1. The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. 2. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 micronM barium plus 500 nM ouabain. 3. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 micronM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. 4. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. 5 Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. 6 It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain.

Mechanisms underlying K+‐induced hyperpolarizations in the presence and absence of phenylephrine were investigated in endothelium‐denuded rat mesenteric arteries (for all mean values, n=4). Myocyte resting membrane potential (m.p.) was −58.8±0.8 mV. Application of 5 mM KCl produced similar hyperpolarizations in the absence (17.6±0.7 mV) or presence (15.8±1.0 mV) of 500 nM ouabain. In the presence of ouabain +30 μM barium, hyperpolarization to 5 mM KCl was essentially abolished. In the presence of 10 μM phenylephrine (m.p. −33.7±3 mV), repolarization to 5 mM KCl did not occur in the presence or absence of 4‐aminopyridine but was restored (−26.9±1.8 mV) on addition of iberiotoxin (100 nM). Under these conditions the K+‐induced repolarization was insensitive to barium (30 μM) but abolished by 500 nM ouabain alone. In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mM K+ was inhibited in the additional presence of 300 nM levcromakalim, an action which was reversed by 10 μM glibenclamide. RT–PCR, Western blotting and immunohistochemical techniques collectively showed the presence of α1‐, α2‐ and α3‐subunits of Na+/K+‐ATPase in the myocytes. In K+‐free solution, re‐introduction of K+ (to 4.6 mM) hyperpolarized myocytes by 20.9±0.5 mV, an effect unchanged by 500 nM ouabain but abolished by 500 μM ouabain. We conclude that under basal conditions, Na+/K+‐ATPases containing α2‐ and/or α3‐subunits are partially responsible for the observed K+‐induced effects. The opening of myocyte K+ channels (by levcromakalim or phenylephrine) creates a ‘K+ cloud’ around the cells which fully activates Na+/K+‐ATPase and thereby abolishes further responses to [K+]o elevation. British Journal of Pharmacology (2002) 136, 918–926. doi:

Videomicroscopic methods with off-line analysis of microcirculatory parameters by multifunctional computer-assisted image analysis systems have significant advantages for in vivo microvascular research. A limitation of these methods is, however, that red blood cell velocities (V_RBC) exceeding 2 mm/s cannot be measured using standard video framing rates. In the present study, a high-speed video camera, recording up to 600 frames per second, was incorporated in the set-up, and V_RBC was measured off-line with the line-shift-diagram method. The aim of this study was to test the reproducibility and validity of the method using a high-speed video camera and to evaluate its applicability in vivo. V_RBC were measured in arterioles of the split hydronephrotic kidney. The intra- and interindividual variability was small for V_RBC below 40 mm/s. The validity of the method was tested using the mass conservation principle and found to be at least as good as that of the dual-slit photometric technique. The present approach extends the application of videomicroscopy coupled to image analysis systems to the analysis of high V_RBC.