Nitric oxide activates Ca2+-activated K+ channels in cultured bovine adrenal chromaffin cells

Neuroscience Letters 248 (1998) 127–129

Nitric oxide activates Ca2+-activated K+ channels in cultured bovine adrenal chromaffin cells Chun-He Chen a, Hitoshi Houchi b, Masahar Ohnaka a, Sadaichi Sakamoto a, Yasuharu Niwa a, Yutaka Nakaya a ,* a

Department of Nutrition, School of Medicine, University of Tokushima, Kuramoto-cho 3, Tokushima 770, Japan b Department of Pharmacology, School of Medicine, University of Tokushima, Tokushima 770, Japan Received 1 November 1997; received in revised form 23 March 1998; accepted 26 March 1998

Abstract The effects of sodium nitroprusside (SNP) on Ca2+-dependent K+ (KCa) channels in cultured bovine adrenal chromaffin cells were investigated using single channel recording patch-clamp techniques. KCa channels were activated by application of 100 mM SNP to the extracellular side of cell-attached patches. Methylene blue (300 mM), an inhibitor of soluble guanylate cyclase, or H-8 (1 mM), a protein kinase inhibitor with relative specificity for cGMP-dependent protein kinase, diminished but did not completely abolish the SNP-induced KCa channel activation. Diethylamine/NO complex (DEA/NO), an NO donor, also activated KCa channels in cell-attached patches. Furthermore, application of 100 mM SNP or 100 nM DEA/NO to the intracellular surface of excised inside-out patches also activated KCa channels in the bath solution which contained 1 mM Ca2+. These results indicate that SNP is capable of activating the KCa channel via cGMP-dependent and -independent mechanisms. These studies demonstrate that NO may serve as an important regulatory mechanism for catecholamine secretion in chromaffin cells via the activation of KCa channels.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: K+ channel; Chromaffin cell; Diethylamine/NO complex; Sodium nitroprusside; cGMP; Patch-clamp recordings

Bovine adrenal medulla chromaffin cells represent a viable model for the investigation of mechanisms for molecular modulatory neurosecretion. Chromaffin cells store and release high concentrations of catecholamine, ATP, and natriuretic peptides. Nitric oxide (NO), an inhibitory neurotransmitter released from peripheral neurons, which was first recognized as an endothelium-derived relaxing factor and implicated in blood-vessel relaxation, has recently been identified as an important intra- and intercellular messenger molecule via its activation of a soluble guanylate cyclase, resulting in formation of guanosine 3′:5′-cyclic monophosphate (cGMP). cGMP appears to stimulate catecholamine synthesis through activation of tyrosine hydroxylase [21,24] the rate-limiting step in catecholamine biosynthesis, whereas it plays an inhibitory role in the regulation of secretion from chromaffin cells [16]. However, the cellular mechanism underlying this NO-mediated inhibition, remains incompletely understood. Ca2+-dependent K+ chan* Corresponding author. Tel: +81 886 337090; fax: +81 886 337113; e-mail: [email protected]

nels (KCa channels) have been identified and characterized in chromaffin cells [1,11,12]. KCa channels are regulated by intracellular Ca2+ ([Ca2+]i) and membrane potential, and in this way may serve as a negative feedback pathway in the control of catecholamine secretion [3,14,22]. Membrane depolarization and catecholamine secretion typically result from inhibition of the KCa channel [5,18]. We hypothesize that NO inhibits catecholamine secretion, at least in part, by activation of KCa channels. To investigate this hypothesis we employed patch-clamp techniques to measure KCa channels in cultured bovine adrenal chromaffin cells to determine the effects of NO on these channels. Isolated bovine adrenal chromaffin cells were enzymatically dispersed as described previously [6]. Briefly, the medulla was manually sliced and the slices were digested in a medium containing 0.1% collagenase, 0.01% soybean trypsin inhibitor, and 0.5% bovine serum albumin in balanced salt solution (BSS) (135 mM NaCl, 5.6 mM KCl, 1.2 mM MgSO4, 2.2 mM CaCl2, 10 mM glucose, and 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)/NaOH; pH 7.40). Individual cells were

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00316- 4

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C.-H. Chen et al. / Neuroscience Letters 248 (1998) 127–129

Fig. 1. Modulation of KCa channel activity by SNP in cell-attached patches. The channel currents were recorded in controls, and after the addition of 100 mM SNP to the bath solution. The KCa channel activation was diminished by methylene blue (A) or H-8 (B). The histogram shows a representative experiment in which KCa channels were modulated by SNP and subsequent addition of methylene blue or H-8 to the bath solution (C). The bath solution contained 100 mM K-aspartate, 40 mM KCl, 10 mM K-MOPS buffer, 1 mM Mg2+, and 2 mM Ca2+, pH 7.2. The pipette solution contained 140 mM KCl, 10 mM K-MOPS, and 10−4 M Ca2+, pH 7.2. The membrane potential (Vm) was +20 mV in these experiments.

then plated in 35 mm culture dishes and maintained for 3–6 days as monolayer cultures in Eagle’s basal medium supplemented with 5% heat-inactivated fetal calf serum. Four or five glass coverslips were placed on the bottom of each culture dish. Membrane currents were recorded in cell-attached and inside-out patches with a patch-clamp amplifier as previously described [7]. Soft glass pipettes prepared by an electrode puller (PP-83; Narishige, Tokyo, Japan) were used following Sylgard coating. The electrical resistance of the patch pipette was 3–5 MQ for single channel recording. Experiments were conducted within a temperature range of 35 to 37°C. pClamp Version 6.0 (Axon Instruments, Foster City, CA, USA) was used for data acquisition and analysis of single channel currents. The length of recording for the NPo calculation was .1 min. The open probability (NPo) was determined from current amplitude histograms and was calculated using the following equation:

solution decreased but did not completely inhibit the SNPinduced channel activities (NPo by 0.201 ± 0.113 to 0.09 ± 0.05, P , 0.05, n = 5) (Fig. 1B). Fig. 1C summarizes the effects of MB or H-8 on the SNP-induced channel activities at a test potential of +20 mV. The subsequent addition of MB or H-8 significantly decreased NPo which was mediated by SNP. We conclude that stimulation of the PKG pathway is necessary, at least in part, for the activation of KCa channels by SNP. In addition, the inhibition of KCa channels by MB or H-8 was not complete, suggesting that a

N

NPo = ∑ (nPn) n=1

where n is the number of channels in the patch and Pn is the integrated channel opening. Statistical comparisons were made using a Student’s paired t-test, and data are expressed as the mean ± SEM. The effects of SNP on cell-attached patches were investigated. SNP activated significantly KCa channels at 100 mM (NPo from 0.006 ± 0.003 to 0.205 ± 0.102, n = 5, P , 0.01). To determine whether the SNP-induced increase of KCa channels was due to activation of the soluble guanylate cyclase and increasing intracellular cGMP, the effects of methylene blue (MB), an inhibitor of soluble guanylate cyclase [10], were examined. MB partially inhibited the effects of SNP on KCa channels (NPo by 0.162 ± 0.102 to 0.102 ± 0.05, P , 0.05, n = 5) (Fig. 1A). To clarify the role of the cGMP pathway in this response, we also tested the effect of H-8, a protein kinase inhibitor with relative specificity for cGMP-dependent protein kinase (PKG) [8], on KCa channels. The addition of 1 mM H-8 to the bath

Fig. 2. KCa channels were activated in a concentration-dependent manner by 1–100 mM SNP in cell-attached (A) and inside-out patches (B). (C,D) Show the effects of DEA/NO on KCa channel in cell-attached and inside-out patches, respectively. The channel currents were recorded in controls (a); and after the addition of 100 nM DEA/NO to the bath solution (b). The bath and pipette solutions were the same as in Fig. 1, except the bath solution contained 3 × 10−7 M Ca2+ conditions.

C.-H. Chen et al. / Neuroscience Letters 248 (1998) 127–129

cGMP-and PKG-independent mechanism is probably involved in the SNP-mediated KCa channel activation. We also investigated the direct effects of SNP on KCa channels in inside-out patches. SNP, at concentrations of 3 to 100 mM on the cytosolic side, also activated KCa channels in a dose-dependent manner. However, addition of MB or H-8 to the bathing solution did not alter the SNP-induced KCa channel activation in inside-out patches (data not shown). Fig. 2 shows that SNP modulated KCa channels in a dose-dependent manner in cell-attached (Fig. 2A) and inside-out (Fig. 2B) configurations. In order to determine if the SNP-induced KCa channel activation was due to the generation of NO or some other mechanism, we also tested the effect of DEA/NO, a pure NO donor, on KCa channel activation. The addition of 100 nM DEA/NO to the bath solution significantly activated the KCa channels (NPo from 0.003 ± 0.001 to 0.234 ± 0.153, n = 4) (Fig. 2C). Moreover, 100 nM DEA/NO also activated KCa channels in inside-out patches (NPo from ,0.001 to 0.356 ± 0.181, n = 4) (Fig. 2D). These results were consistent with the hypothesis that NO modulates the KCa channel directly. Previous investigations have shown that the NO activation of guanylate cyclase to produce cGMP is an autoinhibitory mechanism of catecholamine release in chromaffin cells and that PKG appears to be involved in these inhibitory effects [17]. KCa channels could be activated directly by NO [2,9,13] and indirectly activated following cGMP [4,23]. Recent evidence indicates that NO and PKG activate KCa channels in a variety of smooth muscle cells [15,19, 20]. The most important aspect of the findings herein is that KCa channels are regulated by SNP via cGMP-dependent and -independent pathways. [1] Artalejo, A.R., Garcia, A.G. and Neher, E., Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells, Pflugers Arch., 423 (1993) 97–103. [2] Bolotina, V.M., Najibi, S., Palacino, J.J., Pagano, P.J. and Cohen, R.A., Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle, Nature, 368 (1994) 850–853. [3] Brayden, J.E. and Nelson, M.T., Regulation of arterial tone by activation of calcium-dependent potassium channels, Science, 256 (1992) 532–535. [4] Fujino, K., Nakaya, S., Wakatsuki, T., Miyoshi, Y., Nakaya, Y., Mori, H. and Inoue, I., Effects of nitroglycerin on ATP-induced Ca2+-mobilization, Ca2+-activated K+ channels and contraction of cultured smooth muscle cells of porcine coronary artery, J. Pharmacol. Exp. Ther., 256 (1991) 371–377. [5] Gonzalez, Garcia, C., Cena, V., Keiser, H.R. and Rojas, E., Catecholamine secretion induced by tetraethylammonium from cultured bovine adrenal chromaffin cells, Biochim. Biophys. Acta., 1177 (1993) 99–105. [6] Greenberg, A. and Zinder, O., Alpha- and beta-receptor control of catecholamine secretion from isolated nidrenal medulla cells, Cell. Tissue Res., 226 (1982) 655–665. [7] Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.G., Improved patch-clamp techniques for high-resolution current recording from cells and cell free membrane patches, Pflugers Arch., 391 (1981) 85–100.

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