Proc. Nail. Acad. Sci. USA Vol. 88, pp. 5438-5442, June 1991 Physiology/Pharmacology Hormone-regulated K+ channels in follicle-enclosed oocytes are activated by vasorelaxing K+ channel openers and blocked by antidiabetic sulfonylureas (K+ conductance/folficular cels/Xenopus oocyte/gonadotropin/cromakalim) ERIC HONORt AND MICHEL LAZDUNSKI Institut de Pharmacologie Moldculaire et Cellulaire, UPR 411 du Centre National de la Recherche Scientifique, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Communicated by Josef Fried, February 28, 1991 ABSTRACT Follicular oocytes from Xenopus laevis con- tain K+ channels activated by members of the recently recog- nized class of vasorelaxants that include cromakalim and pinacidil and blocked by antidiabetic sulfonylureas, such as glibenclamide. These channels are situated on the adherent follicular cells and are not present in denuded oocytes. Cro- makalim-activated K+ channels are also activated by increases in intracellular cAMP, and cAMP-activated K+ channels are blocked by glibenclamide. Although cromakalim and cAMP effects are synergistic, cromakalim activation of K+ channels is drastically reduced or abolished by treatments that stimulate protein kinase C (e.g., muscarinic effectors, phorbol esters). Gonadotropins, known to play an essential role in ovarian physiology, also activate cromakalim and sulfonylurea- sensitive K+ channels. Follicular oocytes constitute an excellent system for studying regulation of cromakalim-sensitive K+ channels that are important in relation to a variety of disease processes, such as cardiovascular dysfunction and asthma, as well as brain function. Potassium channels are involved in a wide variety of biolog- ical functions (1). Therefore, it is not surprising that their pharmacology has attracted considerable interest in the past few years (2-4), which has led to the recent development of K+ channel effectors of considerable importance for present and future therapeutic use. Two classes of such agents are antidiabetic sulfonylureas, which block ATP-sensitive K+ (KATP) channels in beta pancreatic cells (3-6) and provoke insulin release (6, 7), and K+ channel openers (KCOs), which hyperpolarize smooth muscle cells (3, 4, 8-12) and have important potential applications in a variety of diseases (3, 4, 8, 13), including hypertension and asthma. The relaxant effects of KCOs are antagonized by sulfonylureas (3, 4, 8, 12), and both families of drugs have the same target channel (3, 4, 8, 12, 14). Potassium channels sensitive to both KCOs and to sulfo- nylureas are also present in the brain (15, 16), where they play diverse important functions (16), including key roles in glu- cose-regulated neurosecretion and in disorders associated with anoxia and ischemia (16, 17). KCOs have been found to have antiepileptic effects (18, 19) as well. In follicle-enclosed oocytes from Xenopus laevis, endoge- neous K+ channels have been demonstrated (20). This sys- tem provides a valuable model for investigating K+ channel regulation because K+ channel activity in follicular oocytes is modulated by cAMP, by protein kinase C-dependent processes, and by a large variety of hormones and transmit- ters, such as catecholamines, adenosine, gonadotropins, vasoactive intestinal peptide, the E series of prostaglandins, atrial natriuretic factor, and muscarinic agonists (20-24). This paper shows that follicular oocytes from X. laevis have a K+ channel that is activated by smooth muscle cell KCOs and blocked by antidiabetic sulfonylureas. Moreover, this channel is modulated both by protein kinase C and by variations in intracellular cAMP. MATERIALS AND METHODS Clusters of oocytes were removed surgically from adult X. laevis females under tricaine anesthesia and were stored in Barth saline solution [88 mM NaCl/1 mM KCI/0.82 mM MgSO4/0.33 mM Ca(NO3)2/0.41 mM CaCl2/2.4 mM NaHCO3/10 mM Hepes, pH 7.4 with NaOH] supplemented with penicillin at 100 international units per ml and strepto- mycin at 100 jug/ml. Oocytes were defolliculated manually in a glass Petri dish in Barth medium (24). Oocyte diameters were measured in at least two directions (at 900) in case they were not strictly spherical. Oocytes were impaled with two glass microelectrodes filled with 3 M KCl (0.5- to 2.0-Mfl resistance) and were voltage-clamped using the Dagan 8500 voltage clamp amplifier. Voltage commands were generated using PCLAMP (Axon Instruments, Burlingame, CA) running on an IBM PC/AT computer. The oocytes were routinely voltage-clamped at -20 mV, which is the predicted equilibrium potential for chloride ions. This protocol tended to minimize any eventual influence of chloride in the ionic currents recorded. Drugs were applied externally by addition to the perfusate by means of a peri- staltic pump at a flow rate of 3 ml/min. Saline solution (ND 96) of the following composition was used in all procedures unless otherwise stated: 96 mM NaCl/2 mM KCI/1.8 mM CaC12/2 mM MgCl2/5 mM Hepes, pH 7.4 with NaOH. All chemicals were obtained from Sigma, unless otherwise stated. The variability of the results was expressed as the SE of the mean; n indicates the number of cells contributing to the mean, and N indicates the number of Xenopus. RESULTS AND DISCUSSION Fig. LA shows that the KCO cromakalim (Ck), also called BRL34915, induces a large hyperpolarization of the mem- brane potential of follicular oocytes. Control oocytes have a mean resting membrane potential of -57 + 6 mV (n = 8, N = 3), whereas Ck (100 ,aM)-treated oocytes have a membrane potential of -88 ± 8 mV (n = 7, N = 3). The Ck-induced Abbreviations: KATP, ATP-sensitive K+ channels; KCO, K+ chan- nel opener; Ck, cromakalim; hCG, human chorionic gonadotropin; 8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate; ICAMP, cAMP-activated K+ conductance. 5438 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Downloaded by guest on June 12, 2020