neurons showed at least one fast excitatory PSP (EPSP) in response to single pulse focal stimulation of interganglionic nerve strands. Of 22 submucosal neurons tested with repetitive strand stimulation, non-cholinergic, nonadrenergic slow inhibitory PSPs (IPSPs) and slow EPSPs were evoked in nine (41%) and 17 (77%) neurons, respectively, while a noradrenergic slow IPSP was elicited in only one (5%) neuron. NPY (100 nM) was applied by superfusion for 40 s to 11 submucosal neurons in eight ganglia. NPY induced a strong hyperpolarization (19 ± 7 mV; range 15–28 mV) in 91% of neurons (10/11 cells) with an accompanying 56 ± 8% decrease in R in (Figs. 1 and 2A); recovery after NPY-withdrawal was slow (7–15 min). NPY-hyperpolarizations showed a voltage-dependency (n = 3 cells; Fig. 1A); by a d.c. membrane hyperpolarization the peak amplitude of NPY-induced hyperpolarizations were diminished and essentially abolished at around EK (−84 mV, assuming Ki was 150 mM), while it was augmented by a membrane depolarization. In view of these electrophysiological characteristics, the NPY-induced hyperpolarization was considered to be due to an increased potassium conductance (GK). Slow EPSPs and idazoxan-insensitive slow IPSPs were suppressed by the NPY-hyperpolarization (Fig. 2A). It can not be predicted whether the NPY-inhibition of slow postsynaptic potentials were presynaptic or postsynaptic, since both the synaptic potentials can be postsynaptically diminished by a conductance increase during the NPY-hyperpolarization. On the other hand, effect of NPY on the fast

Fig. 1. NPY-hyperpolarization. Evidence for voltage-dependency and postsynaptic origin. NPY (100 nM) was superfused for 40 s (black bars, deadspace time; 15–20 s). (A) NPY responses were obtained from the same cell at the resting (−55 mV; middle trace), depolarized (−45 mV; upper trace) and hyperpolarized (−80 mV; lower trace) membrane potentials. Downward deflections are electrotonic potentials elicited by hyperpolarizing current pulses (50 pA, 80 ms, 0.2 Hz). (B) In the same neuron, NPYhyperpolarization was tested in Ca2+-free Krebs solution (compare with (A)). The cell depolarized about 10 mV in the Ca2+-free solution and no postsynaptic response to electrical interganglionic strand stimulation which previously elicited fast and slow EPSPs (not shown) could be recorded (open arrow).

213

Fig. 2. NPY-hyperpolarization and postsynaptic potentials. (A) NPY (100 nM) was superfused for 2 min (black bars, dead-space time; 15–20 s) in normal Krebs solution (upper trace; resting membrane potential −64 mV) and in presence of idazoxan (1 mM; lower trace). Focal electrical stimuli were applied to an interganglionic nerve strand at filled triangles. Downward deflections are electrotonic potentials elicited by hyperpolarizing current pulses (50 pA, 80 ms, 0.2 Hz). (B) Fast EPSPs (upper row) and tonic hyperpolarizing potentials (lower row) were stored in a digital memory and slowly drawn on chart paper before (1) and during NPY-induced hyperpolarization (2). Action potentials fired by supramaximal fast EPSP in control were suppressed during the NPY-hyperpolarization.

EPSPs could not be described clearly, because of action potential firing from suprathreshold fast EPSPs in control which was suppressed during NPY-hyperpolarization (Fig. 2B). The possibility that NPY promoted the local release of endogenous hyperpolarizing substances, such as norepinephrine, somatostatin and enkephalins 12] to elicit the hyperpolarization was investigated. The most likely transmitter candidate for the largest hyperpolarizing postsynaptic potential, slow IPSP, which is present in more than 80% of caecal submucosal neurons 9], is norepinephrine. The slow IPSP is known to be mediated by activation of postsynaptic a2-adrenoceptors 9]. Idazoxan (1 mM), the a2-adrenoceptor antagonist, did not alter the NPY-hyperpolarization (n = 4 cells; Fig. 2A). Since extracellular Ca2+ is essential for neurotransmitter release, the action of NPY was examined in Ca2+-free Krebs solution. The NPY-induced hyperpolarization was not affected, even after all postsynaptic responses to focal electrical stimulations were abolished by perfusion of the Ca2+-free Krebs solution (n = 4 cells; Fig. 1B). It can be concluded that NPY hyperpolarized the submucosal neurons presumably by acting directly on postsynaptic NPY receptors. In the absence of useful competitive antagonists, accepted order of peptide potency for classifying Y1-receptor is NPY = PYY = Pro34-NPY . NPY13–36, whereas that for Y2-receptor activation is PYY ≥ NPY ≥ NPY13– 36 . Pro34-NPY, PYY being much less active than NPY at Y3-receptors 4,5]. In the submucosal neurons of descending colon, peptide YY (PYY; 100 nM) caused a hyperpolarization (18 ± 5 mV; range 10–25 mV), accompanied by a 62 ± 14% decrease in R in (n = 5). The relative potency of NPY-related peptides (100 nM) was compared directly in the same single neurons. In one neuron, NPY, Pro34-NPY and NPY13–36 caused hyperpolarization of 28, 27 and 18

214

K. Hirai et al. / Neuroscience Letters 227 (1997) 212–214

mV, respectively, whereas in another neuron the hyperpolarizations elicited by PYY, Pro34-NPY and NPY13–36 were 21, 20 and 13 mV, respectively. The present results suggest the possibility that the NPY-receptor mediating the hyperpolarization might be of the Y1-subtype. The newly found direct hyperpolarizing action of NPY on the submucosal neurons in colon agrees with the previous reports about inhibition of voltage-dependent calcium channels in sympathetic nerve terminals of cultured rat superior cervical ganglia 13], in hypothalamic synaptosomes 7], and in neuroblastoma cells 6,8,11] or calciumactivated potassium channels in vascular smooth muscle cells 15]. Although activation of both Y1- and Y2-receptors may lead to an inhibition of adenylate cyclase, Y1-receptors are thought to mobilize intracellular Ca2+ by stimulating GO protein 1,5,14]. If an elevation of the intracellular Ca2+-concentration resulted from Y1-receptor activation, it would be possible to hyperpolarize the neurons by opening Ca2+ activated K+ channels. Our results also raise the possibility that NPY, which is present in varicose fiber networks around non-NPY-immunoreactive neuron somata 3], may be one of the transmitter candidates responsible for the small non-noradrenergic, non-cholinergic slow IPSP 10] observed in nine of 22 submucosal neurons of the descending colon in the course of this study. The financial assistance of the Welcome Trust, and a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan is gratefully acknowledged. [1] Beck-Sickinger, A.G. and Jung, G., Structure-activity relationships of neuropeptide Y analogues with respect to Y1 and Y2 receptors, Biopolymers, 37 (1995) 123–142. [2] Browning, K.N., Cunningham, S.M.C., Timmermans, J.-P. and Lees, G.M., Modulation of synaptic transmission in the myenteric plexus of the guinea-pig descending colon. A role for extrinsic neuropeptide Y (NPY) containing fibres?, J. Physiol., 495 (1996) 100P–101P.

[3] Cunningham, S.M.C. and Lees, G.M., Neuropeptide Y in submucosal ganglia: regional differences in the innervation of guinea-pig large intestine, J. Autonomic Nerv. Sys., 55 (1995) 135–145. [4] Cunningham, S.M.C., Mihara, S. and Lees, G.M., Y2-Receptormediated selective inhibition of slow, inhibitory postsynaptic potential in submucous neurons of guinea-pig caecum, Br. J. Pharmacol., 113 (1994) 883–888. [5] Gehlert, D., Subtypes of receptor for neuropeptide Y: implications for the targeting of therapeutics, Life Sci., 55 (1994) 551–562. [6] Lynch, J.W., Lemos, V.S., Bucher, B., Stoclet, J.-C. and Takeda, K., A pertussis toxin-insensitive calcium influx mediated by neuropeptide Y2 receptors in a human neuroblastoma cell line, J. Biol. Chem., 269 (1994) 8226–8233. [7] Martire, M., Pistritto, G., Mores, N., Agnati, L.F. and Fuxe, K., Presynaptic A2-adrenoceptors and neuropeptide Y Y2 receptors inhibit [3H]noradrenaline release from rat hypothalamic synaptosomes via different mechanisms, Neurosci. Lett., 188 (1995) 9–12. [8] McDonald, R.L., Vaughan, P.F.T., Beck-Sickinger, A.G. and Peers, C., Inhibition of Ca2+ channel currents in human neuroblastoma (SHSYSY) cells by neuropeptide Y and a novel cyclic neuropeptide Y analogue, Neuropharmacology, 34 (1995) 1507–1514. [9] Mihara, S., Intracellular recordings from neurones of the submucous plexus, Prog. Neurobiol., 40 (1993) 529–572. [10] Mihara, S., Nishi, S., North, R.A. and Surprenant, A., A non-cholinergic slow inhibitory post-synaptic potential in neurones of the guinea-pig submucous plexus, J. Physiol., 390 (1987) 357–365. [11] Shigeri, Y. and Fujimoto, M., Y2 Receptors for neuropeptide Y are coupled to three intracellular signal transduction pathways in a human neuroblastoma cell line, J. Biol. Chem., 269 (1994) 8842– 8848. [12] Surprenant, A., Shen, K.-Z., North, R.A. and Tatsumi, H., Inhibition of calcium currents by noradrenaline, somatostatin and opioids in guinea-pig submucosal neurons, J. Physiol., 431 (1990) 585–608. [13] Toth, P.T., Bindokas, V.P., Bleakman, D., Colmers, W.F. and Miller, R.J., Mechanism of presynaptic inhibition by neuropeptide Y at sympathetic nerve terminals, Nature, 364 (1993) 635–639. [14] Wiley, J.W., Gross, R.A. and MacDonald, R.L., Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite actions on rat nodose neuron calcium currents, J. Neurophysiol., 70 (1993) 324–330. [15] Xiong, Z. and Cheung, D.W., Neuropeptide Y inhibits Ca2+-activated K+ channels in vascular smooth muscle cells from the rat tail artery, Pflu¨gers Arch., 429 (1994) 280–284.