No abstract

1 Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 mM) induced sustained contractions with no increase in [Ca 2 þ ] i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser 19 . 2 The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 mM). LY333531 (1 mM), a specific inhibitor of PKCb, also inhibited the PDBu-induced contraction in the pregnant myometrium. 3 In the pregnant myometrium permeabilized with a-toxin, PDBu increased the contractions induced at fixed Ca 2 þ concentration (0.3 mM) both in nonpregnant and pregnant myometria, indicating Ca 2 þ sensitization of contractile elements. 4 Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (a, b, g), novel PKC (d, e, y), and atypical PKC (z but not i and l). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of b isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. 5 CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. 6 These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the b PKC isoform and CPI-17.

1 It has been reported that endothelium-dependent relaxation is impaired in pulmonary hypertensive vessels. The underlying mechanisms for this phenomenon, however, have not yet been identi®ed. In this study, the mechanisms responsible for decreased endothelium-dependent relaxation in the pulmonary artery isolated from monocrotaline (MCT)-induced pulmonary hypertensive rat (MCT rat) were examined. MCT (60 mg kg 71 ), or its vehicle was administered by a single subcutaneous injection to 6-week-old male Sprague Dawley rats. 2 Endothelium-dependent relaxation induced by carbachol or ionomycin in the MCT rat artery was signi®cantly smaller than that in vehicle-treated rat (control rat) artery. Cyclic GMP levels, measured by enzyme-immunoassay, under resting or stimulation with carbachol or ionomycin were also smaller in the MCT rat artery. However, sodium nitroprusside-induced cyclic GMP accumulation in the endothelium-denuded artery was similar in control and MCT rats. These results suggest that MCT treatment decreases endothelial nitric oxide (NO) production. 3 Resting endothelial Ca 2+ levels ([Ca 2+ ] i ) in the fura-PE3-loaded MCT rat artery, were not di erent from those in the control rat. However, the increase in endothelial [Ca 2+ ] i elicited by carbachol was attenuated in the MCT rat. 4 In quantitative RT ± PCR analysis, the expression of mRNA encoding endothelial NO synthase was rather increased in the MCT rat artery, suggesting an up-regulation of eNOS expression. 5 These results provide evidence that impaired NO-mediated arterial relaxation in the MCT rat is due to dissociation between eNOS expression and NO production. This dissociation may be derived from an inhibition of receptor-mediated Ca 2+ metabolism and also from the apparent decrease in Ca 2+ sensitivity of eNOS.

1 Xestospongin-C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane-permeable IP 3 receptor inhibitor. In this study we examined the e ects of this compound on smooth muscle from guinea-pig ileum. 2 In guinea-pig ileum permeabilized with a-toxin, xestospongin-C (3 mM) inhibited contractions induced by Ca 2+ mobilized from sarcoplasmic reticulum (SR) with IP 3 or carbachol with GTP, but not with ca eine. 3 In intact smooth muscle tissue, xestospongin-C (3 ± 10 mM) inhibited carbachol-and high-K + -induced increases in [Ca 2+ ] i and contractions at sustained phase. 4 It also inhibited voltage-dependent inward Ba 2+ currents in a concentration-dependent manner with an IC 50 of 0.63 mM. Xestospongin-C (3 ± 10 mM) had no e ect on carbachol-induced inward Ca 2+ currents via non-selective cation channels; but it did reduce voltage-dependent K + currents in a concentration-dependent manner with an IC 50 of 0.13 mM. 5 These results suggest that xestospongin-C inhibits the IP 3 receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin-C appears to inhibit voltage-dependent Ca 2+ and K + currents at a concentration range similar to that at which it inhibits the IP 3 receptor. Xestospongin-C is a selective blocker of the IP 3 receptor in permeabilised cells but not in cells with intact plasma membrane.