Patch clamp methods were used to study the biophysical properties of Ca ²+ -activated potassium (KCa) channels in cerebrovascular smooth muscle cells (CVSMCs) derived from the cerebral arteries of adult rats. Procedures were developed for the enzymatic dissociation of CVSMCs from cerebral arteries. Dissociated cells were maintained at 4°C or cultured at 37°C for 1-3 days prior to use. CVSMCs were identified using a monoclonal antibody specific for smooth muscle a -actin. The calcium-sensitive

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... uorescent probe fura-2 was employed to measure tht resting and serotonin (5-HT) stimulated levels of free intracellular calcium, [Ca ²+ ] in these cells. A resting [Ca ²+ ]i level of 41 ± 5.6 nM was obtained from a total of 110 CVSMCs in culture. Serotonin (5-HT, 10 nM to 100μ M) induced a transient increase of [Ca ²+Ji above resting levels. The effects of three known blockers of voltage-dependent Ca ²+ channels, nifedipine, La³ + and Co²+ were tested by coapplication with 5-HT. It was found the neither 10 mM LaC13 nor 10 mM C0C12 reduced the rise in [Ca ²+ ft evoked by application of 1 μ M 5-HT. Nifedipine (10 μ M) also failed to significantly reduce the rise in [Ca ²+ ]i activated by 1 μ M5-HT. The effects of the partially selective 5-HT₂ receptor antagonist, ketanserin (5 nM) reversibly attenuated the 5-HT response in all cells tested. The effect of 5-HT on [Ca ²+ Ji was clearly dose-dependent and the concentration of 5- HT producing a half-maximal increase in [Ca ²+ ]i was found to be 10 nM.[more abstract]