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The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial ceils (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits [[alpha].sub.2], [[alpha].sub.3], [[alpha].sub.4], [[alpha].sub.5], [[alpha].sub.7], [[beta].sub.2], and [[beta].sub.4] but not [[beta].sub.3]. Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential ([E.sub.rev]) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in [E.sub.rev] of nicotine-induced current as a function of extracellular [Na.sup.+] concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming [K.sup.+]/[Na.sup.+] permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free [Ca.sup.2+] concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by [Na.sup.+], which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine. microvascular endothelium; mRNA expression; acetylcholine-evoked current; nicotinic receptor subunits; intracellular calcium

The effects of internal adenosine triphosphate (ATP) concentration, levcromakalim and glibenclamide on adenosine triphosphate (ATP)-sensitive potassium (KATP) channels were analyzed in isolated rabbit aortic cells. Administration of levcromakalim and glibenclamide in freshly dissociated rabbit aortic endothelial cells activated several distinct conductance channels. Furthermore, five types of KATP conductances were detected based on their sensitivity to ATP and pharmacology.

Patch-clamp experiments, bioassay and spectrofluorimetry of the rabbit aorta to analyze the influence of K(+)-channel blockers on intracellular free Ca2+ level (Ca2+) increase, acteylcholine (Ach)-induced outward K+ current and vascular smooth muscle relaxation reveal that the K(+)-channel blocker tetraethylammonium ions (TEA) suppress Ach-induced secretion of endothelium-derived relaxing factors (EDRFs). TEA, but not glibenclamide, suppresses the Ach-induced increase in Ca(2+)levels and the vascular muscle relaxation. Ca(2+)-dependent K+ channels are essential for increasing the Ca(2+) levels during the release of EDRF.

Imaging fluorescence microscopic study of the endothelium of rabbit cardiac valves reveals cytosolic endothelial calcium ion measurements in an intact monolayer without being subjected to enzyme treatment. Individual cells with intact intercellular links and functional muscarinic acetylcholine receptor presence enhance comprehension of endothelial cell signalling. Pathophysiological states such as mechanical disruption which occurs in angioplasty and catheterization mechanisms are studied using this method.

The effects of caffeine and ACh on intracellular calcium ion mobilization and current activation in vascular endothelial cells were investigated. Arterial endothelial cells from rabbits were isolated using an enzymatic dissociation method to determine the temporal association between global changes in (Ca2+)i and activation of ionic currents and changes in membrane potential. Experimental results showed that caffeine and ACh induce transient increases in (Ca2+)i. It was also found that Ca2+-dependent current activation monitors local (Ca2+)i changes near the plasmalemma.

Presents a fluorescence imaging of intracellular free calcium (Ca2+) concentration in endothelial cells of rabbit cardiac valves. Morphological differences between endocardial and vascular endothelium; Synchronous oscillations in Ca2+.