Pfltigers Arch (1994) 428:476-484 E6i gl iin Journal of Physiology 9 Springer-Verlag 1994 Acetylcholine-stimulated changes of membrane potential and intracellular Ca 2§ concentration recorded in endothelial cells in situ in the isolated rat aorta Thomas D. Carter, David Ogden Divison of Neurophysiology and Neuropharmacology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK Received April 29, 1994/Received after revision June 13, 1994/Accepted June 14, 1994 Abstract. The intracellular free Ca 2+ concentration and membrane potential changes evoked by acetylcholine were recorded from whole-cell patch-clamped endothe- lial cells in situ in the isolated rat aorta. The endothelium had a resting membrane potential of -52 + 3mV (SEM, range -35 mV to -76 mV n = 34) and a low input resistance (32-54 Mr2). The membrane potential hyperpolarised by 3-30 mV on continuous application of acetylcholine at concentrations that produced endo- thelium-dependent relaxations in isolated rat aortic rings (range 1-500 nM). The response often comprised com- plex fluctuations of hyperpolarised membrane potential. Calcium concentration was measured with the fluores- cent indicator furaptra, which has a wide range and min- imises Ca 2+ buffering. Acetylcholine evoked an initial rapid elevation of intracellular Ca z+ concentration, peak- ing in the range 6-35 gM, which declined with a half time of approximately 6 s, followed by repetitive [Ca 2+] spikes of amplitude 2-18 gM in 23 of 34 cells. The initial [Ca 2+] transient and hyperpolarisation were unaf- fected by removal of external Ca 2+, whilst subsequent [Ca2+] spikes and maintained hyperpolarisations re- quired the presence of external Ca 2+. Both the hyperpo- larisation and Ca 2+ responses elicited by acetylcholine were abolished by atropine (1 gM). These results show that endothelial cells in situ exhibit large, fast repetitive [Ca 2+] spikes in response to extracellular acetylcholine. Key words: Endothelium - Intracellular calcium - Furaptra - Membrane potential - Patch-clamp - Ace- tylcholine Introduction Endothelial cells modulate the tone of vascular smooth muscle, and hence blood flow through vascular beds, by releasing a wide variety of vasoactive mediators includ- Correspondence to: T. D. Carter ing prostacyclin (PGI2) and endothelium-derived relax- ing factor (EDRF) [37]. An elevation of endothelial in- tracellular calcium ion concentration, comprising an ini- tial release of Ca 2+ from internal stores followed by Ca > influx from the external medium, is commonly re- garded as the major mechanism by which extracellular agonists evoke the release of these two vasoactive medi- ators. Electrophysiological studies have shown that ago- nists that stimulate PGI2 and EDRF release also cause a hyperpolarisation of the endothelial celt membrane [31, 39]. Changes of membrane potential have. in turn, been shown to affect both the intracellular [Ca 2+] [6] and EDRF release [28] and it is possible that membrane hy- perpolarisation is important in modulating the Ca2+-de - pendent release of EDRF by increasing Ca 2+ influx. The role of intracellular [Ca2+] and membrane poten- tial has been investigated mainly with dissociated cells in tissue culture [8, 17, 20, 31, 39], a situation in which the endothelial cells are removed from close contact with other cell types in the vessel wall. Both the structural and functional characteristics of endothelial cells are controlled to some extent by signals from their environ- ment (e.g. hormones, nutrients, cellular metabolites, sub- cellular matrix, cell-cell contacts, etc.) [13], and the be- haviour of endothelial cells in tissue culture is known to differ from that in situ. An important example is the endothelium-dependent relaxation of blood vessels by acetylcholine (ACh) [16], which contrasts with the in- ability of vascular endothelial cells in tissue culture to produce either an elevation of intracellular [Ca >] or EDRF release in response to ACh [25, 27]. In freshly dissociated endothelial cells muscarinic agonists have been shown to raise intracellular [Ca2+] [4] although at concentrations that were often much higher than those required to produce EDRF release in isolated vessels [16]. The reason for this change may be that endothelial muscarinic receptors are lost quickly in tissue culture [433. Here we describe a preparation in which both changes of intracellular free [Caz+] and membrane po- tential were recorded simultaneously from endothelial