Intracellular calcium concentration ([Ca2+]i) was measured with aequorin in ferret and rat aortic strips contracted with phorbol esters. In ferret aorta, 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 1 microM) induced contractions without significantly increasing [Ca2+]i, whereas 21 mM K+ induced smaller contractions with a significant rise in [Ca2+]i. Ca2+-free 2.5 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-physiological saline solution (PSS) had no effect on DPBA-induced tension, whereas it abolished contractions induced by 66 mM K+. The alpha 1-adrenergic agonist phenylephrine (10(-5) M) induced less than 10% of the tension with no initial [Ca2+]i spike under Ca-free conditions. In rat aorta, both phorbol 12-myristate 13-acetate (PMA, 2 microM) and DPBA (1 microM) induced contractions without increasing [Ca2+]i; Ca2+-free EGTA-PSS or the addition of the calcium channel blocker gallopamil (D600, 1 microM), however, abolished greater than 50% of the tension induced by either phorbol ester with a decrease in [Ca2+]i. These results are consistent with the idea that 1) resting [Ca2+]i is both sufficient and required to support phorbol ester-induced contractions in two vascular smooth muscles, suggesting an increased sensitivity of the contractile apparatus for Ca2+, and 2) there are differences in the mechanisms by which phorbol esters and alpha 1-agonists may activate vascular smooth muscle.

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To determine whether the mechanical properties of vascular smooth muscle are stimulus specific, force, stiffness, and the unloaded shortening velocity (Vmax) were measured during contractions of aortic smooth muscle strips stimulated with phenylephrine or KCl. After activation, muscle force and stiffness rose to a steady-state plateau where they were maintained. In phenylephrine contractions, Vmax peaked during force development and then fell to a lower steady-state level during force maintenance, whereas in the KCl contractions, Vmax did not decline during sustained contractions. Stimulation with KCl, compared with phenylephrine, produced lower steady-state forces. One possible interpretation is that the muscle formed latch cross-bridges during phenylephrine contractions, but not during KCl depolarizations. The slope of the plot of relative muscle force vs. stiffness for phenylephrine contractions, compared with KCl depolarizations, was reduced. This may imply tht the relative force per attached latch crossbridge could be reduced.

Gemfibrozil is a widely used drug that elevates plasma HDL and lowers triglycerides and LDL. The mechanism of action of this pharmacological agent on HDL metabolism is not established. Since the liver is the major organ involved in HDL production and removal, we assessed the effect of gemfibrozil on the modulation of apoA-I (a major protein of HDL)-containing particles by a human hepatoblastoma cell line (Hep G2). Incubation of Hep G2 cells with gemfibrozil resulted in the following statistically significant findings: (1) increased accumulation of apoA-I in the medium without affecting uptake of radiolabeled HDL-protein or HDL-apoA-I; (2) accelerated incorporation of [3H]leucine and [35S]methionine into apoA-I; (3) equivalent increases in [3H]leucine incorporation into HDL particles without and with apoA-II (LpA-I and LpA-I+A-II, respectively); (4) equal efflux of fibroblast cholesterol by harvested LpA-I and LpA-I+A-II particles; (5) increased steady state apoA-I mRNA without affecting apoA-I transcription; and (6) increased apoA-I mRNA half-life (2.2-fold). These data indicate that gemfibrozil stabilizes apoA-I mRNA transcripts, resulting in increased translation of functional apoA-I-containing particles capable of effluxing cellular cholesterol, thus defining a major mechanism by which gemfibrozil increases HDL.

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