RESUMO
Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca2+ ionophore ionomycin produced an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca2+]i in VSM cells leads to an even more rapid activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca(2+)-dependent component of ERK1/2 activation was mediated by CaM kinase II. Transient ERK1/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 mumol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca2+ but prevented CaM kinase II and ERK1/2 activation with almost identical potencies. Consistent with a role for Ca2+ and calmodulin in intracellular Ca(2+)-induced activation of ERK, cells pretreated with calmodulin inhibitors (W-7 or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK1/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to angiotensin II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type delta 2 CaM kinase II in COS-7 cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca(2+)-dependent pathways in VSM cells may be mediated in part by CaM kinase II-dependent activation of ERK1/2.
Assuntos
Aorta/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas Quinases Ativadas por Mitógeno , Mitógenos/farmacologia , Músculo Liso Vascular/metabolismo , Proteínas Quinases/fisiologia , Transdução de Sinais , Animais , Aorta/citologia , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Ativação Enzimática , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Ionomicina/farmacologia , Ionóforos , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Músculo Liso Vascular/citologia , Inibidores de Proteínas Quinases , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) gamma-subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II gammaB- and gammaC-subunits and a novel gamma-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the gammaG-subunit. A clone predicted to encode a 62. 2-kDa gamma-subunit, designated as gammaE, was isolated with a variable domain structure similar to a gammaB-subunit but with a 114-nucleotide insertion in the conserved "association" domain of CaM kinase II subunits. A full-length gammaE-subunit construct expressed in COS cells resulted in multimeric CaM kinase II holoenzymes (470 kDa) with activation and autoregulatory properties similar to expressed holoenzymes composed of gammaB-, gammaC-, or gammaG-subunits. Expression of gammaE and related gamma-subunit mRNAs containing the 114-base insertion was documented in porcine tissues by reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis of partially purified CaM kinase II from carotid arterial smooth muscle and brain using a sequence-specific anti-peptide antibody. Immunoprecipitations of tissue homogenates indicated a comparatively high level of expression of subunits containing the insert in brain and provided evidence for their co-assembly with other more abundant subunits into CaM kinase II heteromultimers. Our analyses indicate the following patterns of gamma-subunit expression: vascular smooth muscle, gammaB > gammaC > gammaE,G; heart, gammaB > gammaE,C > gammaG; brain, gammaE and related subunits >> gammaA,B,C,G.
Assuntos
Encéfalo/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Músculo Liso Vascular/enzimologia , Miocárdio/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cromatografia em Gel , Clonagem Molecular , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) and development of the Ca2+/CaM-independent (autonomous) form of the kinase was investigated in cultured vascular smooth muscle (VSM) cells. Within 15 s of ionomycin (1 microM) exposure 52.7 +/- 4.4% of the kinase became autonomous, a response that was partially maintained for at least 10 min. This correlated with 32P phosphorylation of CaM kinase II delta-subunits in situ and was abolished by pretreatment with the CaM kinase II inhibitor KN-93. The in situ Ca2+ dependence for generating autonomous CaM kinase II was determined in cells selectively permeabilized to Ca2+ and depleted of sarcoplasmic reticulum Ca2+ by pretreatment with thapsigargin. Analysis of the resulting curve revealed an EC50 (concentration producing 50% of maximal response) of 692 +/- 28 nM [Ca2+]i, a maximum of 68 +/- 2% of the total activity becoming autonomous reflecting nearly complete activation of CaM kinase II and a Hill slope of 3, indicating a highly cooperative process. Based on this dependence and measured [Ca2+]i responses in intact cells, increases in autonomous activity stimulated by angiotensin II, vasopressin and platelet-derived growth factor-BB (4.6-, 2-, and 1.7-fold, respectively) were unexpectedly high. In intact cells stimulated by ionomycin, the correlation between autonomous activity and [Ca2+]i resulted in a parallel curve with an EC50 of 304 +/- 23 nM [Ca2+]i. This apparent increase in Ca2+ sensitivity for generating autonomous activity in intact VSM cells was eliminated by thapsigargin pretreatment. We conclude that alteration of [Ca2+]i over a physiological range activates CaM kinase II in VSM and that this process is facilitated by release of Ca2+ from intracellular pools which initiates cooperative autophosphorylation and consequent generation of autonomous CaM kinase II activity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Músculo Liso Vascular/enzimologia , Animais , Células Cultivadas , Ativação Enzimática , Músculo Liso Vascular/citologia , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
Protein kinase C (PKC) has been proposed to be involved in the regulation of vascular smooth muscle (VSM) contractile activity. However, little is known in detail about the activation of this kinase or specific isozymes of this kinase by contractile stimuli in VSM. As an index of PKC activation, Ca(2+)- and phospholipid-dependent histone IIIS kinase activity was measured in the particulate fraction from individual strips of isometrically contracting carotid arterial smooth muscle. Phorbol 12,13-dibutyrate (PDB) increased PKC activity in the particulate fraction (155% over resting value by 15 min) with a time course which paralleled or preceded force development. Stimulation with the agonist histamine (10(-5) M) resulted in rapid increases in both force and particulate fraction PKC activity which was maximal by 2 min (increase of 139%) and partially sustained over 45 min (increase of 41%). KCl (109 mM), which evokes a sustained contractile response, caused a slow increase (124% by 45 min) in particulate fraction PKC activity. No significant increases in activator-independent histone kinase activity were observed in response to any stimulus tested. PKC alpha and PKC beta were identified as the principal Ca2+/phospholipid-dependent PKC isozymes expressed in this tissue. In unstimulated arterial tissue, the ratio of immunodetectable isozyme content (alpha:beta) was estimated to be 1:1 in the particulate and 1.5:1 in the cytosolic fractions. Upon stimulation with each of the three contractile stimuli, particulate fraction PKC content assessed by immunoblotting increased with a time course and to an extent comparable to the observed changes in PKC activity. There was no evidence of differential regulation of the PKC alpha or -beta isozymes by PDB compared to the other contractile stimuli. These results indicate that diverse contractile stimuli are capable of tonically activating PKC in preparations of functional smooth muscle, and are consistent with a functional role for PKC alpha and/or -beta in the regulation of normal smooth muscle contractile activity.
Assuntos
Músculo Liso Vascular/enzimologia , Proteína Quinase C/metabolismo , Vasoconstrição , Animais , Compartimento Celular , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Histamina/farmacologia , Técnicas In Vitro , Isoenzimas/química , Potenciais da Membrana , Contração Muscular , Dibutirato de 12,13-Forbol/farmacologia , Prazosina/farmacologia , Suínos , Fatores de TempoRESUMO
The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.