The effects of cyclic GMP (cGMP) and activation of cGMP-dependent protein kinase (PKG) on the phosphorylation of the inositol 1,4, 5-trisphosphate (IP3) receptor were examined in intact rat aorta using the technique of back phosphorylation. Aorta treated with the nitric oxide donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, or the selective PKG activator, 8-(4-para-chlorophenylthio)-cGMP (8-CPT-cGMP), demonstrated increased IP3 receptor phosphorylation in situ, which was both time- and concentration-dependent with a stoichiometry of 0.5 mol of phosphate/mol of receptor above control. Treatment of aorta with the adenyl cyclase activator, forskolin, also demonstrated increased phosphorylation of the IP3 receptor on the PKG site, although the selective cAMP-dependent protein kinase activator, 8-(4-para-chlorophenylthio)-cAMP (8-CPT-cAMP), did not increase the phosphorylation of the IP3 receptor. Moreover, the PKG selective inhibitor, KT 5823, inhibited both sodium nitroprusside and forskolin-induced IP3 receptor phosphorylation more potently than the selective cAMP-dependent protein kinase inhibitor, KT 5720, suggesting that PKG mediates the increase in IP3 receptor phosphorylation by both cyclic nucleotides in intact aorta. These results provide further support for the notion that PKG is activated by both cAMP and cGMP in intact vascular smooth muscle and that PKG performs a critical role in cyclic nucleotide-dependent relaxation of blood vessels.

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

PURPOSE This investigation examined the trends for gender-based advancement in academic surgery by performing a comparative analysis of the rate of change in the percentage of medical students, surgery residents, and full professors of surgery who are women. METHODS All available Women in Medicine Annual Reports were obtained from the American Association of Medical Colleges (AAMC). The gender compositions of medical graduates, surgery residents, and full professors were plotted. Binomial and linear trendlines were calculated to estimate the year when 50% of surgery full professors would be women. Additionally, the percentage distribution of men and women at each professorial rank was determined from 1995 to 2009 using these reports to demonstrate the rate of academic advancement of each gender. RESULTS The slope of the line of increase for women full professors is significantly less than for female medical students and for female general surgery residents (0.36, compared with 0.75 and 0.99, respectively). This predicts that the earliest time that females will account for 50% of full professors in surgery is the year 2096. When comparing women and men in academic ranks, we find that women are much less likely than men to be full professors. CONCLUSIONS The percentage of full professors in surgery who are women is increasing at a rate disproportionately slower than the increases in female medical students and surgery residents. The rates of increase in female medical students and surgery residents are similar. The disproportionately slow rate of increase in the number of female full professors suggests that multiple factors may be responsible for this discrepancy.

Cyclic GMP (cGMP) mediates vascular smooth muscle relaxation in response to nitric oxide and atrial natriuretic peptides. One mechanism by which cGMP decreases vascular tone is by lowering cytosolic Ca 2+ levels in smooth muscle cells. Although mechanisms by which cGMP regulates cytosolic Ca 2+ are unclear, an important role for the cGMPdependent protein kinase in regulating Ca 2+ has been proposed. Cyclic GMP-dependent protein kinase has been shown to regulate several pathways that control cytosolic Ca 2+ levels: inositol 1,4,5-trisphosphate production and action, Ca 2+ -ATPase activation, and activation of Ca 2+ -activated K + channels. The pleiotropic action of cGMP-dependent protein kinase is proposed to occur through the phosphorylation of I n the 1970s vasodilators such as nitroglycerin and nitroprusside were found to relax blood vessels through the generation of cyclic GMP (cGMP) in smooth muscle cells. 13 Studies from the laboratories of Ignarro (Gruetter et al 4 ) and Murad (Arnold et al 5 ) identified the free radical nitric oxide (NO) as the substance derived from these drugs that elevated cGMP by activating soluble guanylate cyclase. In 1980, Furchgott and colleagues (Reference 6 and Cherry et al 7 ) discovered an endothelium-derived substance that relaxed contracted arterial strips. This substance, termed endothelium-derived relaxing factor (EDRF), elevated cGMP in vascular smooth muscle. 810 The pharmacologic similarities between NO and EDRF led Furchgott, 11 Ignarro et al, 12 and Palmer et al 13 to propose that EDRF was in fact NO. Thus began one of the most fascinating areas of not only vascular biology but intercellular communication in general.The role of cGMP as a mediator of NO signaling has recently focused attention on another area of investigation established in the mid-1970s, ie, the role of cGMPdependent protein kinase and other potential cGMP receptor proteins in cell function. Vascular smooth muscle cells are a rich source of cGMP-dependent protein kinase, 14 a serine-threonine protein kinase selectively activated by cGMP, and a member of the large protein kinase family (see Reference 15 for a recent review). Cyclic GMP-dependent protein kinase is closely related in structure and function to the cyclic AMP (cAMP)-dependent protein kinase, but unlike important proteins that control several signaling pathways in smooth muscle cells. One potential target for cGMP-dependent protein kinase is the class of okadaic acid-sensitive protein phosphatases that appears to regulate K + channels among other potentially important events to reduce cytosolic Ca 2+ and tone. In addition, cytoskeletal proteins are targets for cGMP-dependent protein phosphorylation, and it is now appreciated that the cytoskeleton may play a key role in signal transduction. the cAMP-dependent protein kinase, little is known of the function of the cGMP-dependent protein kinase. Specific substrate proteins are not well characterized in many instances, and the precise mechanism of cGMPdependent protein kinase in t...