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...

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo.The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal-regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC. Vascular smooth muscle cell (VSMC)1 proliferation and migration are associated with several vascular diseases such as atherosclerosis and restenosis following vascular injury (1-5). VSMC from several species when cultured in vitro undergo a change in phenotype from a contractile state to a synthetic state similar to the changes that occur with VSMC in vivo in response to vascular injury (6 -8). Several lines of evidence suggest that nitric oxide (NO) inhibits VSMC proliferation (9 -13), suggesting that signal transduction pathways regulated by NO may be important in VSMC phenotypic modulation. NO stimulates the production of cyclic GMP (14), which, in turn, regulates several functions of VSMC such as smooth muscle relaxation (15). The major receptor protein for cGMP in VSMC is cGMP-dependent protein kinase (PKG), a serine/ threonine kinase that catalyzes the phosphorylation of important proteins that regulate intracellular Ca 2ϩ levels and relaxation of vascular smooth muscle (16). Our laboratory has recently demonstrated that PKG also plays a major role in the regulation of the phenotype and morphology of VSMC (17, 18).The expression of PKG is highly variable in VSMC. When adult rat aortic SMC are subcultured in vitro, PKG expression is reduced to nearly un...

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.

YARA and TAT penetrate in the porcine ear skin in vitro and carry a conjugated model peptide, P20, with them. Thus, the use of PTDs can be a useful strategy to increase topical delivery of peptides for treatment of cutaneous diseases.

Introduction Human saphenous vein (HSV) is the most widely used bypass conduit for peripheral and coronary vascular reconstructions. However, outcomes are limited by a high rate of intimal hyperplasia (IH). HSV undergoes a series of ex vivo surgical manipulations prior to implantation, including hydrostatic distension, marking, and warm ischemia in solution. We investigated the impact of surgical preparation on HSV cellular function and development of IH in organ culture. We hypothesized that oxidative stress is a mediator of HSV dysfunction. Methods HSV was collected from patients undergoing vascular bypass before and after surgical preparation. Smooth muscle and endothelial function were measured using a muscle bath. Endothelial preservation was assessed with immunohistochemical staining. An organ culture model was used to investigate the influence of surgical preparation injury on the development of IH. Superoxide levels were measured using a high-performance liquid chromatography-based assay. The influence of oxidative stress on HSV physiologic responses was investigated by exposing HSV to hydrogen peroxide (H2O2). Results Surgical vein graft preparation resulted in smooth muscle and endothelial dysfunction, endothelial denudation, diminished endothelial nitric oxide synthase staining, development of increased IH, and increased levels of reactive oxygen species. Experimental induction of oxidative stress in unmanipulated HSV by treatment with H2O2 promoted endothelial dysfunction. Duration of storage time in solution did not contribute to smooth muscle or endothelial dysfunction. Conclusions Surgical vein graft preparation causes dysfunction of the smooth muscle and endothelium, endothelial denudation, reduced endothelial nitric oxide synthase expression, and promotes IH in organ culture. Moreover, increased levels of reactive oxygen species are produced and may promote further vein graft dysfunction. These results argue for less injurious means of preparing HSV prior to autologous transplantation into the arterial circulation.