RESUMO
Background Sex-specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries. Methods and Results Enhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura-2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production. Conclusions Our data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.
Assuntos
Canais de Cálcio , Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células Endoteliais/metabolismo , Estradiol/farmacologia , Feminino , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Caracteres SexuaisRESUMO
Sphingolipids, modified by dietary fatty acids, are integral components of plasma membrane and caveolae that are also vasoactive compounds. We hypothesized that dietary fatty acid saturation affects vasoconstriction to sphingosine-1-phosphate (S1P) through caveolar regulation of rho kinase. Wild type (WT) and caveolin-1-deficient (cav-1 KO) mice which lack vascular caveolae were fed a low-fat diet (LF), 60% high-saturated fat diet (lard, HF), or 60% fat diet with equal amounts of lard and n-3 polyunsaturated menhaden oil (MO). Weight gain of WT on HF and MO diets was similar while markedly blunted in cav-1 KO. Neither high-fat diet affected the expression of cav-1, rho, or rho kinase in arteries from WT. In cav-1 KO, MO increased the vascular expression of rho but had no effect on rho kinase. HF had no effect on rho or rho kinase expression in cav-1 KO. S1P produced a concentration-dependent constriction of gracilis arteries from WT on LF that was reduced with HF and restored to normal with MO. Constriction to S1P was reduced in cav-1 KO and no longer affected by a high-saturated fat diet. Inhibition of rho kinase which reduced constriction to PE independent of diet in arteries from WT and cav-1 KO only reduced constriction to S1P in arteries from WT fed MO. The data suggest that dietary fatty acids modify vascular responses to S1P by a caveolar-dependent mechanism which is enhanced by dietary n-3 polyunsaturated fats.
Assuntos
Artérias/metabolismo , Caveolina 1/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Caveolina 1/genética , Dieta com Restrição de Gorduras , Dieta Hiperlipídica , Camundongos , Camundongos Knockout , Esfingosina/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
We tested whether dietary fatty acids alter membrane composition shifting localization of signaling pathways within caveolae to determine their role in vascular function. Wild type (WT) and caveolin-1-deficient mice (cav-1 KO), required for vascular caveolae formation, were fed low fat (LF), high saturated fat (HF, 60% kcal from lard), or high-fat diet with 50:50 lard and n-3 polyunsaturated fatty acid-enriched menhaden oil (MO). HF and MO increased body weight and fat in WT but had less effect in cav-1 KO. MO increased unsaturated fatty acids and the unsaturation index of aorta from WT and cav-1 KO. In LF WT aorta, endothelial nitric oxide synthase (eNOS) was localized to cav-1-enriched low-density fractions which shifted to actin-enriched high-density fractions with acetylcholine (ACh). HF and MO shifted eNOS to high-density fractions in WT aorta which was not affected by ACh. In cav-1 KO aorta, eNOS was localized in low-density non-caveolar fractions but not shifted by ACh or diet. Inducible NOS and cyclooxygenase 1/2 were not localized in low-density fractions or affected by diet, ACh or genotype. ACh-induced dilation of gracilis arteries from HF WT was similar to dilation in LF but the NOS component was reduced. In WT and cav-1 KO, dilation to ACh was enhanced by MO through increased role for NOS and cyclooxygenase. We conclude that dietary fats affect vascular fatty acid composition and membrane localization of eNOS but the contribution of eNOS and cyclooxygenase in ACh-mediated vascular responses is independent of lipid rafts.
Assuntos
Cavéolas/metabolismo , Gorduras na Dieta/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/metabolismo , Acetilcolina/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Glicemia/metabolismo , Composição Corporal/efeitos dos fármacos , Composição Corporal/fisiologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Caveolina 1/deficiência , Caveolina 1/fisiologia , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/metabolismo , Óleos de Peixe/farmacologia , Músculo Grácil/irrigação sanguínea , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/fisiopatologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase important in transducing intracellular Ca2+ signals. While in vitro data regarding the role of CaMKII in the regulation of endothelial nitric oxide synthase (eNOS) are contradictory, its role in endothelial function in vivo remains unknown. Using two novel transgenic models to express CaMKII inhibitor peptides selectively in endothelium, we examined the effect of CaMKII on eNOS activation, NO production, vasomotor tone and blood pressure. Under baseline conditions, CaMKII activation was low in the aortic wall. Consistently, systolic and diastolic blood pressure, heart rate and plasma NO levels were unaltered by endothelial CaMKII inhibition. Moreover, endothelial CaMKII inhibition had no significant effect on NO-dependent vasodilation. These results were confirmed in studies of aortic rings transduced with adenovirus expressing a CaMKII inhibitor peptide. In cultured endothelial cells, bradykinin treatment produced the anticipated rapid influx of Ca2+ and transient CaMKII and eNOS activation, whereas CaMKII inhibition blocked eNOS phosphorylation on Ser-1179 and dephosphorylation at Thr-497. Ca2+/CaM binding to eNOS and resultant NO production in vitro were decreased under CaMKII inhibition. Our results demonstrate that CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro, but does not regulate NO production, vasorelaxation or blood pressure in vivo under baseline conditions.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animais , Linhagem Celular , Humanos , FosforilaçãoRESUMO
Angiotensin-II (Ang-II) is a well-established mediator of vascular remodeling. The multifunctional calcium-calmodulin-dependent kinase II (CaMKII) is activated by Ang-II and regulates Erk1/2 and Akt-dependent signaling in cultured smooth muscle cells in vitro. Its role in Ang-II-dependent vascular remodeling in vivo is far less defined. Using a model of transgenic CaMKII inhibition selectively in smooth muscle cells, we found that CaMKII inhibition exaggerated remodeling after chronic Ang-II treatment and agonist-dependent vasoconstriction in second-order mesenteric arteries. These findings were associated with increased mRNA and protein expression of smooth muscle structural proteins. As a potential mechanism, CaMKII reduced serum response factor-dependent transcriptional activity. In summary, our findings identify CaMKII as an important regulator of smooth muscle function in Ang-II hypertension in vivo.
Assuntos
Angiotensina II/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos de Músculo Liso/metabolismo , Remodelação Vascular/fisiologia , Animais , Feminino , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , RNA Mensageiro/metabolismo , Vasoconstrição/fisiologiaRESUMO
Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Miométrio/efeitos dos fármacos , Receptores do FSH/metabolismo , Contração Uterina/efeitos dos fármacos , Adolescente , Adulto , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Miografia , Miométrio/fisiologia , Gravidez , Transdução de Sinais/fisiologia , Contração Uterina/fisiologia , Adulto JovemRESUMO
BACKGROUND: Multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMCs), but its function in experimental hypertension has not been explored. The aim of this study was to determine the impact of CaMKII inhibition selectively in VSMCs on Ang II hypertension. METHODS AND RESULTS: Transgenic expression of a CaMKII peptide inhibitor in VSMCs (TG SM-CaMKIIN model) reduced the blood pressure response to chronic Ang II infusion. The aortic depressor nerve activity was reset in hypertensive versus normotensive wild-type animals but not in TG SM-CaMKIIN mice, suggesting that changes in baroreceptor activity account for the blood pressure difference between genotypes. Accordingly, aortic pulse wave velocity, a measure of arterial wall stiffness and a determinant of baroreceptor activity, increased in hypertensive versus normotensive wild-type animals but did not change in TG SM-CaMKIIN mice. Moreover, examination of blood pressure and heart rate under ganglionic blockade revealed that VSMC CaMKII inhibition abolished the augmented efferent sympathetic outflow and renal and splanchnic nerve activity in Ang II hypertension. Consequently, we hypothesized that VSMC CaMKII controls baroreceptor activity by modifying arterial wall remodeling in Ang II hypertension. Gene expression analysis in aortas from normotensive and Ang II-infused mice revealed that TG SM-CaMKIIN aortas were protected from Ang II-induced upregulation of genes that control extracellular matrix production, including collagen. VSMC CaMKII inhibition also strongly altered the expression of muscle contractile genes under Ang II. CONCLUSIONS: CaMKII in VSMCs regulates blood pressure under Ang II hypertension by controlling structural gene expression, wall stiffness, and baroreceptor activity.
Assuntos
Angiotensina II/farmacologia , Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Músculo Liso Vascular/efeitos dos fármacos , Pressorreceptores/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacos , Animais , Aorta/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Ecocardiografia , Hipertensão/induzido quimicamente , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/fisiopatologia , Norepinefrina/sangue , Análise de Sequência com Séries de Oligonucleotídeos , Pressorreceptores/fisiologia , Remodelação Vascular/fisiologiaRESUMO
Fat intake alters mitochondrial lipid composition which can affect function. We used novel methodology to assess bioenergetics, including simultaneous ATP and reactive oxygen species (ROS) production, in liver and heart mitochondria of C57BL/6 mice fed diets of variant fatty acid content and saturation. Our methodology allowed us to clamp ADP concentration and membrane potential (ΔΨ) at fixed levels. Mice received a control diet for 1719 weeks, a high-fat (HF) diet (60% lard) for 1719 weeks, or HF for 12 weeks followed by 67 weeks of HF with 50% of fat as menhaden oil (MO) which is rich in n-3 fatty acids. ATP production was determined as conversion of 2-deoxyglucose to 2-deoxyglucose phosphate by NMR spectroscopy. Respiration and ATP production were significantly reduced at all levels of ADP and resultant clamped ΔΨ in liver mitochondria from mice fed HF compared to controls. At given ΔΨ, ROS production per mg mitochondrial protein, per unit respiration, or per ATP generated were greater for liver mitochondria of HF-fed mice compared to control or MO-fed mice. Moreover, these ROS metrics began to increase at a lower ΔΨ threshold. Similar, but less marked, changes were observed in heart mitochondria of HF-fed mice compared to controls. No changes in mitochondrial bioenergetics were observed in studies of separate mice fed HF versus control for only 12 weeks. In summary, HF feeding of sufficient duration impairs mitochondrial bioenergetics and is associated with a greater ROS "cost" of ATP production compared to controls. These effects are, in part, mitigated by MO.
Assuntos
Gorduras na Dieta/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Animais , Respiração Celular/fisiologia , Metabolismo Energético , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismoRESUMO
The multifunctional Ca(2+)/calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca(2+) channel current (ICa), cytoplasmic and sarcoplasmic reticulum Ca(2+), and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual ICa 50 ms after peak amplitude, indicative of loss of L-type Ca(2+) channel-dependent ICa facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca(2+) concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca(2+) concentration was abolished by pretreatment with nifedipine, an L-type Ca(2+) channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca(2+) content was reduced as a result of diminished sarcoplasmic reticulum Ca(2+) ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca(2+) ATPase inhibitor phospholamban. Despite the differences in intracellular Ca(2+) concentration, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin II, and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cálcio/metabolismo , Homeostase , Angiotensina II/farmacologia , Animais , Benzilaminas/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sulfonamidas/farmacologiaRESUMO
Vascular smooth muscle contraction occurs following an initial response to an increase in intracellular calcium concentration and a sustained response following increases in the sensitivity of contractile proteins to calcium (calcium sensitization). This latter process is regulated by the rhoA/rho kinase pathway and activated by serotonin. In multiple cell types, signaling molecules compartmentalize within caveolae to regulate their activation. We hypothesized that serotonin differentially compartmentalizes rhoA within caveolar versus noncaveolar lipid rafts to regulate sustained vascular contractions. To test this hypothesis, we measured aortic contractions in response to serotonin in wild-type (WT) and cav-1-deficient mice (cav-1 KO). RhoA-dependent contractions in response to serotonin were markedly augmented in arteries from cav-1 KO mice despite a modest reduction in rhoA expression compared with WT. We found that under basal conditions, rhoA in WT arteries was primarily localized within high-density sucrose gradient fractions but temporally shifted to low-density fractions in response to serotonin. In contrast, rhoA in cav-1 KO arteries was primarily in low-density fractions and shifted to high-density fractions in a similar timeframe as that seen in WT mice. We conclude that localization of rhoA to caveolar versus noncaveolar lipid rafts differentially regulates its activation and contractions to rhoA-dependent agonists with greater activation associated with its localization to noncaveolar rafts. Disruption of rhoA localization within caveolae may contribute to increased activation and enhanced vascular contractions in cardiovascular disease.
Assuntos
Caveolina 1/metabolismo , Músculo Liso Vascular/metabolismo , Serotonina/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Artérias/citologia , Artérias/metabolismo , Caveolina 1/deficiência , Caveolina 1/genética , Masculino , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso Vascular/citologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
The maternal cardiovascular system undergoes hemodynamic changes during pregnancy via angiogenesis and vasodilation to ensure adequate perfusion of the placenta. Improper vascularization at the maternal-fetal interface can cause pregnancy complications and poor fetal outcomes. Recent evidence indicates that small conductance Ca(2+)-activated K(+) channel subtype 3 (SK3) contributes to vascular remodeling during pregnancy, and we hypothesized that abnormal SK3 channel expression would alter the ability of the maternal cardiovascular system to adapt to pregnancy demands and lead to poor fetal outcomes. We investigated this hypothesis using transgenic Kcnn3(tm1Jpad)/Kcnn3(tm1Jpad) (SK3(T/T)) mice that overexpress the channel. Isolated pressurized uterine arteries from nonpregnant transgenic SK3(T/T) mice had larger basal diameters and decreased agonist-induced constriction than those from their wild-type counterparts; however, non-receptor-mediated depolarization remained intact. In addition to vascular changes, heart rates and ejection fraction were increased, whereas end systolic volume was reduced in SK3(T/T) mice compared with their wild-type littermates. Uterine sonography of the fetuses on pregnancy day 14 showed a significant decrease in fetal size in SK3(T/T) compared with wild-type mice; thus, SK3(T/T) mice displayed an intrauterine growth-restricted phenotype. The SK3(T/T) mice showed decreased placental thicknesses and higher incidence of fetal loss, losing over half of their complement of pups by midgestation. These results establish that the SK3 channel contributes to both maternal and fetal outcomes during pregnancy and point to the importance of SK3 channel regulation in maintaining a healthy pregnancy.
Assuntos
Morte Fetal/metabolismo , Retardo do Crescimento Fetal/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/biossíntese , Animais , Feminino , Morte Fetal/genética , Retardo do Crescimento Fetal/genética , Frequência Cardíaca/genética , Frequência Cardíaca/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Placenta/anatomia & histologia , Placenta/diagnóstico por imagem , Gravidez , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Volume Sistólico/genética , Volume Sistólico/fisiologia , Ultrassonografia Pré-Natal/métodos , Artéria Uterina/anatomia & histologia , Artéria Uterina/diagnóstico por imagem , Útero/irrigação sanguínea , Útero/diagnóstico por imagemRESUMO
Although 5HT(2A) receptors mediate contractions of normal arteries to serotonin (5HT), in some cardiovascular diseases, other receptor subtypes contribute to the marked increase in serotonin contractions. We hypothesized that enhanced contractions of arteries from diabetics to 5HT are mediated by an increased contribution from multiple 5HT receptor subtypes. We compared responses to selective 5HT receptor agonists and expression of 5HT receptor isoforms (5HT(1B), 5HT(2A), and 5HT(2B)) in aorta from nondiabetic (ND) compared to type 2 diabetic mice (DB, BKS.Cg-Dock7(m)+/+Lepr(db)/J). 5HT, 5HT(2A) (TCB2 and BRL54443), and 5HT(2B) (norfenfluramine and BW723C86) receptor agonists produced concentration-dependent contractions of ND arteries that were markedly increased in DB arteries. Neither ND nor DB arteries contracted to a 5HT(1B) receptor agonist. MDL11939, a 5HT(2A) receptor antagonist, and LY272015, a 5HT(2B) receptor antagonist, reduced contractions of arteries from DB to 5HT more than ND. Expression of 5HT(1B), 5HT(2A), and 5HT(2B) receptor subtypes was similar in ND and DB. Inhibition of rho kinase decreased contractions to 5HT and 5HT(2A) and 5HT(2B) receptor agonists in ND and DB. We conclude that in contrast to other cardiovascular diseases, enhanced contraction of arteries from diabetics to 5HT is not due to a change in expression of multiple 5HT receptor subtypes.
Assuntos
Aorta/metabolismo , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Serotonina/metabolismo , Vasoconstrição , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Receptor 5-HT2A de Serotonina/deficiência , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2B de Serotonina/deficiência , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Receptor 5-HT2B de Serotonina/genética , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
In late gestation, enhanced myometrial contractility is mediated in part through increased Rho/Rho kinase. Since leptin, which is elevated in pregnancy and obesity, can directly depress myometrial function, we hypothesized that in leptin receptor-deficient mice, myometrial contractility would be greater in late pregnancy due to increased Rho/Rho kinase activity. To test this, we correlated RhoA and Rho kinase expression to contractility in myometrium from nonpregnant (NP) and late-pregnant (P18) heterozygous leptin receptor-deficient mice (db/+) vs. wild-type (WT) mice. In NP mice, KCl-induced contractions were similar between WT and db/+ myometrium. However, the Rho kinase-dependent component of the contractions was greater in db/+ mice, along with an increased expression of Rho kinase. KCl-induced contractions increased in strength in myometrium from P18 WT and db/+ compared with NP. Although the contribution of Rho kinase to contractions was unchanged in P18 WT mice, it was decreased in P18 db/+ mice. The decrease in Rho kinase-dependent contractions in P18 db/+ mice coincided with reduced RhoA and Rho kinase expression relative to NP db/+. Addition of high-fat-induced abnormal glucose utilization prevented changes in Rho kinase function. We conclude that abnormal leptin signaling increases expression and function of Rho kinase to maintain contractile function in NP myometrium and that during pregnancy the contribution of RhoA and Rho kinase expression to myometrial function is reduced despite an increase in myometrial contractility. Thus, other signaling mechanisms appear to compensate when leptin signaling is reduced to maintain contractile function during pregnancy.
Assuntos
Diabetes Gestacional/metabolismo , Contração Muscular/fisiologia , Miométrio/metabolismo , Receptores para Leptina/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Peso Corporal/fisiologia , Diabetes Gestacional/fisiopatologia , Modelos Animais de Doenças , Feminino , Teste de Tolerância a Glucose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Obesidade/metabolismo , Obesidade/fisiopatologia , Gravidez , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTPRESUMO
We hypothesized that rho/rho kinase plays a role in sex differences in vascular dysfunction of diabetics. Contractions to serotonin were greater in isolated aortic rings from nondiabetic males versus females and increased further in streptozotocin-induced diabetic males but not females. The increased contractions to serotonin in males were reduced by inhibitors of rho kinase (fasudil, Y27632 and H1152) despite no change in expression of rhoA or rho kinase. Contractions to U46619 were not altered by fasudil or Y27632 or the presence of diabetes. In contrast to acute effects of fasudil, chronic treatment with fasudil increased contractions to serotonin in aorta from both non-diabetic and diabetic males. In summary, serotonin-induced contractions were increased in aorta from diabetic males but not females. Although administration of rho kinase inhibitors acutely decreased contractions to serotonin, long-term treatment with fasudil increased contractions. Long-term fasudil treatment may increase compensatory mechanisms to enhance vasoconstrictions.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Angiopatias Diabéticas/etiologia , Quinases Associadas a rho/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Caracteres Sexuais , Estreptozocina , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
The objective of this study was to determine if mechanisms involved in vascular dysfunction in type 2 diabetes differ with sex. Vascular reactivity, expression, and activation of rhoA and rho kinase were measured in aorta from male and female nondiabetic C57BLKS/J and diabetic BKS.Cg-m(+/+) Lepr(db)/J (db/db) mice, a model of type 2 diabetes. Relaxation to acetylcholine and nitroprusside was similar in aorta from nondiabetic male and female mice. Relaxation to acetylcholine was reduced approximately 50% in both male and female diabetic mice. Although inhibition of rho kinase with H-1152 increased relaxation to acetylcholine and nitroprusside in nondiabetic males, it had no effect on the response in either nondiabetic or diabetic females or diabetic males. Contraction to serotonin was increased similarly in male and female diabetic mice compared with nondiabetic mice and was reduced following inhibition of rho kinase with either fasudil or H-1152. Activation of rhoA and its downstream effector, rho kinase, was greater in aorta from diabetic males compared with nondiabetic males. In contrast, there were no differences in vascular activation of rhoA or rho kinase in diabetic females. The increased activity of rhoA and rho kinase in diabetic mice was not due to a change in protein expression of rhoA or rho kinase (ROCK1 and ROCK2) in vessels from either males or females. Although contractile dysfunction in vessels occurs in both male and female diabetic mice, the dysfunction in diabetic males is dependent upon activation of rhoA and rho kinase. Alternative mechanisms affecting rho kinase activation may be involved in females.
Assuntos
Aorta/enzimologia , Doenças da Aorta/etiologia , Diabetes Mellitus Tipo 2/enzimologia , Angiopatias Diabéticas/etiologia , Vasodilatação , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Doenças da Aorta/enzimologia , Doenças da Aorta/fisiopatologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Serotonina/metabolismo , Fatores Sexuais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTPRESUMO
The mechanisms that control the timing of labor have yet to be fully characterized. In a previous study, the overexpression of small conductance calcium-activated K(+) channel isoform 3 in transgenic mice, Kcnn3(tm1Jpad)/Kcnn3(tm1Jpad) (also known as SK3(T/T)), led to compromised parturition, which indicates that KCNN3 (also known as SK3) plays an important role in the delivery process. Based on these findings, we hypothesized that SK3 channel expression must be downregulated late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. Thus, we investigated the effects of SK3 channel expression on gestation and parturition, comparing SK3(T/T) mice to wild type (WT) mice. Here, we show in WT mice that SK3 transcript and protein are significantly reduced during pregnancy. We also found the force produced by uterine strips from Pregnancy Day 19 (P19) SK3(T/T) mice was significantly less than that measured in WT or SK3 knockout control (SK3(DOX)) uterine strips, and this effect was reversed by application of the SK3 channel inhibitor apamin. Moreover, two treatments that induce labor in mice failed to result in complete delivery in SK3(T/T) mice within 48 h after injection. Thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when SK3 channel activity is in excess. Our data indicate that SK3 channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice.
Assuntos
Trabalho de Parto Prematuro/prevenção & controle , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Contração Uterina/genética , Contração Uterina/fisiologia , Animais , Sequência de Bases , DNA Complementar/genética , Regulação para Baixo , Feminino , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miométrio/metabolismo , Trabalho de Parto Prematuro/genética , Trabalho de Parto Prematuro/fisiopatologia , GravidezRESUMO
OBJECTIVE: Studies have suggested that sex differences in endothelial function in part account for the lower incidence of cardiovascular disease in premenopausal women compared with men. Less is known about the role of smooth muscle. We hypothesized that signaling mechanisms that regulate calcium sensitivity in vascular muscle also play a role in determining sex differences in contractile function. METHODS AND RESULTS: In aorta, concentration-dependent contractions to serotonin were greater in male versus female mice whereas contractions to KCl and U46619 were similar. Nitric oxide or other endothelial-derived factors did not account for the difference in responses to serotonin because inhibition of nitric oxide synthase (NOS) with N(G)-nitro-L-arginine, genetic deficiency of endothelial NOS, and removal of endothelium increased contractions but did not abolish the enhanced contractions in aorta from males. Contractions in aorta from both males and females were abolished by a serotonergic 5HT2A receptor antagonist (ketanserin), however there was no sex difference in 5HT2A receptor expression. Activation of RhoA and Rho-kinase by serotonin was greater in aorta from males compared with females, but this was not related to greater expression of RhoA or Rho-kinase isoforms (ROCK1 and ROCK2). The sex difference in aortic contractions to serotonin was abolished by an inhibitor of Rho-kinase, Y27632. CONCLUSION: We conclude that increased contractions to serotonin in aorta from male mice are attributable to differences in RhoA/Rho-kinase activation in smooth muscle independent of differences in the expression of RhoA or Rho-kinase.
Assuntos
Aorta/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Contração Muscular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Serotonina/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Feminino , Masculino , Camundongos , Músculo Liso Vascular/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III , Fatores Sexuais , Quinases Associadas a rhoRESUMO
To test the hypothesis that protein kinase C (PKC)beta-induced reactive oxygen species (ROS) underlie the vascular dysfunction in diabetes, we examined the effects of (S)-13[(dimethylamino)-methyl]-10,11-14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadi-azacyclohexadecene-1,3(2H)-dione (LY333531; LY), a specific PKCbeta inhibitor, on arachidonic acid (AA)-mediated dilation in small coronary arteries from streptozotocin-induced diabetic rats. This study was designed to determine whether diabetes impairs AA-induced vasodilation of small coronary arteries and whether this defect could be blunted by dietary treatment with LY. Coronary diameter was measured using videomicroscopy in isolated pressurized vessels. In controls, AA dose dependently dilated coronary arteries, with 1 muM producing 54.7 +/- 3.1% and 30 microM producing 72.0 +/- 3.0% dilation (n = 9). In diabetic rats, 1 microM AA only produced 31.4 +/- 3.8% (n = 8; p < 0.01 versus control) and 30 microM 43.8 +/- 3.7% dilation (n = 8; p < 0.001 versus control). Nitroprusside-mediated vasodilations were similar in control and diabetic rats. In contrast, in diabetic rats receiving LY, AA-mediated coronary dilations were normal. In controls, AA-mediated vasodilation was inhibited by miconazole (an inhibitor of cytochrome P450 epoxygenase) and by iberiotoxin (IBTX, an inhibitor of the large conductance Ca(2+)-activated K(+) channel), but miconazole and IBTX had no effects in diabetic vessels. In diabetic rats receiving LY, the effects of miconazole and IBTX were similar to control. Superoxide dismutase restored responses to AA in diabetic vessels but had no effect in vessels from control or diabetic rats on LY. These results suggest that AA-mediated vasodilation in rat coronary arteries are impaired in diabetic rats due to increases in generation of ROS. LY protects against these defects in diabetes through inhibition of PKCbeta-mediated production of ROS.
Assuntos
Ácido Araquidônico/farmacologia , Vasos Coronários/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Indóis/farmacologia , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Vasos Coronários/fisiologia , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Masculino , Estresse Oxidativo , Oxigenases/fisiologia , Proteína Quinase C beta , Ratos , Ratos Sprague-Dawley , Estreptozocina , Superóxido Dismutase/farmacologiaRESUMO
Very little is known regarding the mechanisms of action of angiotensin II (Ang II) or the consequences of Ang II-dependent hypertension in the cerebral circulation. We tested the hypothesis that Ang II produces constriction of cerebral arteries that is mediated by activation of AT1A receptors and Rho-kinase. Basilar arteries (baseline diameter approximately 130 microm) from mice were isolated, cannulated and pressurized to measure the vessel diameter. Angiotensin II was a potent constrictor in arteries from male, but not female, mice. Vasoconstriction in response to Ang II was prevented by an inhibitor of Rho-kinase (Y-27632) in control mice, and was reduced by approximately 85% in mice deficient in expression of AT1A receptors. We also examined the chronic effects of Ang II using a model of Ang II-dependent hypertension, mice which overexpress human renin (R+) and angiotensinogen (A+). Responses to the endothelium-dependent agonist acetylcholine were markedly impaired in R+A+ mice (P<0.01) compared with controls, but were restored to normal by a superoxide scavenger (PEG-SOD). A-23187 (another endothelium-dependent agonist) produced vasodilation in control mice, but no response or vasoconstriction in R+A+ mice. In contrast, dilation of the basilar artery in response to a NO donor (NONOate) was similar in R+A+ mice and controls. Thus, Ang II produces potent constriction of cerebral arteries via activation of AT1A receptors and Rho-kinase. There are marked gender differences in cerebral vascular responses to Ang II. Endothelial function is greatly impaired in a genetic model of Ang II-dependent hypertension via a mechanism that involves superoxide.