RESUMO
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Assuntos
Animais , Masculino , Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Vasoconstrição/fisiologia , Aorta Torácica , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Azepinas/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fenilefrina/agonistas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos Wistar , Ribonucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Assuntos
Músculo Liso Vascular/fisiologia , Cadeias Leves de Miosina/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Vasoconstrição/fisiologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Aorta Torácica , Azepinas/farmacologia , Western Blotting , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Masculino , Toxinas Marinhas , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Oxazóis/farmacologia , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Fenilefrina/agonistas , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos Wistar , Ribonucleotídeos/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidoresRESUMO
AIMS: Cytokines interfere with signaling pathways and mediators of vascular contraction. Endothelin-1 (ET-1) plays a major role on vascular dysfunction in conditions characterized by increased circulating levels of adipokines. In the present study we tested the hypothesis that the adipokine chemerin increases vascular contractile responses via activation of ET-1/ET-1 receptors-mediated pathways. MAIN METHODS: Male, 10-12 week-old Wistar rats were used. Endothelium-intact and endothelium-denuded aortic rings were incubated with chemerin (0.5 ng/mL or 5 ng/mL, for 1 or 24h), and isometric contraction was recorded. Protein expression was determined by Western blotting. KEY FINDINGS: Constrictor responses to phenylephrine (PE) and ET-1 were increased in vessels treated for 1h with chemerin. Chemerin incubation for 24h decreased PE contractile response whereas it increased the sensitivity to ET-1. Endothelium removal significantly potentiated chemerin effects on vascular contractile responses to PE and ET-1. Incubation with either an ERK1/2 inhibitor (PD98059) or ETA antagonist (BQ123) abolished chemerin effects on PE- and ET-1-induced vasoconstriction. Phosphorylation of MEK1/2 and ERK1/2 was significantly increased in vessels treated with chemerin for 1 and 24h. Phosphorylation of these proteins was further increased in vessels incubated with ET-1 plus chemerin. ET-1 increased MEK1/2, ERK1/2 and MKP1 protein expression to values observed in vessels treated with chemerin. SIGNIFICANCE: Chemerin increases contractile responses to PE and ET-1 via ERK1/2 activation. Our study contributes to a better understanding of the mechanisms by which the adipose tissue affects vascular function and, consequently, the vascular alterations present in obesity and related diseases.
Assuntos
Adipocinas/administração & dosagem , Aorta Torácica/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Adipocinas/metabolismo , Animais , Aorta Torácica/metabolismo , Western Blotting , Quimiocinas , Relação Dose-Resposta a Droga , Endotelina-1/administração & dosagem , Endotelina-1/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Flavonoides/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Masculino , Peptídeos Cíclicos/farmacologia , Fenilefrina/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.
Assuntos
Diabetes Mellitus Experimental/complicações , Disfunção Erétil/tratamento farmacológico , Óxido Nítrico Sintase Tipo I/metabolismo , Pênis/efeitos dos fármacos , Peptídeos/uso terapêutico , Venenos de Aranha/uso terapêutico , Animais , GMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Disfunção Erétil/complicações , Disfunção Erétil/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NG-Nitroarginina Metil Éster , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Venenos de Aranha/farmacologia , ômega-Conotoxina GVIARESUMO
Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.
Assuntos
Humanos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Doenças Cardiovasculares/metabolismo , Pneumopatias/metabolismoRESUMO
Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Doenças Cardiovasculares/metabolismo , Humanos , Pneumopatias/metabolismo , Proteína ORAI1 , Molécula 1 de Interação EstromalRESUMO
Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.
Assuntos
Animais , Masculino , Camundongos , Endotélio Vascular/enzimologia , Hipertensão/fisiopatologia , Artérias Mesentéricas/enzimologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Resistência Vascular/fisiologia , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Hipertensão/induzido quimicamente , Artérias Mesentéricas/fisiologia , Ouabaína/farmacologiaRESUMO
Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 microM) or indomethacin (10 microM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 microM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 microM, a nonselective K+ channel blocker), Tram-34 (10 microM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 microM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20 degrees C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of alpha-1 and alpha-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.
Assuntos
Endotélio Vascular/enzimologia , Hipertensão/fisiopatologia , Artérias Mesentéricas/enzimologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Resistência Vascular/fisiologia , Animais , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Hipertensão/induzido quimicamente , Masculino , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Ouabaína/farmacologiaRESUMO
The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.
Assuntos
Neuropeptídeos/farmacologia , Neurotoxinas/farmacologia , Ereção Peniana/efeitos dos fármacos , Pênis/efeitos dos fármacos , Venenos de Aranha/farmacologia , Aranhas , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Estimulação Elétrica , Disfunção Erétil/complicações , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/fisiopatologia , Hipertensão/induzido quimicamente , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Neuropeptídeos/isolamento & purificação , Neurotoxinas/isolamento & purificação , Óxido Nítrico/metabolismo , Ereção Peniana/fisiologia , Pênis/inervação , Pênis/metabolismo , Ratos , Ratos WistarRESUMO
This study tests the hypothesis that contractile responses in aortae of hypertensive rats are more dependent on gap junctional communication compared to those from normotensive rats. The experimental approach was pharmacological, using inhibitors of gap junctional activity (heptanol and octanol). Two models of experimental hypertension were characterized: (1) mineralocorticoid (DOCA)-hypertensive rats and (2) stroke-prone spontaneous hypertensive rats (SHRSP). Vessels from DOCA-hypertensive rats showed a greater relaxation to heptanol and octanol, particularly when precontracted with phenylephrine, compared to sham-operated animals. Octanol-induced relaxation in aortic segments from SHRSP did not differ from normotensive values regardless of the agonist used to cause contraction. These results suggest that in DOCA hypertension, gap junctional communication and voltage-operated calcium channels are differentially regulated, which could explain in part the changes in vascular reactivity observed in mineralocorticoid hypertension.
Assuntos
Aorta/efeitos dos fármacos , Heptanol/farmacologia , Hipertensão/fisiopatologia , Octanóis/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta/fisiologia , Desoxicorticosterona , Relação Dose-Resposta a Droga , Hipertensão/induzido quimicamente , Hipertensão/genética , Técnicas In Vitro , Masculino , Nifedipino/farmacologia , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Calcium ions (Ca2+) trigger the contraction of vascular myocytes and the level of free intracellular Ca2+ within the myocyte is precisely regulated by sequestration and extrusion mechanisms. Extensive evidence indicates that a defect in the regulation of intracellular Ca2+ plays a role in the augmented vascular reactivity characteristic of clinical and experimental hypertension. For example, arteries from spontaneously hypertensive rats (SHR) have an increased contractile sensitivity to extracellular Ca2+ and intracellular Ca2+ levels are elevated in aortic smooth muscle cells of SHR. We hypothesize that these changes are due to an increase in membrane Ca2+ channel density and possibly function in vascular myocytes from hypertensive animals. Several observations using various experimental approaches support this hypothesis: 1) the contractile activity in response to depolarizing stimuli is increased in arteries from hypertensive animals demonstrating increased voltage-dependent Ca2+ channel activity in hypertension; 2) Ca2+ channel agonists such as Bay K 8644 produce contractions in isolated arterial segments from hypertensive rats and minimal contraction in those from normotensive rats; 3) intracellular Ca2+ concentration is abnormally increased in vascular myocytes from hypertensive animals following treatment with Ca2+ channel agonists and depolarizing interventions, and 4) using the voltage-clamp technique, the inward Ca2+ current in arterial myocytes from hypertensive rats is nearly twice as large as that from myocytes of normotensive rats. We suggest that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density, resulting from increased channel synthesis or reduced turnover, underlies the increased vascular reactivity characteristic of hypertension.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Resistência VascularRESUMO
Calcium ions (Ca2+) trigger the contraction of vascular myocytes and the level of free intracellular Ca2+ within the myocyte is precisely regulated by sequestration and extrusion mechanisms. Extensive evidence indicates that a defect in the regulation of intracellular Ca2+ plays a role in the augmented vascular reactivity characteristic of clinical and experimental hypertension. For example, arteries from spontaneously hypertensive rats (SHR) have an increased contractile sensitivity to extracellular Ca2+ and intracellular Ca2+ levels are elevated in aortic smooth muscle cells of SHR. We hypothesize that these changes are due to an increase in membrane Ca2+ channel density and possibly function in vascular myocytes from hypertensive animals. Several observations using various experimental approaches support this hypothesis: 1) the contractile activity in response to depolarizing stimuli is increased in arteries from hypertensive animals demonstrating increased voltage-dependent Ca2+ channel activity in hypertension; 2) Ca2+ channel agonists such as Bay K 8644 produce contractions in isolated arterial segments from hypertensive rats and minimal contraction in those from normotensive rats; 3) intracellular Ca2+ concentration is abnormally increased in vascular myocytes from hypertensive animals following treatment with Ca2+ channel agonists and depolarizing interventions, and 4) using the voltage-clamp technique, the inward Ca2+ current in arterial myocytes from hypertensive rats is nearly twice as large as that from myocytes of normotensive rats. We suggest that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density, resulting from increased channel synthesis or reduced turnover, underlies the increased vascular reactivity characteristic of hypertension.
Assuntos
Ratos , Animais , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiologia , Resistência Vascular/fisiologia , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos Endogâmicos SHR , Ratos WistarRESUMO
We tested the hypothesis that cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, increases intracellular Ca2+ concentration ([Ca2+]) in aortic myocytes and that the increase in [Ca2+]i is higher in aortic cells from deoxycorticosterone acetate (DOCA)-hypertensive rats. Male Sprague-Dawley rats, 250-300 g, underwent uninephrectomy, received a silastic implant containing DOCA (200 mg/kg) and had free access to water supplemented with 1.0% NaCl and 0.2% KCl. Control rats were also uninephrectomized, received normal tap water, but no implant. Intracellular Ca2+ measurements were performed in aortic myocytes isolated from normotensive (Systolic blood pressure = 120 +/- 3 mmHg; body weight = 478 +/- 7 g, N = 7) and DOCA-hypertensive rats (195 +/- 10 mmHg; 358 +/- 16 g, N = 7). The effects of CPA on resting [Ca2+]i and on caffeine-induced increase in [Ca2+]i after [Ca2+]i depletion and reloading were compared in aortic cells from DOCA and normotensive rats. The phasic increase in [Ca2+]i induced by 20 mM caffeine in Ca(2+)-free buffer was significantly higher in DOCA aortic cells (329 +/- 36 nM, N = 5) compared to that in normotensive cells (249 +/- 16 nM, N = 7, P < 0.05). CPA (3 microM) inhibited caffeine-induced increases in [Ca2+]i in both groups. When the cells were placed in normal buffer (1.6 mM Ca2+, loading period), after treatment with Ca(2+)-free buffer (depletion period), an increase in [Ca2+]i was observed in DOCA aortic cells (45 +/- 11 nM, N = 5) while no changes were observed in normotensive cells. CPA (3 microM) potentiated the increase in [Ca2+]i (122 +/- 30 nM, N = 5) observed in DOCA cells during the loading period while only a modest increase in [Ca2+]i (23 +/- 10 nM, N = 5) was observed in normotensive cells. CPA-induced increase in [Ca2+]i did not occur in the absence of extracellular Ca2+ or in the presence of nifedipine. These data show that CPA induces Ca2+ influx in aorta from both normotensive and DOCA-hypertensive rats. However, the increase in [Ca2+]i is higher in DOCA aortic cells possibly due to an impairment in the mechanisms that control [Ca2+]i. The large increase in [Ca2+]i in response to caffeine in DOCA cells probably reflects a greater storage of Ca2+ in the SR.
Assuntos
Cálcio/metabolismo , Desoxicorticosterona/farmacologia , Hipertensão/induzido quimicamente , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Cafeína/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Inibidores Enzimáticos/farmacologia , Líquido Intracelular , Transporte de Íons/efeitos dos fármacos , Masculino , Nifedipino/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
We tested the hypothesis that cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ -ATPase, increases intracellular Ca2+ concentration ([Ca2+]i) in aortic myocytes and that the increase in [Ca2+]i is higher in aortic cells from deoxycorticosterone acetate (DOCA)-hypertensive rats. Male Sprague-Dawley rats, 250-300 g, underwent uninephrectomy, received a silastic implant containing DOCA (200 mg/kg) and had free access to water supplemented with 1.0 per cent NaCl and 0.2 per cent KCl. Control rats were also uninephrectomized, received normal tap water, but no implant. Intracellular Ca2+ measurements were performed in aortic myocytes isolated from normotensive (Systolic blood pressure = 120 + 3 mmHg; body weight = 478 ñ 7 g, N = 7) and DOCA-hypertensive rats (195 ñ 1O mmHg; 358 ñ 16 g, N = 7). The effects of CPA on resting [Ca2+]i and on caffeine-induced increase in [Ca2+]i after [Ca2+]i depletion and reloading were compared in aortic cells from DOCA and normotensive rats. The phasic increase in [Ca2+]i induced by 20 mM caffeine in Ca2+ -free buffer was significantly higher in DOCA aortic cells (329 ñ 36 nM, N = 5) compared to that in normotensive cells (249 ñ 16 nM, N = 7, P<0.05). CPA (3 muM) inhibited caffeine-induced increases in [Ca2+]i in both groups. When the cells were placed in normal buffer (1.6 mM Ca2+, loading period), after treatment with Ca2+ -free buffer (depletion period), an increase in [Ca2+]i was observed in DOCA aortic cells (45 ñ 11 nM, N = 5) while no changes were observed in normotensive cells. CPA (3 muM) potentiated the increase in [Ca2+]i (l22 ñ 3O nM, N = 5) observed in DOCA cells during the loading period while only a modest increase in [Ca2+]i, (23 ñ 10 nM, N = 5) was observed in normotensive cells. CPA-induced increase in [Ca2+]i did not occur in the absence of extracellular Ca2+ or in the presence of nifedipine. These data show that CPA induces Ca2+ influx in aorta from both normotensive and DOCA-hypertensive rats. However, the increase in [Ca2+]i is higher in DOCA aortic cells possibly due to an impairment in the mechanisms that control [Ca2+]i. The large increase in [Ca2+]i in response to caffeine in DOCA cells probably reflects a greater storage of Ca2+ in the SR.
Assuntos
Ratos , Animais , Masculino , Cafeína/farmacologia , Cálcio/metabolismo , Desoxicorticosterona/farmacologia , Inibidores Enzimáticos/farmacologia , Hipertensão/induzido quimicamente , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Nifedipino/farmacologia , Transporte de Íons/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
Strips of tail artery from stroke-prone spontaneously hypertensive rats (SHRSP), but not from normotensive Wistar Kyoto (WKY) rats, exhibit oscillatory activity after stimulation with norepinephrine. In addition, oscillatory activity is observed in response to tetraethylammonium (TEA) in vessels from both SHRSP and WKY rats. Mechanistically, the oscillatory contractions are associated with calcium (Ca2+)-driven action potentials. We have tested the hypothesis that intracellular Ca2+ stores participate in the generation of norepinephrine-induced oscillatory contractions in tail arteries from SHRSP. Additionally, the role of intracellular Ca2+ stores on TEA-induced contractions were evaluated. Contractile force in strips of tail artery from SHRSP and WKY rats was measured, using standard muscle bath procedures, and the effect of interventions that affect the storage of intracellular Ca2+ on the oscillatory contractions was evaluated. Depletion of intracellular Ca2+ stores, with ryanodine, or inhibition of Ca2+ uptake into the sarcoplasmic reticulum (SR), with thapsigargin and cyclopiazonic acid (CPA), did not inhibit oscillatory contractions induced by norepinephrine in SHRSP vessels. However, these agents inhibited the amplitude of TEA-induced contractions in WKY strips. Bay K 8644 and A23187 inhibited TEA-induced oscillatory contractions in WKY vessels. In SHRSP tail artery Bay K 8644 inhibited both norepinephrine and TEA-induced contractions, while A23187 did not have any effect. The phospholipase C inhibitor, NCDC (3X 10(-5) M), blocked oscillatory activity induced by norepinephrine in SHRSP tail artery and TEA-induced oscillations both in SHRSP and WKY vessels. These observations suggest that Ca2+ release and Ca2+ uptake into intracellular Ca2+ stores are not involved in the contraction-relaxation cycles that characterize norepinephrine-induced oscillatory activity in SHRSP tail artery. Similarly, SR Ca2+ stores may modulate but are not essential for TEA-induced oscillatory contractions.
Assuntos
Cálcio/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/enzimologia , Periodicidade , Fenilcarbamatos , Vasoconstrição/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Artérias/química , Artérias/efeitos dos fármacos , Artérias/fisiologia , Pressão Sanguínea , Calcimicina/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Carbamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Hipertensão/genética , Indóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Ionóforos/farmacologia , Masculino , Músculo Liso Vascular/química , Músculo Liso Vascular/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores Citoplasmáticos e Nucleares/metabolismo , Rianodina/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Cauda/irrigação sanguínea , Tetraetilamônio/farmacologia , Tapsigargina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Vasoconstrição/efeitos dos fármacosRESUMO
Ca2+ plays a major role in vascular contraction, and a defect in intracellular Ca2+ regulation has been associated with increased vascular reactivity in hypertension. To test the hypothesis that the sarcoplasmic reticulum does not adequately buffer Ca2+ in deoxycorticosterone acetate (DOCA) hypertension, contractile experiments were performed with a specific inhibitor of the sarcoplasmic reticulum Ca2+ ATPase, cyclopiazonic acid (CPA). Contractile force in aortic strips from DOCA and control rats was measured, using standard muscle bath procedures, to evaluate (i) Ca2+ handling, assessing caffeine and serotonin (5HT) induced contractions in Ca(2+)-free buffer and (ii) relaxation rate after 5HT washout. Contractile responses elicited with 5HT (3 x 10(-6) mol/L) and caffeine (20 mmol/L) were greater in DOCA than in control arteries. CPA (1 x 10(-7) to 3 x 10(-5) mol/L) reduced phasic contractions to 5HT and caffeine in DOCA and control aorta, and no differences in the IC50 values were observed. Aortae from DOCA rats contracted when placed in normal buffer, subsequent to treatment with Ca(2+)-free buffer, but control aortae did not. CPA potentiated these responses in DOCA aorta and only caused a modest contraction in control aorta. CPA-induced contraction did not occur in Ca(2+)-free buffer, and it was inhibited by nifedipine (IC50 = 4 x 10(-9) mol/L). The relaxation rate, after 5HT washout (3 x 10(-6) mol/L), was increased in DOCA aorta (2.6 +/- 0.3 min) compared with control (1.7 +/- 0.2 min), and CPA (10(-5) mol/L) increased the relaxation rate in both groups. The results support the hypothesis of defective Ca2+ handling in DOCA hypertension. However, an increased Ca2+ influx, and not a decreased buffering ability of the sarcoplasmic reticulum, contributes to the enhanced vascular reactivity observed in DOCA hypertension.