Hyperpolarization of isolated capillaries from guinea‐pig heart induced by K⁺ channel openers and glucose deprivation

Abstract

The present study was designed to test if microvascular coronary endothelial cells express ATP–sensitive K+ channels (KATP channels). We performed microfluorometric measurements of the membrane potential of freshly isolated guinea‐pig coronary capillaries equilibrated with the voltage–sensitive dye bis‐oxonol (bis‐[1,3–dibutylbarbituric acid] trimethineoxonol, [DiBAC4(3)]). The resting membrane potential of capillaries in physiological salt solution was −46±4.2 mV (n= 8) at room temperature (22 °C) as determined after calibration of the fluorescence using the Na+–K+ ionophore gramicidin in the presence of different K+ concentrations. Spontaneous membrane potential fluctuations of 10–20 mV amplitude were often observed. A reversible, sustained hyperpolarization to a new membrane potential close to the K+ equilibrium potential (EK) could be induced by application of the K+ channel openers HOE 234 (100 nm to 1 μM), diazoxide (10 pm to 100 nM) or pinacidil (100 nM). Subsequent addition of glibenclamide (200 nm to 2 μM) reversed this hyperpolarization. A glibenclamide–sensitive hyperpolarization of coronary capillaries to values near EK was also observed upon omission of D‐glucose (10 mM) from the superfusing solution or by substituting L‐glucose for D‐glucose. Maximum hyperpolarization was reached in less than 10 min. Our results suggest that microvascular coronary endothelial cells express KATP channels which may be activated during hypoglycaemia.

Glucose Stimulates Ca2+ Influx and Insulin Secretion in 2-Week-old β-Cells Lacking ATP-sensitive K+ Channels

“…Upon depolarization, DiBAC 4 (3) enters the cell and binds to intracellular membranes and proteins, which results in an increase in fluorescence (33). Conversely, hyperpolarization causes extrusion of the dye and decrease in fluorescence (32,34). These experiments were performed with albumin-free solutions.…”

The Small G-protein Rac Mediates Depolarization-induced Superoxide Formation in Human Endothelial Cells

“…The fluorescence intensity (excitation, 488 nm; emission, Ͼ515 nm) was recorded every 20 s using a confocal microscope (Zeiss LSM 410). The fluorescence intensities were calibrated in terms of changes in membrane potential using the cationic ionophore gramicidin in the presence of different potassium concentrations according to Langheinrich and Daut (30).…”

Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and their Use in Ion Channel Screening Assays

“…The CHO cell membrane was hyperpolarized with the potassium ionophore gramicidin D, which, according to the Nernst equation (see the Materials and Methods section), induces a net K + efflux. 21,22 Increasing the concentration of extracellular potassium leads to a decrease in the gramicidin D-induced efflux and consequently to a reduced membrane hyperpolarization. Figure 5 shows the results obtained from such hyperpolarization experiments in the presence of 1 µM gramicidin D. The inset in Figure 5 clearly shows that membrane hyperpolarization with gramicidin leads to a decrease of the oxonol fluorescence, which, according to the Nernst equation, is dependent on the extracellular potassium ion concentration.…”