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Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca(2+)-activated K(+) channels (K(Ca) channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance ( approximately 200 pS) K(Ca) (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K(Ca) channels exhibiting a much smaller conductance ( approximately 32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K(+) channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K(Ca) channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K(+)-selective channel exhibiting intermediate-conductance (37 pS at -60 mV) and potent activation by Ca(2+) (K(d) 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K(Ca) channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K(Ca) channels in functionally distinct smooth muscle cell types suggests that K(Ca) channels play a role in defining the physiological properties of vascular smooth muscle.

Genomic mechanisms of steroid action have been increasingly elucidated over the past four decades. In contrast, rapid steroid actions have been widely recognized only recently, and detailed analysis of the mechanisms involved are still lacking. The present article describes rapid effects of mineralocorticoid hormones on free intracellular calcium in vascular smooth muscle cells as determined by fura 2 spectrofluorometry in single cultured cells from rat aorta. These effects are almost immediate and reach a plateau after only 3 to 5 minutes and are characterized by high specificity for mineralocorticoids versus glucocorticoids. The potent mineralocorticoids aldosterone and fludrocortisone are agonists with estimated apparent EC50 values of approximately 0.1 to 0.5 nmol/L; deoxycorticosterone acetate is an agonist with an EC50 of approximately 5 nmol/L; and progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC50 values of approximately 0.5 to 5 mumol/L). The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The Ca2+ effect appears to involve the release of intracellular Ca2+, as shown by the inhibitory effect of thapsigargin; intriguingly, a relatively small maximum effect (approximately 40 nmol/L increase) is consistently seen. This mechanism operates at physiological subnanomolar aldosterone concentrations and appears to be a likely candidate for rapid fine tuning of cardiovascular responsivity. It may also contribute to known clinical features of mineralocorticoid action that are difficult to explain by the traditional genomic mechanism alone.

Vascular smooth muscle cells (SMCs) in the blood vessel wall are frequently heterogeneous in nature, differing in their gross morphology, size, and shape, subcellular organelles, cytoskeleton, and contractile protein composition. In adult rat arterial vessels, two populations of SMCs have been shown to predominate: elongated bipolar cells, representing the majority of cells, and epithelial-like SMCs. We examined the ionic responses of these two types of SMCs, isolated by multiple subculture, to vasoactive stimuli. Elevations in intracellular Na+ and Ca2+ were measured with SBFI and fura 2, respectively, and changes in membrane potential were measured using the potential-sensitive fluorescent probe bis-oxonol. The resting membrane potential of the elongated bipolar cells was less negative than that of the epithelial-like SMCs. Exposure of the elongated SMCs to endothelin 1, alpha-thrombin, or arginine vasopressin induced elevations in [Ca2+]i and [Na+]i and membrane depolarization. Depolarization occurred because of entry of both Na+ and Ca2+, and pharmacological blockade of Cl- or K+ channels did not attenuate the depolarization. In contrast, when [Ca2+]i was elevated by the same agonists in the epithelial-like SMCs there was a pronounced hyperpolarization that appeared to be the consequence of enhanced activity of charybdotoxin-sensitive Ca(2+)-activated K+ channels because it was abolished by charybdotoxin (20 nmol/L), partially attenuated by tetraethylammonium chloride (10 mmol/L), and unaffected by apamin (1 mumol/L), glibenclamide (1 mumol/L), or 4-aminopyridine (5 mmol/L). Chelation of [Ca2+]i also abolished the hyperpolarization; instead, a small depolarization was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated in a physiological salt solution (PSS) containing HCO3- the intracellular pH (pHi) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and 22Na+ influx rates. The recovery of pHi from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO2/HCO3(-)-buffered PSS than in HEPES-buffered PSS. Recovery of pHi was dependent on extracellular Na+ (Km, 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO3(-)-dependent mechanisms. Recovery instead appeared mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism. Differentiation between this HCO3(-)-dependent mechanism and Na+/H+ exchange was achieved after depletion of cellular ATP. Under these conditions, the NH4Cl-induced 22Na+ influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO2/HCO3(-)-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na+, the EIPA inhibition curve for the intracellular acidosis-induced 22Na+ influx was monophasic (IC50, 39 nM), whereas in an identical CO2/HCO3(-)-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 microM). Taken together, the results indicate that Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism play an important role in regulating the pHi of human vascular smooth muscle. The involvement of the latter mechanism depends on the severity of the intracellular acidosis, varying from approximately 25% in severe intracellular acidosis up to 50% at lesser, more physiological, levels of induced acidosis.