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Single smooth muscle cells were isolated from the basilar artery of the rat by enzymatic dispersion. The membrane properties of the cells were assessed using the patch-electrode voltage-clamp technique, and cell viability was monitored using fluorescein diacetate uptake. Exposure of the cells to oxyhemoglobin (5 microM) resulted in 1) contraction, 2) the appearance of membrane blebs, 3) an increase in the outward potassium currents, 4) a decrease in the membrane resistance, and 5) cell death. In contrast, no effect of oxyhemoglobin on cultured murine neuroblastoma cells was observed. Methemoglobin (100 microM) had no effects on the smooth muscle cells. Catalase (300 units/ml) or dimethyl sulfoxide (0.5%) protected against the effects of oxyhemoglobin; superoxide dismutase (100-1,000 units/ml) provided only partial protection. Exposure of the cells to superoxide anions generated by xanthine (1 mM) plus xanthine oxidase (10 units/l) or to hydrogen peroxide (500 microM) caused an increase in the outward potassium currents without affecting membrane resistance. Generation of hydroxyl radicals by metal ions plus hydrogen peroxide caused the same effects as oxyhemoglobin, that is, an increase in the potassium currents, followed by a decrease in the membrane resistance and cell death. In conclusion, it appears that oxyhemoglobin exerts its effects on vascular smooth muscle cells by the generation of free radicals, chiefly hydroxyl radicals.

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Isolated single fibres from the anterior (a.l.d.) and the posterior (p.l.d.) lattissimus dorsi muscles of embryonic and young chicks were used to study in vivo development of membrane electrical properties. Isolated fibres were obtained by an enzymatic dissociation procedure. Intracellular micro‐electrode recordings from isolated fibres and from fibres in intact muscles showed that the dissociation procedure did not significantly alter resting membrane potentials, input resistances or membrane time constants (tau m). The 14 day embryonic fibres of a.l.d. and p.l.d. did not have a measurable resting conductance to Cl‐. At hatching, about 70% of the resting conductance in p.l.d. fibres was due to Cl‐. Membrane electrical properties were estimated from the analysis of voltage responses to intracellular injection of rectangular pulses of current. At 14 days in ovo, membrane resistance (Rm) was approximately 20 k omega cm2 and membrane capacitance (Cm) was 1‐2 microF/cm2 for both a.l.d. and p.l.d. The mean membrane length constants (lambda) were 1.7 mm for a.l.d. and 1.5 mm for p.l.d. For p.l.d., the values of Rm, tau m and lambda decreased as development proceeded. For a.l.d., there was no change in these values by the time of hatching (21 days). The decreases in the electrical constants for p.l.d. fibres were partly explained by the appearance of a resting Cl‐ conductance during the last week of embryonic development.

Membrane chloride currents in chick skeletal muscle cells grown in tissue culture were studied by use of the whole cell variation of the patch electrode voltage clamp technique. Small diameter myoballs were obtained by adding colchicine to the growth media. To isolate the currents through the chloride channels, the currents through the sodium, calcium and potassium channels were minimized. With symmetrical chloride concentrations bathing the membrane, inward currents were activated by depolarizations above -45 mV. Above 0 mV, the currents became outward. The reversal potential for the currents shifted with the chloride concentration gradient in a manner consistent with the Nernst relation, indicating that the currents were predominantly carried by chloride ions. The instantaneous current-voltage relation obtained from tail current data was linear. The relationship between conductance and membrane potential was sigmoid. The conductance activated above -45 mV, increased steeply between -45 and -10 mV and saturated above +20 mV. Over the range of potentials where the conductance was just beginning to activate, the conductance increased e-fold for a 7 mV depolarization. The currents activated with an exponential time course and did not decline during step depolarizations. Tail currents declined slowly as the sum of two exponential components. The currents were reversibly suppressed by 100 microM SITS and were irreversibly suppressed by 10 microM DIDS.