Inhibition of phosphodiesterase has an additive effect on estrogen's ability to inhibit collagen synthesis in vascular smooth muscle cells.

Several studies have shown that estrogen has the ability to decrease collagen synthetic rates in vascular smooth muscle cells (VSMCs) by increasing cellular cyclic AMP (cAMP) levels. Phosphodiesterase inhibitors have also been shown to inhibit collagen synthesis in VSMCs presumably by preventing the degradation of cAMP. Since estrogens and phosphodiesterase inhibitors are used clinically, it is important to determine the potential for phosphodiesterase inhibitors to potentiate estrogen's ability to inhibit collagen synthesis in VSMCs. The results of the present study demonstrate that the phosphodiesterase inhibitors cilostamide and Ro-20-1724 had an additive effect on estrogen's ability to inhibit collagen synthesis in VSMC. Also, the data suggests that phosphodiesterase inhibitors mediated this additive effect by increasing cellular levels of cAMP.

Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle.

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.

Treatment of vascular smooth muscle cells with estradiol and beta-adrenergic agonists has an additive effect on cAMP levels, but no additive effect on inhibition of collagen synthesis.

Several studies have suggested that increased cell levels of cAMP result in decreased rates of collagen synthesis. Oestrogen treatment of vascular smooth muscle cells (VSMCs) has been shown to cause increased levels of cAMP and decreased rates of collagen synthesis. Beta-adrenergic agonists are also known to increase cellular levels of cAMP in VSMCs, although the effect of beta-adrenergic agonists on collagen synthetic rates in VSMCs is unknown. Since beta-agonists and oestrogens are commonly used clinical agents these studies were conducted to determine the potential of these agents to have an additive effect on cell cAMP levels and inhibition of collagen synthetic rates. When VSMCs were treated with both oestrogen and isoproterenol there was an additive effect on cellular cAMP levels although the observed decrease in collagen synthetic rates was the same as observed in cells treated with just oestrogen. Treatment of VSMCs with propranolol inhibited isoproterenol-induced changes in cAMP but had no effect on either oestrogen-induced increases in cAMP levels or inhibition of collagen synthesis. The cellular location of cAMP following beta-adrenergic agonist treatment was different from the distribution of cAMP in control or oestrogen-treated VSMCs. This difference in cellular distribution of cAMP may partially explain the absence of collagen synthesis inhibition following beta-adrenergic agonist treatment of VSMCs.