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Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+)]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+)]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

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 phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.

Outcomes from vein graft bypass are limited by graft failure, leading causes of which include intimal hyperplasia and vasospasm. intimal hyperplasia remains the most common cause of graft failure, but no therapeutic modalities have been shown to prevent intimal hyperplasia in humans. The small heat shock proteins are a class of naturally occurring proteins in vascular smooth muscle. These proteins have an integral role in maintenance of vascular tone and in cellular defense against various stressors. Transduction domains have enabled intracellular therapeutic delivery of peptide analogs of heat shock proteins, as well as peptide inhibitors of the kinases that phosphorylate these proteins. These cell-permeant peptides have been shown to prevent vasospasm and intimal hyperplasia in vitro. Since vascular bypass using vein grafts is analogous to autologous organ transplantation, ex vivo treatment of the vein graft with cell-permeant peptide inhibitors of vasospasm and intimal hyperplasia prior to implantation provides a unique opportunity for targeted treatment of the graft to improve patency.

Cyclic GMP (cGMP) inhibits intracellular calcium ([Ca2+]i) mobilization in vascular smooth muscle cells by a mechanism that is not well understood. Because several studies suggest that cGMP inhibits inositol 1,4,5-trisphosphate (IP3) action, we examined the effects of cGMP-dependent protein kinase on IP3 receptor phosphorylation. The purified IP3 receptor was phosphorylated using either the cGMP- or cAMP-dependent protein kinase in vitro. Phosphorylation was time-dependent and stoichiometric using both kinases. Two-dimensional phosphopeptide mapping, high performance liquid chromatography analysis, and amino acid analysis showed that identical sites were phosphorylated using either kinase, and identified serine 1755 as the site of phosphorylation. The synthetic peptide corresponding to serine 1755 (GRRESLTSFG) was phosphorylated with aKm in the range of 30-40 microM by both kinases. The kinetic analysis revealed that this peptide substrate is the best substrate described for cGMP kinase to date. Vascular smooth muscle cells prelabeled with [32P]orthophosphate and treated with atrial natriuretic peptide or sodium nitroprusside to elevate cGMP also resulted in increased labeling of the IP3 receptor. Phosphorylation of IP3 receptor by cGMP kinase may regulate the function of IP3 receptor in vascular smooth muscle cells and contribute to the effect of cGMP to regulate intracellular calcium levels.

Marking human saphenous vein graft (HSV) with a surgical skin marker to prevent twisting on implantation is a common practice in peripheral and coronary artery bypass procedures. This study is designed to examine the effects of surgical skin markers on the HSV smooth muscle and endothelial functional responses. De-identified HSV remnants were collected during peripheral and coronary artery bypass procedures. Physiologic responses of the HSV were measured using a muscle bath. Veins that were marked with surgical skin markers intraoperatively generated significantly less contractile force to depolarizing KCl (110 mM) and receptor-mediated contractile agonists than unmarked HSV, suggesting that surgical skin markers impaired HSV smooth muscle contractility. To directly access the effects of chemical components in the surgical skin markers, unmarked HSV was exposed to isopropyl alcohol (a solvent commonly used in surgical skin markers) or methylene blue (a dye). Smooth muscle contractility was significantly reduced by isopropyl alcohol and methylene blue. Endothelial-dependent relaxation to carbachol was significantly reduced after exposure to surgical skin markers. Our data demonstrated that marking HSV with surgical skin markers reduces smooth muscle and endothelial functional viability.

Vein graft intimal hyperplasia remains the leading cause of graft failure, despite many pharmacological approaches that have failed to translate to human therapy. We investigated whether local suppression of inflammation and fibrosis with MMI-0100, a novel peptide inhibitor of Mitogen Activated Protein Kinase Activated Protein Kinase II (MK2), would be an alternative strategy to reduce cell proliferation and intimal hyperplasia. The cell permeable peptide MMI-0100 was synthesized using standard Fmoc chemistry. Pharmacological doses of MMI-0100 induced minimal human endothelial and smooth muscle cell proliferation (30% and 12% respectively). MMI-0100 suppressed IL-6 expression to control levels, without effect on IL-8 expression. MMI-0100 caused sodium nitroprusside induced smooth muscle cell relaxation and inhibited intimal thickening in human saphenous vein rings in a dose-dependent fashion. In a murine aortic bypass model, MMI-0100 reduced intimal thickness in vein grafts by 72%, and there were fewer F4/80-reactive cells in vein grafts treated with MMI-0100. MMI-0100 prevents vein graft intimal thickening ex vivo and in vivo. These results suggest that inhibition of MK2 with the cell-permeant peptide MMI-0100 may be a novel strategy to suppress fibrotic processes such as vein graft disease.

Arabinogalactan-proteins (AGPs) that bind to beta-glycosyl Yariv antigens have been purified from the culture medium and plasma membrane of "Paul's Scarlet" rose cells. Starting from culture medium or from plasma membrane vesicles prepared by aqueous two-phase partitioning, the purification procedure involved Yariv antigen-induced precipitation and subsequent chromatographic steps. Two fractions, AGP-(a) and AGP-(b), were obtained from the culture medium, and one AGP fraction was obtained from the plasma membrane. The glycosyl compositions of all three fractions were dominated by arabinosyl and galactosyl residues and included glucuronosyl and other minor residues. Methylation analysis showed that AGP-(a) and AGP-(b) were both highly branched 3,6-galactans with terminal arabinofuranosyl substituents. The amino acid compositions of all three AGPs were high in alanine, hydroxyproline, and serine and/or threonine. The amino-terminal sequence of AGP-(b) contained an alanine-hydroxyproline repeat. While sharing general structural similarity, the AGPs from the plasma membrane and the culture medium were distinguishable by composition and by size and charge, with the plasma membrane AGPs being larger and more negatively charged than the culture medium AGPs.

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.

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57% ± 4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.

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.

We examined the ability of a protein transduction domain (PTD), YARA, to penetrate in the skin and carry a conjugated peptide, P20. The results with YARA were compared to those of a well-known PTD (TAT) and a control, nontransducing peptide (YKAc). The combined action of PTDs and lipid penetration enhancers was also tested.YARA, TAT, YKAc, P20, YARA-P20, and TAT-P20 were synthesized by Fmoc chemistry. Porcine ear skin mounted in a Franz diffusion cell was used to assess the topical and transdermal delivery of fluorescently tagged peptides in the presence or absence of lipid penetration enhancers (monoolein or oleic acid). The peptide concentrations in the skin (topical delivery) and receptor phase (transdermal delivery) were assessed by spectrofluorimetry. Fluorescence microscopy was used to visualize the peptides in different skin layers.YARA and TAT, but not YKAc, penetrated abundantly in the skin and permeated modestly across this tissue. Monoolein and oleic acid did not enhance the topical and transdermal delivery of TAT or YARA but increased the topical delivery of YKAc. Importantly, YARA and TAT carried a conjugated peptide, P20, into the skin, but the transdermal delivery was very small. Fluorescence microscopy confirmed that free and conjugated PTDs reached viable layers of the skin.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.

Background Vasospasm occurs in conduits used for vascular reconstructions. The small heat shock proteins, HSP20 and HSP27, coordinately regulate vascular smooth muscle tone. Phosphorylated HSP20 is associated with vasorelaxation, and phosphorylated HSP27 inhibits the phosphorylation of HSP20 and relaxation. We hypothesized that the relationship between the phosphorylated states of these two proteins might dictate the tone of a vessel and may contribute to vasospasm. Study design Sodium nitroprusside relaxation of vascular smooth muscle was recorded using pig coronary artery and human saphenous vein. Segments were frozen and homogenized, and extracted proteins were separated by one- and two-dimensional gel electrophoresis, transferred to Immobilon (Millipore), and probed with anti-cGMP-dependent protein kinase (anti-PKG), -HSP20, -HSP27, and -phosphoHSP27 antibodies. Band intensity was estimated using densitometry. Results Pig coronary artery completely relaxed (100%) with SNP (10 −7 M), but human saphenous vein only partially relaxed (20%). The levels of cGMP-dependent protein kinase and HSP20 were similar in the two tissue types. Human saphenous vein had significantly higher levels of HSP27 versus pig coronary artery (30.14 ± 0.8 versus 6.62 ± 0.2 pixels/mg; p ≤ 0.001) and phosphoHSP27 (8.29 ± 3.43 versus 0.012 ± 0.008 pixels/mg; p ≤ 0.001). Conclusions Human saphenous vein contained significantly higher levels of HSP27 and pHSP27. Increased levels of phosphorylated HSP27 might contribute to vasospasm in human saphenous vein.

To the Editor: In the article, “Phosphoproteome analysis of cardiomyocytes subjected to β-adrenergic stimulation,” Chu et al concluded that “a de novo phosphorylation of a cardiac heat shock protein p20 was identified, for the first time , in mouse cardiomyocytes, after prolonged activation of the β-adrenergic signaling pathway.1 However, cyclic nucleotide-dependent phosphorylation of p20, also referred to as heat shock-related protein 20 (HSP20), has been well characterized in smooth, skeletal, and cardiac …

Sildenafil is an oral phosphodiesterase type 5 inhibitor that is a vasodilator used in the treatment of erectile dysfunction. Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of the heat shock related protein 20 (HSP20). The purpose of this study was to determine if sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20.Peptides containing an 11 amino acid enhanced protein transduction domain (PTD) and the functional 13 amino acid sequence of HSP20 with a phosphoserine (PTD-pHSP20) were synthesized using F-MOC technology. Rings of porcine coronary artery were suspended in a muscle bath and sub-maximally contracted with serotonin. Increasing concentrations of sodium nitroprusside (SNP; 0.01-10 microM), sildenafil (0.01-100 microM), or PTD-pHSP20 (0.1-1.0 mM) were added to the baths and the percent relaxation was recorded. To determine if sildenafil-induced vasorelaxation was associated with increases in the phosphorylation of HSP20, rings of porcine coronary artery were untreated (control) or treated with SNP (10 microM) or sildenafil (100 microM) for 2, 5, and 10 min and then snap frozen. Extracted proteins were then separated using two-dimensional SDS-PAGE, transferred to a membrane, and probed for HSP20.Sildenafil induced vasorelaxation of pre-contracted coronary artery in a dose-dependent manner. Sildenafil-induced vasorelaxation was associated with an increase in the phosphorylation of HSP20. Transduction of peptide analogues of pHSP20 led to a dose-dependent relaxation of pre-contracted porcine coronary artery.These findings suggest that sildenafil-induced vasorelaxation is associated with increases in the phosphorylation of HSP20 and that transduction of phosphopeptide analogues of HSP20 is sufficient for relaxation of vascular smooth muscle.

Background— The small heat shock proteins HSP20, HSP25, αB-crystallin, and myotonic dystrophy kinase binding protein (MKBP) may regulate dynamic changes in the cytoskeleton. For example, the phosphorylation of HSP20 has been associated with relaxation of vascular smooth muscle. This study examined the function of HSP20 in heart muscle. Methods and Results— Western blotting identified immunoreactive HSP20, αB-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, αB-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and αB-crystallin were associated in an aggregate of ≈200 kDa, and αB-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, αB-crystallin, and actin staining was predominantly in transverse bands. Treatment with sodium nitroprusside led to increases in the phosphorylation of HSP20, as determined with 2-dimensional immunoblots. Incubation of transiently permeabilized myocytes with phosphopeptide analogues of HSP20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the rate of lengthening and a more rapid decay of the calcium transient. Conclusions— HSP20 is associated with αB-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.

Vascular diseases, such as atherosclerosis and restenosis following angioplasty or transplantation, are due to abnormal vascular smooth muscle growth and gene expression. The smooth muscle cells (SMC) in response to injury lose their contractile function, become highly proliferative and synthesize and secrete extracellular matrix proteins. Similar changes in the phenotypic properties of vascular SMC occur during in vitro culture. In this report, we examined whether restoration of the expression of the major receptor protein for nitric oxide (NO) signaling in smooth muscle, the guanosine 3′:5′ cyclic monophosphate (cGMP)-dependent protein kinase (PKG), reestablished contractile function to cultured rat aortic SMC. Contractile function was monitored using the silicone polymer wrinkle assay used previously to determine contractility in cultured mesangial cells. Noncontractile rat aortic smooth muscle cells transfected with the cDNA encoding the type I isoform of PKG, but not those transfected with empty vector, formed discreet wrinkles on the substratum in response to serum indicative of contraction. Treatment of the PKG-expressing SMC with sodium nitroprusside (SNP), an NO donor, and with cGMP analogs, or with the adenylyl cyclase activator, forskolin, and with adenosine 3′:5′ cyclic monophosphate (cAMP) analogs reduced wrinkling. The expression of a major PKG substrate protein involved in smooth muscle relaxation, heat shock-related protein-20 (HSP20), was also reestablished in PKG-expressing SMC. Treatment of the PKG-expressing SMC with nitroprusside resulted in phosphorylation of HSP20. Collectively, these results indicate that PKG expression is important to establish contractility to SMC in culture.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O2consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.

Publisher Summary Nitric oxide (NO) is an important cellular regulator that exerts major effects on processes as diverse as the regulation of vascular tone, and synaptic transmission and plasticity. Although NO—composed of only two atoms—is one of the simpler chemical structures in biology, its effects on cells are unusually complex. This chapter examines the most widely utilized NO signal transduction pathway, the cyclic GMP/cGMP-dependent protein kinase (PKG) system, in the regulation of smooth muscle tone. The role of PKG in smooth muscle is emphasized because this kinase is a critical control point for intracellular calcium regulation, contractile protein function, and gene expression. Special consideration is given to the pitfalls regarding interpretation of the published information from current studies.

Publisher Summary Studies on the role of cyclic nucleotides, that is cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in smooth muscle relaxation began in the mid 1960s. If the role of cAMP (cyclic nucleotides) in smooth muscle relaxation appears to be relatively straight-forward, the role of cGMP (cyclic nucleotides) was less clear. The initial studies suggested a role for cGMP in mediating smooth muscle contraction. These conclusions were based on correlations between cGMP content and contraction in response to contractile agonists such as acetylcholine. A more thorough analysis, however, revealed that the elevations in cGMP observed with acetylcholine actually occurred after the onset of contraction, suggesting that cGMP was produced in response to intracellular Ca2+ elevations. It was not until the late 1970s that several investigators demonstrated that nitrogen oxide containing vasodilators such as nitroprusside and nitroglycerine elevated cGMP in smooth muscle tissues. These findings suggested that cGMP mediated relaxation of smooth muscle, a finding that was eventually supported by the discovery of the potent smooth muscle relaxing effects of cGMP analogs. Nevertheless, there have been dissociations reported between cyclic nucleotide elevating compounds or analogs on one hand, and the relaxation of different smooth muscle preparations on the other. Uterine smooth muscle relaxation, for example, is unaffected by elevations in cGMP. Vascular and bronchial smooth muscle, however, are highly responsive to the relaxing effects of cGMP. The diversity of responses to cyclic nucleotides in the various smooth muscle types may reflect different mechanisms that lead to the decrease in tone in the different types of smooth muscle.

Although nitric oxide (NO)-mediated relaxation of vascular smooth muscle was described several years ago, the downstream effects of NO and the mechanism of relaxation of vascular smooth muscle are still not well understood. NO produced endogenously or from nitrovasodilators such as nitroglycerin or sodium nitro-prusside generates cGMP (Arnold et al. 1977; DeRubertis and Craven 1977; Gruetter et al. 1980) which in turn produces vascular smooth muscle relaxation. There have been several reviews of the roles of NO and cGMP in cellular regulation (Walter 1989; Nathan 1992; Marietta 1993; Lincoln 1994; Lincoln et al. 1996). This article is focused on the effects of cGMP-dependent protein kinase (cGMP kinase), the major receptor protein for cGMP in vascular smooth muscle, and our current understanding on how this enzyme mediates relaxation by lowering intracellular Ca2+ ([Ca2+];).

Cyclic GMP-dependent protein kinase is now implicated in a number of important cellular signaling events. The role of PKG in processes as diverse as the regulation of intracellular Ca 2+ levels in smooth muscle tissues to its potential role in gene expression has been the subject of investigations over the past few years. Despite the importance of this enzyme in cellular regulation, few details of the molecular mechanisms of action of PKG are available. There are a number of important issues to consider, however, when studying the role of NO, cGMP, and PKG in cellular function. In the first case, it is important to acknowledge the diversity of effects of NO on cellular processes. At submicromolar concentrations of NO-generating drugs such as nitroprusside, NO is known to activate soluble guanylate cyclase. Predictably, this leads to the activation of PKG and the phosphorylation of proteins relevant to the signaling cascade under investigation. At higher concentrations of NO-generating drugs, however, other effects of NO occur that may be unrelated to PKG activation. These include cross-activation of PKA by cGMP, and the modification of proteins by the NO radical. Another important consideration when investigating the role of cGMP and PKG in cell regulation is the nonspecific actions of cyclic nucleotide analogs (e.g., 8-Br-cyclic nucleotides) and drugs used to inhibit protein kinase activity. For example, high concentrations of cyclic nucleotide analogs may cross-activate both cyclic nucleotide-dependent protein kinases when incubated with cultured cells at high concentrations for prolonged periods of time. And finally, the specificity of protein phosphorylation catalyzed by protein kinases must be considered. Both PKA and PKG, for example, catalyze the phosphorylation of identical residues in protein substrates in vitro . In the intact cell, the pattern of protein phosphorylation may be affected by the localization of the kinases or the presence of adaptor or anchoring proteins. Many of these experimental problems may be addressed with appropriate pharmacological protocols (dose-response curves and time courses), and there are now available specific cDNAs for expressing catalytic domains or subunits of protein kinases. In this way, the specific role of PKG may be addressed through transfection studies.

Introduction: HSP27 and HSP20, small heat shock proteins, have been implicated in the regulation of vascular smooth muscle (VSM) tone. Stress induced hypertension leads to increased expression of HSP27 in VSM. Transduction of VSM with HSP20 leads to vasorelaxation. Phosphorylated HSP27 (pHSP27) inhibits the activation of HSP20. We hypothesize that expression of HSP27 is greater in VSM refractory to cyclic nucleotide vasorelaxation. Methods: Sodium nitroprusside (SNP 10-7M) induced relaxation of VSM was measured using segments of pig coronary artery (PCA) and human saphenous vein (HSV) in a muscle bath. The levels of PKG, HSP20, phosphorylated HPS20 (pHSP20), HSP27 and pHSP27 in tissue extracts were determined by one- and two-dimensional gel electrophoresis and western blotting. Band intensity was estimated by densitometry. Results: PCA completely relaxed with SNP (100%), however, HSV did not (60%). Both tissue types had similar levels of expression of PKG and HSP20. The levels of HSP27 were 5 fold higher in the HSV (6.62 + 0.15 vs. 30.14 + 0.80) and pHSP27 was undetectable in PCA (0 vs.2.25 + 0.1). The levels of pHSP20 were undetectable in HSV. Conclusions: HSV segments refractory to relaxation with SNP had similar levels of expression of PKG and HSP20 as PCA segments demonstrating that the mediators of cyclic nucleotide vasorelaxation were present in both tissues. Increased levels of pHSP27 inhibit the phosphorylation of HSP20, and therefore, vasorelaxation of the HSV segments. Intracellular pHSP20 may be necessary and sufficient for VSM relaxation.

Publisher Summary The signaling pathways by which nitric oxide (NO) affects cell function, are by no means limited to the stimulation of guanylate cyclase. Concentrations of NO that activate guanylate cyclase may also have other effects on cells. This is because, at least in part, of the fact that NO binds with high affinity to heme moieties in proteins—guanylate cyclase being the only example of a heme-containing enzyme. At high concentrations of NO, that is, those that might be realized as a consequence of the induction on NO synthase by cytokines and other biological modifier molecules, enzymes containing iron-sulfur groups bind NO. One of the recently described mechanisms of NO signaling, at least in terms of its pathophysiological effects on cells, is the formation of peroxynitrite. NO reacts with superoxide generated in response to cellular responses to oxidative injury to form the free radical peroxynitrite. Peroxynitrite, in turn, may have a variety of effects on cells, including orthonitration of tyrosine residues on proteins. The significance of this effect of NO is not clear at this time, but peroxynitrite production and protein “nitration” have been correlated with tissue injury and pathological responses of the tissues to insult.

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 Ca2+ levels in smooth muscle cells. Although mechanisms by which cGMP regulates cytosolic Ca2+ are unclear, an important role for the cGMP-dependent dependent protein kinase in regulating Ca2+ has been proposed. Cyclic GMP-dependent protein kinase has been shown to regulate several pathways that control cytosolic Ca2+ levels: inositol 1,4,5-trisphosphate production and action, Ca(2+)-ATPase 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 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 Ca2+ 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.

Background: Sildenafil is an oral phosphodiesterase type 5 inhibitor used in the treatment of erectile dysfunction. Phosphorylated heat shock protein 20 (pHSP20) mediates cyclic nucleotide induced vasorelaxation. The purpose of this study was to determine if sildenafil induced vasorelaxation is mediated through pHSP20. Methods: Peptides containing an 11 amino acid enhanced protein transduction domain (PTD) and the functional 13 amino acid sequence of HSP20 with a phosphoserine (PTD-pHSP20) were synthesized using F-MOC technology. Rings of porcine coronary artery were suspended in a muscle bath and maximally contracted with serotonin. Increasing concentrations of PTD-HSP20 (0.1 – 1.0mM), sodium nitroprusside (SNP; 0.1 - 10μM), or sildenafil (0.1 – 100 μM) were added to the baths and the percent relaxation was recorded. To determine if sildenafil induced vasorelaxation is associated with an increase in HSP20 phosphorylation, strips of porcine coronary artery were untreated (control), or treated with SNP (100 μM) or sildenafil (100 μM) for 5 minutes and then snap frozen. Extracted proteins were then separated using 2-dimensional SDS-PAGE and probed for HSP20. Results: Transduction of peptide analogues of pHSP20 led to a dose dependent relaxation of pre-contracted porcine coronary artery (open triangles). Sildenafil induced vasorelaxation of pre-contracted coronary artery in a dose dependent manner (closed triangles). Sildenafil induced vasorelaxation was associated with an increase in the phosphorylation of HSP20 (See graph). Conclusions: Transduction of PTD-pHSP20 results in a dose dependent relaxation of porcine coronary artery similar to the relaxation induced by Sildenafil. Sildenafil treatment led to increases in the phosphorylation of HSP20. Taken together, these findings suggest that protein transduction of PTD-pHSP20 may be an effective method of treating erectile dysfunction.

Treatment of cell walls isolated from carrot, cotton, tobacco, and tomato with anhydrous hydrogen fluoride (HF) at −23° generated a high yield of disaccharides of galacturonic acid and rhamnose from the backbone of the rhamnose-rich region of the pectins. 1 H-N.m.r. analysis showed ∼30 percent of the disaccharides to be acetylated at a unique location. Two-dimensional 1 H- 1 H homonuclear-correlation n.m.r. spectroscopy indicated this to be O-3 of galacturonic acid. During reaction in the HF, the disaccharides were quantitatively converted into the 3- O -acetylated and nonacetylated α- d -galactopyranosyluronic acid β- l -rhamnopyranose 1,2′:2,1′-dianhydride.