IONIC CURRENTS IN RAT PULMONARY AND MESENTERIC ARTERIAL MYOCYTES IN PRIMARY CULTURE AND SUBCULTURE

The electrophysiological properties of cultured single vascular smooth muscle (VSM) cells from rat pulmonary (PA) and mesenteric (MA) arteries were studied using the whole cell patch-clamp technique. Cells were studied at 3-7 days as primary cultures, or were replated after 10-20 days and subcultured for 2-5 days. In the standard physiological bath solution (containing 1.8 mM Ca2+), and with 125 mM K+ + 10 mM ethylene glycol-bis(beta-aminoethyl ether)N, N,N',N'-tetraacetic acid (EGTA)-filled pipettes, both PA and MA primary cultured cells had high input resistances (mean =2-3 GOMEGA) and resting membrane potentials of about -40 mV. The cells were clamped at a holding potential of -70 mV. Depolarization to -20 mV or more evoked a transient inward current (I(in)) that was eliminated in Ca2+-free bath solution; this indicates that I(in) was carried by Ca2+. I(in) was substantially smaller in subcultured cells from both PA and MA. Depolarization also activated three components of outward current (I(out)) in primary cultured PA and MA cells: a rapidly inactivating transient component (I(rt)), a slowly inactivating transient component (I(st)), and a steady-state (noninactivating) component (I(ss)). All three components of I(out) were inhibited to varying degrees by 5 mM 4-aminopyridine and were eliminated by replacing intracellular K+ with Cs+, but were only minimally affected by removal of extracellular Ca2+. These results suggest that this I(out) was carried by K+ and was voltage gated. Little external Ca2+-dependent I(out) was observed under these conditions, but a substantial Ca2+-dependent component was seen when the EGTA concentration in the pipettes was reduced to 0.1 mM. In primary cultured cells of both PA and MA, I(rt) was comparable in amplitude to I(ss); in subcultured PA and MA cells, however, I(ss) was significantly smaller than I(rt), and I(st) was not detectable. These data demonstrate that the electrophysiological properties of arterial smooth muscle cells can change markedly in culture. Nevertheless, short-term primary cultured PA and MA cells retain many of the electrophysiological properties of native or acutely isolated VSM and are therefore good model systems for further electrophysiological investigation.