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C M Brophy

Publications and source records attributed to C M Brophy.

At least 19 recordsLinked to original sources
PubMed2001

The paradox of smooth muscle physiology.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (contraction) and the release of force (relaxation). The signaling events that activate contraction include Ca(2+)-dependent myosin light chain phosphorylation. The signaling events that mediate relaxation include the removal of a contractile agonist (passive relaxation) and activation of cyclic nucleotide-dependent signaling pathways in the continued presence of a contractile agonist (active relaxation). The major questions that remain in contractile physiology include (1) how is tonic force maintained when intracellular Ca(2+) levels and myosin light chain phosphorylation have returned to basal levels; and (2) what is the mechanism of cyclic nucleotide-dependent relaxation? This review focuses on these specific controversies surrounding the molecular mechanisms of contraction and relaxation of vascular smooth muscle.

Animals↗
PubMed2001

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

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 O(2) consumption, 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.

Animals↗
PubMed2000

Cellular stress inhibits vascular smooth muscle relaxation.

PURPOSE: Cellular stress has been shown to induce a group of proteins called heat shock proteins (HSPs). Recent evidence suggests that a group of small HSPs may modulate vascular smooth muscle contraction (HSP27) and/or relaxation (HSP20). In this investigation, we hypothesized that cellular stress would alter contraction and/or relaxation of intact vascular smooth muscles and would lead to changes in the induction and/or phosphorylation of the small HSPs. METHODS: Bovine carotid arteries were obtained from an abattoir, and physiologic contractile responses were determined in a muscle bath. Phosphorylation state-specific antibodies were produced and characterized against HSP27. Phosphorylation events were determined with phosphorylation state-specific antibodies or whole-cell phosphorylation and two-dimensional gel electrophoresis. RESULTS: Cellular stress induced by arsenite or heat shock did not alter basal tone or the magnitude of contractions induced by serotonin or high extracellular potassium chloride. However, cellular stress led to inhibition of forskolin and sodium nitroprusside-induced vasorelaxation. This impaired vasorelaxation was associated with increases in the phosphorylation of HSP27 and decreases in forskolin-induced phosphorylation of HSP20. CONCLUSION: Cellular stress, which leads to increases in the phosphorylation of HSP27, inhibits cyclic nucleotide-dependent vascular relaxation and cyclic nucleotide-dependent increases in the phosphorylation of HSP20.

Animals↗
PubMed2000

The macromolecular associations of heat shock protein-27 in vascular smooth muscle.

BACKGROUND: Behavioral stress is a risk factor for hypertension and atherosclerosis. Stress leads to increases in the expression and phosphorylation of heat shock proteins (HSPs) in vascular smooth muscle. Two small heat shock proteins, HSP27 and HSP20, have been implicated in the regulation of the contraction and relaxation of smooth muscle. We hypothesized that stress-induced alterations in the phosphorylation of HSP27 would effect the macromolecular associations of the small HSPs. METHODS: Bovine carotid artery smooth muscle was treated with buffer alone or with the chemical stressor, arsenite. HSP27 phosphorylation was determined with isoelectric focusing immunoblotting. Macromolecular interactions were determined with subcellular fractionation, molecular sieving, and glutaraldehyde cross-linking and immunoblotting. RESULTS: Arsenite treatment led to increases in the phosphorylation of HSP27, a redistribution of some HSP27 from a cytosolic to a particulate fraction and to the formation of larger macromolecular aggregates of HSP27. Glutaraldehyde cross-linking and immunoblotting demonstrated that HSP27 existed in monomeric and dimeric forms, which suggested that the large aggregates were not simply aggregates of HSP27 but contained other proteins. CONCLUSIONS: Cellular stress leads to increases in the phosphorylation of HSP27 and to changes in the macromolecular associations of HSP27 in intact vascular smooth muscles. The functions of the small HSPs in the vascular smooth muscle may be dependent on both phosphorylation and macromolecular associations.

Animals↗
PubMed2000

Functional expression of NOS 1 in vascular smooth muscle.

Substances that increase intracellular calcium concentration ([Ca(2+)](i)), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca(2+)](i) also activate Ca(2+)/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca(2+)-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeled L-arginine to L-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.

Animals↗
PubMed1999

The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation.

The small heat shock-related protein 20 (HSP20) is present in four isoforms in bovine carotid artery smooth muscles. Three of the isoforms are phosphorylated and one is not. Increases in the phosphorylation of two isoforms of HSP20 (isoform 3, pI 5.9; and 8, pI 5.7) are associated with cyclic nucleotide-dependent relaxation of bovine carotid artery smooth muscles. Increases in the phosphorylation of another isoform (isoform 4, pI 6.0) are associated with phorbol ester-induced contraction of bovine carotid artery smooth muscles. In this investigation we determined that isoforms 3 and 8 are phosphorylated on Ser16 of the HSP20 molecule during activation of cAMP-dependent signaling pathways. Phosphorylation state-specific antibodies produced against a peptide containing phosphorylated Ser16 recognized isoforms 3 and 8 but not isoform 4. In human vascular tissue, only isoform 3 is present. Incubation of transiently permeabilized strips of bovine carotid artery smooth muscle with synthetic peptides in which Ser16 is phosphorylated, inhibits contractile responses to high extracellular KCl and to serotonin. These data suggest that phosphorylation of HSP20 on Ser16 modulates cAMP-dependent vasorelaxation.

Amino Acid Sequence↗
PubMed1999

Phosphorylation of the small heat shock-related protein, HSP20, in vascular smooth muscles is associated with changes in the macromolecular associations of HSP20.

Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20. We hypothesized that phosphorylation of HSP20 in vascular smooth muscles is associated with alterations in the macromolecular associations of HSP20. Treatment of bovine carotid artery smooth muscles with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and the adenylate cyclase activator, forskolin, led to increases in the phosphorylation of HSP20 and dissociation of macromolecular aggregates of HSP20. However, 3-isobutyl-1-methylxanthine and forskolin treatment of a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle, did not result in increases in the phosphorylation of HSP20 or to dissociation of macromolecular aggregates. HSP20 can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA) in both carotid and umbilical arteries and this phosphorylation of HSP20 is associated with dissociation of macromolecular aggregates of HSP20. Activation of cyclic nucleotide-dependent signaling pathways does not lead to changes in the macromolecular associations of another small heat shock protein, HSP27. Interestingly, the myosin light chains (MLC20) are in similar fractions as the HSP20, and phosphorylation of HSP20 is associated with changes in the macromolecular associations of MLC20. These data suggest that increases in the phosphorylation of HSP20 are associated with changes in the macromolecular associations of HSP20. HSP20 may regulate vasorelaxation through a direct interaction with specific contractile regulatory proteins.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins.

Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20, and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins.

Animals↗
PubMed1999

Balloon angioplasty induces heat shock protein 70 in human blood vessels.

Balloon angioplasty produces a mechanically induced injury to the blood vessel wall. Heat shock protein 70 (HSP70) is a molecular chaperone whose expression can be induced by chemical or thermal stress. Thus, we hypothesized that the mechanical injury associated with balloon angioplasty would lead to increases in the expression of HSP70 in vascular smooth muscle. Segments of popliteal and trifurcation vessels from above-the-knee amputations were subject to transluminal balloon angioplasty, excised, and placed in organ cultures. Neighboring vessel that was not subjected to balloon angioplasty served as controls. Some vessels were treated with sodium arsenite (positive control, known to induce HSP70 expression). The vessels were homogenized and the proteins were separated by gel electrophoresis and transferred to Immobilon. Western blots with an antibody specific for the inducible form of HSP70 were analyzed by densitometry. Our results showed that HSP70 expression can be induced by the mechanical injury associated with balloon angioplasty in human atherosclerotic vessels.

Analysis of Variance↗
PubMed1999

Endothelial-dependent vasodilation is associated with increases in the phosphorylation of a small heat shock protein (HSP20).

PURPOSE: Increases in the phosphorylation of a small heat shock protein (HSP20) are associated with cyclic nucleotide-dependent vasorelaxation. The effect of pressure and flow on vessel diameter was studied. We hypothesized that physiologic conditions that induce vasorelaxation would lead to increases in HSP20 phosphorylation. METHODS: Flow-dependent changes in vessel diameter, at different intraluminal pressures, were measured with a laser optical micrometer in intact bovine carotid arteries. Experiments were performed in the presence and absence of norepinephrine (10(-5) mol/L). Increases in the phosphorylation of HSP20 were determined with isoelectric focusing immunoblots. RESULTS: The increase in vessel diameter was most significant at low intraluminal pressures (20 mm Hg), high flow rates (200 mL/min), and in the presence of the vasoconstrictor norepinephrine (10(-5) mol/L). The addition of methylene blue (a guanylate cyclase inhibitor) completely inhibited flow-induced vasodilation. Under conditions in which maximal flow induced vasodilation occurred, there were significant increases in the phosphorylation of HSP20. CONCLUSION: Flow-dependent vasodilation in isolated perfused segments of bovine carotid arteries was maximal when the intraluminal pressures were low and when the vessels were precontracted with norepinephrine. Flow-dependent vasodilation was inhibited by methylene blue and was associated with increases in the phosphorylation of HSP20, suggesting that the vasodilation was mediated by endothelial production of nitric oxide.

Animals↗
PubMed1999

The small heat shock-related protein-20 is an actin-associated protein.

PURPOSE: The activation of cyclic nucleotide-dependent signaling pathways in vascular smooth muscle is important for the prevention of vein graft spasm and neointimal hyperplasia. Cyclic nucleotide-dependent relaxation is associated with an increase in the phosphorylation of a small heat shock-related protein (HSP20). In this investigation, we examined the mechanisms by which HSP20 may modulate relaxation. METHODS: The relaxation responses of the bovine carotid artery smooth muscles were determined in a muscle bath. HSP20 phosphorylation was quantitated with isoelectric-focusing immunoblots. The association with actin was determined with coimmunoprecipitation and cosedimentation. Molecular sieving columns were used to examine the macromolecular associations of HSP20. RESULTS: The activation of cyclic nucleotide signaling pathways leads to the complete relaxation of carotid smooth muscle. This relaxation response is associated with an increase in the phosphorylation of HSP20. Actin coimmunoprecipitated with HSP20, and the association of actin with recombinant HSP20 in vitro was phosphorylation-state dependent. Finally, HSP20 exists in large (>100 kDa) aggregates, which dissociate with the activation of cyclic nucleotide signaling pathways. CONCLUSION: These data support a role of HSP20 phosphorylation in mediating smooth muscle relaxation, possibly via a direct interaction of large aggregates of HSP20 with the contractile elements.

Actins↗
PubMed1999

Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction.

Activation of cyclic nucleotide-dependent signaling pathways leads to relaxation of bovine carotid artery smooth muscle contractions and is associated with increased phosphorylation of the small heat shock-related protein (HSP20). Previous reports have shown that human umbilical artery smooth muscle is uniquely resistant to cyclic nucleotide-dependent relaxation, and HSP20 is not phosphorylated. In this investigation, we determined the phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. In carotid artery, activation of cyclic nucleotide-dependent signaling pathways inhibited contractile responses to serotonin but did not inhibit myosin light chain phosphorylation or oxygen consumption. The inhibition of contraction was associated with increases in HSP20 phosphorylation. In umbilical artery, activation of cyclic nucleotide-dependent signaling pathways did not inhibit serotonin-induced contraction or myosin light chain phosphorylation. The lack of contractile inhibition in umbilical artery was not associated with significant increases in HSP20 phosphorylation. In conclusion, cyclic nucleotide-dependent contractile inhibition is independent of the inhibition of myosin light chain phosphorylation or oxygen consumption but does correlate with increased HSP20 phosphorylation.

Animals↗
PubMed1999

Thrombin contraction of vascular smooth muscle: implications for vasospasm.

Thrombin contributes to hemostasis by activating platelets, the formation of fibrin, and contraction of the injured vessel. These effects are mediated through the proteolytic activity of thrombin. We hypothesized that thrombin may have a role in vasospasm after arterial injury and examined the physiologic and cellular signaling events of thrombin in intact vascular smooth muscles. Thrombin stimulation of strips of bovine carotid artery smooth muscle led to contractions which relaxed with the addition of the nitric oxide donor, sodium nitroprusside. However, washout of the thrombin and SNP resulted in the re-generation of force. This was not observed with other agonists such as endothelin, thromboxane analogues, or serotonin. Using two-dimensional immunoblotting we demonstrate that thrombin stimulation leads to increases in the tyrosine phosphorylation of 4 proteins, three different isoforms of P44 mitogen activated protein kinase (MAPK) and one isoform of P38 stress activated protein kinase (SAPK). Activation of P38 SAPK leads to activation of MAPKAP kinase-2 and a major substrate protein of MAPKAP kinase-2 is the small heat shock protein, HSP27. HSP27 has been implicated in mediating smooth muscle contraction. These data suggest that in the setting of arterial injury, thrombin-induced contraction may supercede over short acting vasorelaxants such as NO resulting in vasospasm. In addition to stress, physiologic substances such as thrombin, activate SAPKs leading to increases in the phosphorylation of HSP27. Thus, thrombin may play a central role in hemostasis after vascular injury and in the pathologic responses to plaque rupture and thrombosis in atherosclerosis.

Animals↗
PubMed1998

Protein kinase C activation during Ca2+-independent vascular smooth muscle contraction.

The cellular signaling mechanisms that modulate the sustained vascular smooth muscle contractions that occur in vasospasm are not known. We and others have hypothesized that a kinase cascade involving protein kinase C (PKC) modulates sustained vascular smooth muscle contraction. The purpose of this investigation was to develop a model in which the traditional contractile pathways involving myosin light chain phosphorylation are not activated and determine if the PKC pathway is activated under these conditions. The phosphorylation of caldesmon, myosin light chain (MLC20), and the specific PKC substrate, MARCKS (myristoylated, alanine-rich C-kinase substrate) was measured in bovine carotid arterial smoothmuscle (BCASM) stimulated with phorbol 12,13-dibutyrate (PDBu) under Ca2+-containing and Ca2+-free conditions. PDBu stimulation led to increases in caldesmon and MARCKS phosphorylation to the same degree in the presence or absence of Ca2+. PDBu stimulation but did not lead to increases in MLC20 phosphorylation over basal levels in Ca2+-free conditions. Immunoblot analysis of BCASM using PKC isoform-specific antibodies demonstrated the presence of one "Ca2+- dependent" PKC isoform: alpha, and two of the "Ca2+-independent" isoforms: epsilon and zeta. These data suggest that Ca2+-independent isoforms of PKC may play a role in the sustained phase of BCASM contractions through a kinase cascade that involves caldesmon and MARCKS phosphorylation but not MLC20 phosphorylation.

Animals↗
PubMed1998

Thrombin activates MAPKAP2 kinase in vascular smooth muscle.

PURPOSE: Thrombin mediates hemostasis by promoting thrombus development and vasospasm, which reduces the size of the arterial injury. Thrombin stimulation of vascular smooth muscle is associated with activation of mitogen-associated protein kinase. The purpose of this investigation was to determine the subsequent cellular signaling events in thrombin-stimulated vascular smooth muscle contraction. METHODS: Contractile responses of bovine carotid artery smooth muscle were determined in a muscle bath and compared with phosphorylation events with two-dimensional gel electrophoresis. The activity of a novel kinase, mitogen-activated protein kinase-activated protein-2 kinase (MAPKAP2 kinase), was determined by immunoprecipitation and a phosphotransferase assay. A small heat shock protein, HSP27, was identified with immunoblotting. RESULTS: Thrombin induces contraction of vascular smooth muscle and is associated with increased activity of MAPKAP2 kinase and increased phosphorylation of HSP27. Multiple isoforms of HSP27 are the predominant phosphoproteins in vascular smooth muscle, and peptide mapping suggests that the isoforms of HSP27 are structurally related and phosphorylated within similar peptide sequences. CONCLUSIONS: Activation of the MAPKAP2 kinase pathway and phosphorylation of HSP27 are associated with thrombin-induced contraction of vascular smooth muscle.

Animals↗
PubMed1998

Heat shock protein expression in umbilical artery smooth muscle.

Postpartum vasospasm in the umbilical arteries may be due to impaired vasorelaxation secondary to alterations in the expression of heat shock proteins. The contractile responses of pre- and full-term bovine umbilical artery smooth muscles were determined in a muscle bath. Heat shock protein expression was determined in bovine and human arterial tissues using western blotting with specific antisera. Full-term bovine and human umbilical artery smooth muscle was refractory to relaxation induced by the nitric oxide donor, sodium nitroprusside. This impaired vasorelaxation was associated with the expression of the inducible form of the heat shock protein, HSP70i, and increases in the expression of the small heat shock protein, HSP27. Small heat shock proteins have been implicated in modulating contraction and relaxation responses in vascular smooth muscles. Thus, alterations in heat shock protein expression may play a role in umbilical artery vasospasm.

Analysis of Variance↗
PubMed1998

Cyclosporine, but not FK506, selectively induces renal and coronary artery smooth muscle contraction.

BACKGROUND: Cyclosporine immunosuppression for organ transplantation is associated with hypertension and nephrotoxicity. Because the effects of cyclosporine as an immunosuppressant are mediated by the effect of cyclosporine as a phosphatase inhibitor, and phosphatase inhibitors are potent vascular smooth muscle contractile agents, we hypothesized that cyclosporine might induce contraction of the renal artery vascular smooth muscle directly. METHODS: Strips of bovine renal, carotid, superior mesenteric, or coronary arteries were obtained fresh from an abattoir. The strips were equilibrated in a muscle bath, and the contractile responses to cyclosporine and FK506 were determined. RESULTS: Cyclosporine (50 to 5000 micrograms/ml), but not FK506, induced rapidly developing, sustained contractions of renal and coronary artery smooth muscle. The magnitude of the cyclosporine-induced contractions of carotid and superior mesenteric artery smooth muscles was significantly less. The magnitude of renal artery smooth muscle contractions induced by cyclosporine was enhanced in the presence of an intact endothelium. CONCLUSIONS: Although these effects occurred in vitro to relatively high doses of cyclosporine, these data suggest that cyclosporine may selectively induce renal artery smooth muscle contraction through activation of the Ca(2+)-dependent phosphatase (calcineurin) in the smooth muscle, and these contractions may be enhanced by the release of endothelial-derived contracting factors.

Animals↗