ENZYMATIC ISOLATION AND CHARACTERIZATION OF SINGLE VASCULAR SMOOTH-MUSCLE CELLS FROM CREMASTERIC ARTERIOLES

Objective: The goal of the present study was to develop a method to is olate viable arteriolar muscle cells from single cremasteric arteriole s, which retain the contractile and electrophysiological phenotype of the donor microvessels. Methods: Arterioles were hand-dissected from r at and hamster cremaster muscles and dissociated by incubation in papa in and dithioerythritol for 35 min followed by incubation in collagena se, elastase, and soybean trypsin inhibitor for 10 to 25 min in soluti ons containing 100 mu M Ca2+, 10 mu M sodium nitroprusside, and 1 mg/m l albumin at 37 degrees C. Results: Populations of single smooth muscl e cells enzymatically isolated from cremasteric arterioles showed elon gated fusiform morphology and intact plasmalemmal membranes as indicat ed by retention of calcein, by exclusion of ethidium homodimer-1, and by high membrane resistances (11 +/- 0.8 G Omega, n = 36 for rat cells ; 8 +/- 0.6 G Omega, n = 21 for hamster cell; p < 0.05). Muscle cells contracted in a concentration-dependent fashion in response to pipette application ion of norepinephrine (10 nM-100 mu M). Cell shortening i n response to 1 mu M norepinephrine was inhibited by 10 mu M phentolam ine, 1 mu M sodium nitroprusside, and 1 mu M nifedipine or nominally C a2+-free media. Resting membrane potential recorded in patch-clamped c ells by perforated patch methods was -48 +/- 1 mV (n = 47) for rat cel ls and -44 +/- 2.8 mV (n = 14) for hamster cells (p > 0.05). Families of voltage-dependent K+ currents were observed during stepwise depolar izing pulses from -60 mV to more positive potentials. Blockers of volt age-gated and ATP-sensitive K+ channels (4-aminopyridine [3 mM] and gl ibenclamide [1 mu M], respectively) inhibited membrane K+ conductance, increased membrane resistance, and depolarized cells by 20 +/- 4 mV ( n = 8) and 14 +/- 3 mV (n = 6), respectively. Conclusions: The present method permits isolation of smooth muscle cells from a single cremast eric arteriole. These cells seem to :retain tile contractile phenotype , alpha-adrenergic signaling cascade, membrane potential, and K+ condu ctances described for the donor arteriole. Correlating the functional and electrophysiological propel-ties of these smooth muscle cells to i n situ and in vitro studies of their donor arterioles should provide a useful extension for understanding the physiology, pathophysiology, b iophysics, and cell biology of the microcirculation in skeletal muscle .