No abstract

The regulation of L‐type Ca2+ current (ICa) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 μm to 1 mm) and (±)‐S‐nitroso‐N‐acetylpenicillamine (SNAP, 3 or 10 μm) was investigated in frog ventricular myocytes using double voltage clamp and double‐barrelled microperfusion techniques. SNP and SNAP depressed the isoprenaline (ISO, 10‐100 nm)‐ or forskolin (FSK, 1 μm)‐mediated stimulation of ICa via cGMP activation of the cGMP‐stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nm) response was observed at 1 mm SNP. When SNP was applied locally, i.e. to one‐half of the cell, and ISO to the whole cell, the response of ICa to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mm SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mm SNP). The NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (carboxy‐PTIO, 1 mm) reversed the local effect of SNAP (3 μm) on FSK‐stimulated ICa when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. A local application of erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA, 30 μm), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 μm) or SNAP (10 μm) on ICa but had no effect on the distant response. When EHNA was applied on the distant side, with SNP (1 mm) and ISO (100 nm) applied locally, the distant effect of SNP was fully reversed. Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L‐type Ca2+ channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L‐type Ca2+ channels and PDE2.

1. Inwardly rectifying K+ ('K(IR)) currents of freshly dissociated coronary endothelial cells from guinea-pig heart were investigated with the perforated-patch technique. 2. The whole-cell current-voltage relationship of endothelial cells showed strong inward rectification. Increasing the extracellular K+ resulted in an increase of inward currents. The slope conductance of the cells in the potential range negative to the calculated potassium equilibrium potential (EK) with 5, 60 and 150 mm external potassium was 0-18 + 0'14, 0.55 + 0 50 and 0-63 + 0-29 nS (mean + S.D.), respectively. 3. To quantify the steepness of inward rectification, the voltage dependence of the chord conductance of the cells was fitted with a Boltzmann function. The slope factor k describing the steepness of the relationship was 6-8 + 1'5 mV. 4. Extracellular barium induced a potential-and time-dependent block of inward currents through endothelial KIR channels. Half-maximum inhibition of IK(IR) currents was achieved with < 1 AM barium at a membrane potential of -70 mV in a solution containing 60 mM K+.5. Whole-cell inward currents revealed the opening and closing of single KIR channels. The single-channel conductance was 26 + 3 pS with 60 mm external K+ and 33 + 6 pS with 150 mm external K+. 6. Our results suggest that the electrical properties of freshly dissociated endothelial cells are to a large extent determined by five to sixty active strong inwardly rectifying K+ (KIR) channels.