Padmini Komalavilas

Pleiotropic regulation of vascular smooth muscle tone by cyclic GMP-dependent protein kinase.

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.

Activation of Mitogen-activated Protein Kinase Pathways by Cyclic GMP and Cyclic GMP-dependent Protein Kinase in Contractile Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro andin 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.

cGMP signaling through cAMP- and cGMP-dependent protein kinases.

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.

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics

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.

Comparative Study of the Skin Penetration of Protein Transduction Domains and a Conjugated Peptide

Purpose.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.Methods.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.Results.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.Conclusions.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.

Localization, Macromolecular Associations, and Function of the Small Heat Shock–Related Protein HSP20 in Rat Heart

Background—The small heat shock proteins HSP20, HSP25, &agr;B-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, &agr;B-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, &agr;B-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and &agr;B-crystallin were associated in an aggregate of ≈200 kDa, and &agr;B-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, &agr;B-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 &agr;B-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.

Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10−7 M), NO (10−10–10−6 M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura‐2 fluorimetry. Zaprinast (10−4 M), increased NO‐stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast. The guanylyl cyclase inhibitor, H‐(1,2,4) oxadiazolo(4,3‐a) quinoxallin‐1‐one (ODQ, 10−5 M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp‐8‐Br‐cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10−10–10−8 M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10−7–10−6 M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10−7–10−5 M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp‐8‐Br‐cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO. Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP‐independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO.

Cyclic GMP-dependent protein kinase in nitric oxide signaling.

Abstract 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.

The small heat shock protein, HSPB6, in muscle function and disease

The small heat shock protein, HSPB6, is a 17-kDa protein that belongs to the small heat shock protein family. HSPB6 was identified in the mid-1990s when it was recognized as a by-product of the purification of HSPB1 and HSPB5. HSPB6 is highly and constitutively expressed in smooth, cardiac, and skeletal muscle and plays a role in muscle function. This review will focus on the physiologic and biochemical properties of HSPB6 in smooth, cardiac, and skeletal muscle; the putative mechanisms of action; and therapeutic implications.

The small heat shock protein (HSP) 20 is dynamically associated with the actin cross-linking protein actinin.

BACKGROUND The heat shock-related protein (HSP) 20 is associated with actin and modulates smooth-muscle relaxation. We hypothesized that HSP20 mediates vasorelaxation via dynamic interactions with cytoskeletal proteins, such as actin, or actin binding proteins, such as alpha-actinin. METHODS Physiological responses of strips of bovine carotid artery were analyzed with a muscle bath. In other experiments, the arteries were homogenized, and imunoprecipitations were performed. Immunohistochemistry with anti-HSP20 and anti-actinin antibodies was used to determine co-localization of the two proteins. RESULTS Bovine carotid arteries contracted in response to serotonin and rapidly relaxed in response to forskolin. HSP20 co-immunoprecipitated with both actin and alpha-actinin, but not with HSP27 or paxillin. Immunostaining with HSP20 and alpha-actinin antibodies demonstrated that HSP20 and alpha-actinin co-localized. The amount of HSP20 that immunoprecipitated with alpha -actinin was markedly diminished in muscles that were treated with the vasorelaxant forskolin. CONCLUSIONS HSP20 is associated with both actin and alpha-actinin. Activation of cyclic nucleotide-dependent signaling pathways leads to increases in the phosphorylation of HSP20 and a decrease in the association of HSP20 with alpha-actinin. These data suggest that phosphorylation of HSP20 may lead to relaxation of vascular smooth muscles through a dynamic association with cytoskeletal elements.