]i was reversibly inhibited by 27 Ϯ 7% (n ϭ 21, P Ͻ 0.005) in rat and by 55 Ϯ 15% (n ϭ 9, P Ͻ 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca 2ϩ ]i. However, the combination of 8-bromocGMP with SNAP reduced the Ca 2ϩ peak by 42 Ϯ 9% (n ϭ 22, P Ͻ 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca 2ϩ release from intracellular stores. Reduction of intracellular Ca 2ϩ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor THE TONE OF VASCULAR SMOOTH MUSCLE in the corpus cavernosum regulates penile tumescence: tonic contraction maintains the flaccid state, and relaxation leads to erection. Contractility of penile smooth muscle cells (SMCs) is modulated by various neurotransmitters and locally produced vasoactive substances. Considerable evidence suggests that norepinephrine is primarily responsible for tonic contraction of corpus cavernosum SMCs through activation of ␣-adrenergic receptors (1). Agonist binding to ␣-adrenergic receptors activates a G proteincoupled pathway increasing intracellular inositol 1,4,5-trisphosphate (IP 3 ) and activation of IP 3 receptors, followed by Ca 2ϩ release (3). Nitric oxide (NO), which is released by both endothelial cells and nonadrenergic, noncholinergic nerves, leads to relaxation of corpus cavernosum and is necessary for erection (6,28). NO has a variety of cellular effects including direct effects on ion channels and activation of soluble guanylyl cyclase (sGC), which converts GTP to cGMP (4, 26). cGMP in turn activates cGMP-dependent ion channels, cGMP-dependent protein kinase (PKG), and cGMP-regulated phosphodiesterases (PDEs). It has been suggested that, in vascular smooth muscle, most of the cGMP effects are mediated by PKG, because in PKG-1-deficient mice, aortic and corpus cavernosum smooth muscles fail to relax upon activation of the NO/cGMP pathway (16,27). PKG has a variety of effects in smooth muscle, including inhibition of the IP 3 receptor (30) and regulation of the Ca 2ϩ sensitivity of contractile proteins (23). Specific PKG mechanisms contributing to the regulation of SMC tone almost certainly vary from tissue to tissue, and the identity of specific targets involved in relaxation of corpus cavernosum SMCs remains uncertain.PDE types 2, 3, 5, and 11 are expressed in corpus cavernosum SMCs, although the main PDE activity is due to PDE5, which hydrolyzes cGMP (5, 10, 25). Sildenafil citrate (Viagra) acts by enhancing NO-mediated smooth muscle relaxation by competitive inhibition of PDE5, thereby maintaining elevated intracellular cGMP levels (2, 5).We investigated the effect of NO on adrenergically stimulated changes in intracellular Ca 2ϩ concentrat...

We compared the effects of genistein with its structural derivatives daidzein and equol on excitation of pulmonary artery and vein. The concentration of genistein necessary to inhibit contractions evoked by U46619 (1nM-100 microM) ranged from 10 to 100 microM. Genistein (55 microM) reduced KCl-responses by approximately 50% and essentially abolished those evoked by U46619. Daidzein was much less effective against either agonist, and equol was ineffective against U46619. A23187-evoked contractions were markedly reduced by all 3 isoflavones, but caffeine-evoked contractions were not. Using the Western blot technique, we found many proteins were tyrosine phosphorylated within 30 seconds after stimulation with U46619, reaching a peak at 120 seconds and then falling at 300 seconds. One band at 110 kD was increased nearly 300% above baseline, while 3 others ranging from 60 to 80 kD were more than doubled in intensity. Genistein had little effect on baseline levels of phosphorylation but largely prevented the U46619-induced change; daidzein was much less effective in this respect, and equol did not significantly affect this phosphorylation. We conclude that these isoflavones provide powerful tools in the study of excitation-contraction coupling of pulmonary vasculature and that inhibition of tyrosine kinase activity may be useful clinically against pulmonary hypertension.