RESUMEN
The present study investigated whether pregnancy and circulatory ovarian hormones increase the sensitivity of the mesenteric artery to calcitonin gene-related peptide (CGRP)-induced relaxation and possible mechanisms involved in this process. Mesenteric arteries from young adult male rats or female rats (during estrous cycle, after ovariectomy, at Day 20 of gestation, or Postpartum Day 2) were isolated, and the responsiveness of the vessels to CGRP was examined with a small vessel myograph. The CGRP (10(-10) to 10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in mesenteric arteries of all groups. Arterial relaxation sensitivity to CGRP was significantly (P < 0.05) greater in female rats compared with male rats. Pregnancy increased the sensitivity to CGRP significantly (P < 0.05) compared to ovariectomized and Postpartum Day 2 rats. In pregnant rats, CGRP-receptor antagonist, CGRP(8-37), inhibited the relaxation responses produced by CGRP. The CGRP-induced relaxation was not affected by N(G)-nitro-l-arginine methyl ester (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by an inhibitor of guanylate cyclase (1H-[1 , 2 , 4 ]oxadizaolo[4 , 3 -a]quinoxalin-1-one, 10(-5) M). Relaxation responses of CGRP on mesenteric arteries were blocked (P < 0.05) by a cAMP-dependent protein kinase A inhibitor, Rp-cAMPs (10(-5) M). The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10(-3) M), but not ATP-sensitive (glybenclamide, 10(-5) M), potassium channel blocker. Therefore, the results of the present study suggest that mesenteric vascular sensitivity to CGRP is higher during pregnancy and that cAMP, cGMP, and calcium-dependent potassium channels appear to be involved. Therefore, we propose that CGRP-mediated vasodilation may be important to maintain vascular adaptations during pregnancy.
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Péptido Relacionado con Gen de Calcitonina/farmacología , Hormonas Esteroides Gonadales/fisiología , Arterias Mesentéricas/fisiología , Preñez/fisiología , Vasodilatación/efectos de los fármacos , Envejecimiento/fisiología , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Femenino , Masculino , Periodo Posparto/fisiología , Canales de Potasio/metabolismo , Embarazo , Ratas , Ratas Sprague-Dawley , Caracteres SexualesRESUMEN
Human and rodent studies have demonstrated that calcitonin gene-related peptide (CGRP), a potent vasodilator, relaxes uterine tissue during pregnancy but not during labor. The vascular sensitivity to CGRP is enhanced during pregnancy, compared to nonpregnant human uterine arteries. In the present study, we hypothesized that uterine artery relaxation effects of CGRP are enhanced in pregnant rats compared to nonpregnant diestrus rats (NP-DE) and that several secondary messenger systems are involved in this process. We also hypothesized that the expression of CGRP-A receptor components, calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein (RAMP1), and CGRP-B receptors are greater in pregnant rats. For vascular relaxation studies, uterine arteries from either NP-DE or Day 18 pregnant rats were isolated, and responsiveness of the vessels to CGRP was examined with a small vessel myograph. CGRP-A and CGRP-B receptor expressions were assessed by RT-PCR and Western immunoblotting, respectively. CGRP (10(-10)--10(-7) M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in both NP-DE and Day 18 pregnant rat uterine arteries. Pregnancy increased the vasodilator sensitivity to CGRP significantly (P < 0.05) compared to NP-DE rats. CGRP receptor antagonist, CGRP8-37, inhibited CGRP-induced relaxation of pregnant uterine arteries. The CGRP-induced relaxation was not affected by NG-nitro-l-arginine methyl ester (L-NAME) (nitric oxide inhibitor, 10(-4) M) but was significantly (P < 0.05) attenuated by inhibitors of guanylate cyclase (ODQ, 10(-5) M) and adenylate cyclase (SQ 22536, 10(-5) M). CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by potassium channel blockers KATP (glybenclamide, 10(-5) M) and K(CA) (tetraethylammonium, 10(-3) M). The expression of CRLR and RAMP1 was significantly (P < 0.05) elevated during pregnancy compared to nonpregnant diestrus state (NP-DE). However, CGRP-B receptor proteins in uterine arteries were not altered with pregnancy compared to those of NP-DE. These studies suggest that CGRP-induced increases in uterine artery relaxation may play a role in regulating blood flow to the uterus during pregnancy and, therefore, in fetal growth and survival.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Preñez/fisiología , Útero/irrigación sanguínea , Inhibidores de Adenilato Ciclasa , Animales , Arterias/efectos de los fármacos , Western Blotting , Inhibidores Enzimáticos/farmacología , Femenino , Guanilato Ciclasa/antagonistas & inhibidores , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/biosíntesis , Relajación Muscular/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Inhibidores de la Ornitina Descarboxilasa , Fragmentos de Péptidos/farmacología , Embarazo , Preñez/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteína 1 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Péptido Relacionado con el Gen de Calcitonina/biosíntesis , Receptores de Péptido Relacionado con el Gen de Calcitonina/efectos de los fármacosRESUMEN
Calcitonin gene-related peptide (CGRP) levels in plasma and the dorsal root ganglia (DRG) are increased during pregnancy and in ovariectomized rats injected with ovarian hormones. Vasodilatory responses to CGRP are also increased in these animals. In the present study, we hypothesized that pregnancy and ovarian hormones elevate the contents of CGRP in perivascular nerves. We assessed CGRP-dependent mesenteric vascular relaxation induced by electrical field stimulation (EFS) and arterial content of CGRP. Because the mesenteric artery represents resistance vessels, segments of mesenteric arteries collected from female rats at different stages of the estrous cycle, pregnancy, or postpartum and from male rats were used in this study. The EFS-induced relaxation in the presence and absence of CGRP(8-37), an antagonist of CGRP, was used to measure CGRP-dependent relaxation, and radioimmunoassay (RIA) of tissue homogenates was used to assess changes in CGRP content in mesenteric branch arteries. The results show that CGRP-dependent, EFS-induced relaxation response was lower in female rats at diestrus and proestrus than in male rats, and no statistically significant differences were observed between Gestational Day 20 and Postpartum Day 2. The RIA revealed significantly lower mesenteric artery CGRP levels in female rats at proestrus, gestation, and postpartum than in female rats at diestrus or in male rats. We conclude that no correlation exists between CGRP-dependent, EFS-induced relaxation and CGRP content in the mesenteric arteries of these animal groups. Because both CGRP levels in DRG and serum are reported to be elevated, the reduced CGRP content in the vasculature during pregnancy and proestrus implicate enhanced basal release of CGRP at the nerve terminal in these animals.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Arterias Mesentéricas/metabolismo , Embarazo/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , Péptido Relacionado con Gen de Calcitonina/farmacología , Estimulación Eléctrica , Femenino , Ganglios Espinales/metabolismo , Hormonas Esteroides Gonadales/fisiología , Técnicas In Vitro , Masculino , Arterias Mesentéricas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Periodo Posparto , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Esteroides/fisiología , Resistencia Vascular/efectos de los fármacos , Resistencia Vascular/fisiología , Sistema Vasomotor/efectos de los fármacos , Sistema Vasomotor/fisiologíaRESUMEN
Recent studies of rat mesenteric arteries using a wire myograph detected decreased Ca2+ and acetylcholine-induced relaxation responses. Preliminary experiments indicated the reduced responses were associated with the tungsten wire used in the myograph system. Compared with earlier observations, arteries mounted on aged 28-microm tungsten wire showed decreased maximal Ca2+-induced relaxation responses of arteries precontracted with phenylephrine (91.9 +/- 1.5 versus 54.8 +/- 4.5%, p < 0.001) and reduced sensitivity to Ca2+ (ED50 = 1.65 +/- 0.07 versus 4.58 +/- 0.16 mM, p < 0.001). Similar shifts were seen for acetylcholine. When the surface of the wire was cleaned by abrasion with fine sandpaper, both the ED50 for Ca2+ and maximal relaxation significantly improved. An enhanced sensitivity to Ca2+ was also seen when arteries were mounted on newly purchased 14-microm tungsten or 14-microm 24K gold wire with the rank order: 14-microm gold > 14-microm tungsten >> 28-microm aged tungsten wire. Laser Raman spectral analysis of the aged 28-microm tungsten wire showed that the surface was in an oxidized state that shared spectral characteristics with the paratungstate [W12O42](-12) anion. The effect of the paratungstate anion on arterial relaxation was therefore tested. Paratungstate, but not the structurally dissimilar tungstate and metatungstate anions, significantly reduced the sensitivity and magnitude of relaxation induced by Ca2+ and to a lesser extent, relaxation induced by acetylcholine. To learn whether paratungstate inhibits relaxation through the generation of oxygen radicals, the effect of the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (1 mM) was assessed and found to have no effect. Since Ca2+-induced relaxation is inhibited by iberiotoxin, the effect of paratungstate on K+ channel activity was assessed. Paratungstate had no effect on currents through large conductance, Ca2+-activated K+ channels in whole-cell recordings from vascular smooth muscle cells, ruling out an action at the BK(Ca) channel. We conclude that: 1) surface oxidation of tungsten wire commonly used in wire myography significantly and adversely affects vascular responses to vasodilator compounds, 2) the effect is likely mediated by the paratungstate anion, and 3) the effects of the anion are not associated with free radical generation or K+ channel inhibition.
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Calcio/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Compuestos de Tungsteno/farmacología , Acetilcolina/farmacología , Animales , Calcio/farmacología , Cricetinae , Contracción Isométrica/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Arterias Mesentéricas/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Oxidación-Reducción , Técnicas de Placa-Clamp , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.
Asunto(s)
Guanilato Ciclasa/genética , Repeticiones de Microsatélite/genética , Receptores del Factor Natriurético Atrial/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alelos , Cromosomas Artificiales de Bacteriófagos P1/genética , Mapeo Contig , ADN/química , ADN/genética , Elementos Transponibles de ADN/genética , Frecuencia de los Genes , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Eliminación de SecuenciaAsunto(s)
Presión Sanguínea/efectos de los fármacos , Calcio de la Dieta/farmacología , Animales , Calcio/metabolismo , Espacio Extracelular/metabolismo , Neuronas Aferentes/fisiología , Receptores Sensibles al Calcio , Receptores de Superficie Celular/fisiología , Vasodilatación/fisiología , Sistema Vasomotor/fisiologíaRESUMEN
Renal interstitial fluid Ca(2+) concentration ([Ca(2+)](isf)) was measured in anesthetized Wistar rats by using in situ microdialysis. During perfusion of 20 cm of the proximal small intestine with Ca(2+)-free buffer, renal [Ca(2+)](isf) was 1.63 +/- 0.19 mmol/l in the cortex (n = 6) and 1.93 +/- 0.12 mmol/l in the medulla (n = 5, P = 0.223). When Ca(2+) in the intestinal lumen was increased to 3 mmol/l, no change was seen in total or ionized serum Ca(2+) (S(Ca)), urinary Ca(2+) excretion (U(Ca)), or Ca(2+) in a microdialysate of the kidney cortex. Increasing intestinal Ca(2+) further, to 6 mmol/l, was without effect on S(Ca) but significantly increased U(Ca) by 38% and microdialysate Ca(2+) by 36% (1.25 +/- 0.0.09 vs. 1.70 +/- 0. 14 mmol/l, n = 4, P < 0.05). Intravenous infusion of 28 ng. kg(-1). min(-1) of parathyroid hormone for 1 h during perfusion of the intestinal lumen with 1 mmol/ Ca(2+)caused a 7-10% rise in S(Ca), a 40% fall in U(Ca), and a 32% increase in microdialysate Ca(2+) (1.32 +/- 0.13 vs. 1.74 +/- 0.13 mmol/l, n = 6, P < 0.05). Interlobar arteries with a mean diameter of 120 microm were studied by using a wire myograph to determine whether changes in extracellular Ca(2+) affect muscle tone. When precontracted with 5 micromol/l serotonin, the arteries relaxed in response to cumulative addition of Ca(2+) (1-5 mmol/l) with an ED(50) value for Ca(2+) of 3.30 +/- 0.08 mmol/l, n = 3. These data demonstrate that [Ca(2+)](isf) changes dynamically during manipulation of whole-animal Ca(2+) homeostasis and that intrarenal arteries relax in response to extracellular Ca(2+) varied over the range measured in vivo.
Asunto(s)
Calcio/metabolismo , Espacio Extracelular/metabolismo , Riñón/metabolismo , Animales , Calcio/fisiología , Técnicas In Vitro , Microdiálisis , Concentración Osmolar , Hormona Paratiroidea/farmacología , Ratas , Ratas Wistar , Arteria Renal/metabolismo , Vasodilatación/fisiologíaRESUMEN
We recently showed that perivascular sensory nerves of mesenteric resistance arteries (MRA) express a receptor for extracellular Ca2+ (CaR) and proposed that activation of the CaR by Ca2+ causes nerve-dependent vascular relaxation. We now describe a novel procedure for acutely denervating isolated arteries and have used this method to test the hypothesis that Ca2+-induced relaxation of MRA is nerve dependent. MRA were studied using a wire myograph equipped with electrodes for electrical field stimulation (EFS) which caused sympathetic nerve-mediated contraction, and when applied in the presence of guanethidine, induced nerve-mediated relaxation. Ca2+-induced relaxation was produced by the cumulative addition of Ca2+ to MRA precontracted with norepinephrine. Exposure of MRA to 6.5% phenol in ethanol for 20 sec significantly attenuated EFS-induced contraction and relaxation, and Ca2+-induced relaxation. The magnitude of the relaxation response to EFS correlated significantly with the decrease in Ca2+-induced relaxation. In contrast, endothelium-dependent relaxation induced by acetylcholine was slightly, but nonsignificantly decreased by phenol treatment and did not correlate with Ca2+-induced relaxation. These data indicate that brief exposure of isolated MRA to phenol significantly impairs perivascular nerve function and support the hypothesis that Ca2+-induced relaxation is neurally mediated.
Asunto(s)
Calcio/farmacología , Arterias Mesentéricas/inervación , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/inervación , Neuronas/fisiología , Fenol/farmacología , Sistema Vasomotor/fisiología , Acetilcolina/farmacología , Animales , Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/fisiología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inervación , Endotelio Vascular/fisiología , Técnicas In Vitro , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Contracción Muscular/efectos de los fármacos , Desnervación Muscular , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Neuronas/efectos de los fármacos , Norepinefrina/farmacología , Fentolamina/farmacología , Ratas , Ratas Wistar , Tetrodotoxina/farmacologíaRESUMEN
We tested the hypothesis that an endogenous cannabinoid (CB) receptor agonist, such as N-arachidonylethanolamine (anandamide), is the transmitter that mediates perivascular sensory nerve-dependent Ca2+-induced relaxation. Rat mesenteric branch arteries were studied using wire myography; relaxation was determined after inducing contraction with norepinephrine. Cumulative addition of Ca2+ caused dose-dependent relaxation (ED50 = 2.2 +/- 0.09 mM). The relaxation was inhibited by 10 mM TEA and 100 nM iberiotoxin, a blocker of large conductance Ca2+-activated K+ channels, but not by 5 microM glibenclamide, 1 mM 4-aminopyridine, or 30 nM apamin. Ca2+-induced relaxation was also blocked by the selective CB receptor antagonist SR141716A and was enhanced by pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (pefabloc; 30 microM), an inhibitor of anandamide metabolism. Anandamide also caused dose-dependent relaxation (ED50 =.72 +/- 0.3 microM). The relaxation was not inhibited by endothelial denudation, 10 microM indomethacin, or 1 microM miconazole, but was blocked by 3 microM SR141716A, 10 mM TEA, precontraction with 100 mM K+, and 100 nM iberiotoxin, and was enhanced by treatment with 30 microM pefabloc. Mesenteric branch arteries were 200-fold more sensitive to the relaxing action of anandamide than arachidonic acid (ED50 = 160 +/- 7 microM). These data show that: 1) Ca2+ and anandamide cause hyperpolarization-mediated relaxation of mesenteric branch arteries, which is dependent on an iberiotoxin-sensitive Ca2+-activated K+ channel, 2) relaxation induced by both Ca2+ and anandamide is inhibited by CB receptor blockade, and 3) relaxation induced by anandamide is not dependent on its breakdown to arachidonic acid and subsequent metabolism. These findings support the hypothesis that anandamide, or a similar cannabinoid receptor agonist, mediates nerve-dependent Ca2+-induced relaxation in the rat.
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Ácidos Araquidónicos/farmacología , Señalización del Calcio/efectos de los fármacos , Cannabinoides/farmacología , Músculo Liso Vascular/efectos de los fármacos , Neuronas Aferentes/fisiología , Neurotransmisores/farmacología , Receptores de Droga/agonistas , Animales , Endocannabinoides , Técnicas In Vitro , Contracción Isométrica/efectos de los fármacos , Masculino , Arterias Mesentéricas/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Norepinefrina/farmacología , Pinacidilo/farmacología , Piperidinas/farmacología , Alcamidas Poliinsaturadas , Bloqueadores de los Canales de Potasio , Pirazoles/farmacología , Ratas , Ratas Wistar , Receptores de Cannabinoides , Receptores de Droga/antagonistas & inhibidores , Rimonabant , Vasoconstrictores/farmacologíaRESUMEN
We recently described a perivascular sensory nerve-linked dilator system that can be activated by interstitial Ca2+ (Ca2+isf). The present study tested the hypothesis that Ca2+isf in the rat duodenal submucosa varies through a range that is sufficient to activate this pathway. An in situ microdialysis method was used to estimate Ca2+isf. When the duodenal lumen was perfused with Ca2+-free buffer, Ca2+isf was 1.0 +/- 0.13 mmol/l. Ca2+isf increased to 1.52 +/- 0.04, 1.78 +/- 0.10, and 1.89 +/- 0.1 when the lumen was perfused with buffer containing 3, 6, and 10 mmol/l Ca2+, respectively (P < 0.05). Ca2+isf was 1.1 +/- 0.06 mmol/l in fasted animals and increased to 1. 4 +/- 0.06 mmol/l in free-feeding rats (P < 0.05). Wire myography was used to study isometric tension responses of isolated mesenteric resistance arteries. Cumulative addition of extracellular Ca2+-relaxed serotonin- and methoxamine-precontracted arteries with half-maximal effective doses of 1.54 +/- 0.05 and 1.67 +/- 0.08 mmol/l, respectively (n = 5). These data show that duodenal Ca2+isf undergoes dynamic changes over a range that activates the sensory nerve-linked dilator system and indicate that this system can link changes in local Ca2+ transport with alterations in regional resistance and organ blood flow.
Asunto(s)
Calcio/metabolismo , Duodeno/irrigación sanguínea , Duodeno/metabolismo , Espacio Extracelular/metabolismo , Fenómenos Fisiológicos del Sistema Nervioso , Vasodilatación/fisiología , Animales , Duodeno/inervación , Mucosa Intestinal/irrigación sanguínea , Mucosa Intestinal/metabolismo , Masculino , Microdiálisis , Ratas , Ratas Wistar , Sensación/fisiologíaRESUMEN
Traumatic brain injury (TBI) reduces cerebral vascular pressure autoregulation in experimental animals and in patients. In order to understand better the mechanisms of impaired autoregulation, we measured myogenic responses to changes in intraluminal pressure in vitro in pressurized, rodent middle cerebral arteries (MCAs) harvested after TBI. In an approved study, male Sprague-Dawley rats (275-400 g) were anesthetized, intubated, ventilated with 2.0% isoflurane in O2/air, and prepared for fluid percussion TBI. The isoflurane concentration was reduced to 1.5%, and rats (n = 6 per group) were randomly assigned to receive sham TBI followed by decapitation 5 or 30 min later or moderate TBI (2.0 atm) followed by decapitation 5 or 30 min later. After decapitation, MCA segments were removed, mounted on an arteriograph, and pressurized. MCA diameters were measured as transmural pressure was sequentially reduced. MCA diameters remained constant or increased in the sham groups as intraluminal pressure was reduced from 100 to 40 mm Hg. In both TBI groups, diameter decreased with each reduction in pressure. In summary, MCAs removed from uninjured, isoflurane-anesthetized rats had normal vasodilatory responses to decreased intraluminal pressure. In contrast, after TBI, myogenic vasodilatory responses were significantly reduced within 5 min of TBI and the impaired myogenic responses persisted for at least 30 min after TBI.
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Lesiones Encefálicas/fisiopatología , Arterias Cerebrales/fisiopatología , Músculo Liso Vascular/fisiopatología , Animales , Homeostasis , Masculino , Presión , Ratas , Ratas Sprague-Dawley , Vasodilatación , Heridas no Penetrantes/fisiopatologíaRESUMEN
We recently reported that Ca(2+)-induced relaxation could be linked to a Ca2+ receptor (CaR) present in perivascular nerves. The present study assessed the effect of chronic sensory denervation on Ca(2+)-induced relaxation. Mesenteric resistance arteries were isolated from rats treated as neonates with capsaicin (50 mg/kg), vehicle, or saline. The effect of cumulative addition of Ca2+ was assessed in vessels precontracted with 5 microM norepinephrine. Immunocytochemical studies showed that capsaicin treatment significantly reduced the density of nerves staining positively for calcitonin gene-related peptide (CGRP) and for the CaR (CGRP density: control, 51.1 +/- 3.9 microns2/mm2; capsaicin treated, 31.4 +/- 2.8 microns2/mm2, P = 0.01; control CaR density, 46 +/- 4 microns2/mm2, n = 7; capsaicin-treated CaR density, 24 +/- 4 microns2/mm2, n = 8, P = 0.002). Dose-dependent relaxation to Ca2+ (1-5 mM) was significantly depressed in vessels from capsaicin-treated rats (overall P < 0.001, n = 6 or 7), whereas the relaxation response to acetylcholine remained intact. These data support the hypothesis that Ca(2+)-induced relaxation is mediated by activation of the CaR associated with capsaicin-sensitive perivascular neurons.
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Calcio/fisiología , Arterias Mesentéricas/inervación , Arterias Mesentéricas/fisiología , Neuronas Aferentes/fisiología , Resistencia Vascular/fisiología , Animales , Desnervación Autonómica , Calcio/farmacología , Capsaicina , Neuronas Aferentes/patología , Ratas , Ratas Wistar , Resistencia Vascular/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
We have previously demonstrated that arachidonic acid activates extracellular signal-regulated protein kinases (ERKs) group of mitogen-activated protein kinases (MAPKs) in vascular smooth muscle cells (VSMC). To understand the role of arachidonic acid in cellular signaling events, we have now studied its effect on jun N-terminal kinases (JNKs) group of MAPKs in VSMC. Arachidonic acid activated JNK1 in a time- and concentration-dependent manner with maximum effects at 10 min and 50 microM. Induced activation of JNK1 by arachidonic acid is specific as other fatty acids such as linoleic and stearic acids had no such effect. Indomethacin and nordihydroguaiaretic acid (NDGA), potent inhibitors of the cyclooxygenase (COX) and the lipoxygenase (LOX)/monooxygenase (MOX) pathways, respectively, had no effect on arachidonic acid activation of JNK1 suggesting that the observed phenomenon is independent of its metabolism through either pathway. However, 12-hydroperoxyeicosatetraenoic acid (12-HpETE), the LOX metabolite of arachidonic acid significantly induced JNK1 activity. Protein kinase C (PKC) depletion by prolonged treatment of VSMC with phorbol 12-myristate 13-acetate (PMA) resulted in partial decrease in the responsiveness of JNK1 to arachidonic acid suggesting a role for both PKC-dependent and -independent mechanisms in the activation of JNK1 by this important fatty acid. On the other hand, the responsiveness of JNK1 to 12-HpETE was completely abolished in PKC-depleted cells, suggesting a major role for PKC in 12-HpETE-induced JNK1 activation. IL-1beta and TNF-alpha activated JNK1 in a time-dependent manner with maximum effect at 10 min. Desensitization of JNK1 by arachidonic acid significantly reduced its responsiveness to both the cytokines. In addition, 4-bromophenacyl bromide (4-BPB), a potent and selective inhibitor of phospholipase A2 (PLA2), significantly attenuated the cytokine-induced activation of JNK1. Together, these results show that (1) arachidonic acid and its LOX metabolite, 12-HpETE, activate JNK1 in VSMC, (2) PKC-dependent and -independent mechanisms play a role in the activation of JNK1 by arachidonic acid and 12-HpETE, and (3) arachidonic acid mediates, at least partially, the cytokine-induced activation of JNK1.
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Ácido Araquidónico/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Músculo Liso Vascular/enzimología , Animales , Ácido Araquidónico/farmacología , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Indometacina/farmacología , Interleucina-1/metabolismo , Interleucina-1/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Leucotrienos/metabolismo , Lipooxigenasa/metabolismo , Masoprocol/farmacología , Músculo Liso Vascular/citología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We recently showed that perivascular sensory nerves of mesenteric branch arteries express a receptor for extracellular Ca2+ (CaR), and reported data indicating that this CaR mediates relaxation induced by physiologic levels of Ca2+. We have now tested whether the perivascular sensory nerve CaR-linked dilator system is a local phenomenon restricted to the mesentery, or is present in other circulations. Vessels from the mesenteric, renal, coronary, and cerebral circulations were studied. Immunocytochemical analysis was performed using anti-CaR and anti-neural cell adhesion molecule (NCAM) antibodies. Wire myography was used to assess contraction and relaxation. Although perivascular nerves of all arteries stained for CaR protein, there were regional differences. A morphometric method used to estimate CaR positive nerve density revealed the following rank order: mesenteric branch artery > basilar artery = renal interlobar artery > main renal trunk artery > left anterior descending coronary artery. Vessels from the mesentery, renal, coronary, and cerebral circulations showed nerve-dependent relaxation in response to electrical field stimulation (EFS) when precontracted with serotonin in the presence of guanethidine. The degree of Ca2+-induced relaxation of mesenteric, renal, and cerebral arteries positively correlated with the magnitude of EFS-induced relaxation. In contrast, coronary arteries contracted at Ca2+ levels between 1.5 and 3 mmol L(-1), and relaxed to a small degree to 5 mmol L(-1) Ca2+. Thus, a functional perivascular sensory nerve CaR-linked dilator system is present to varying degrees in the mesenteric, renal, and cerebral circulations, but only to a very limited extent in the coronary circulation.
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Arterias/metabolismo , Proteínas de Unión al Calcio/análisis , Neuronas Aferentes/metabolismo , Animales , Arterias/inervación , Arteria Basilar/inervación , Arteria Basilar/metabolismo , Proteínas de Unión al Calcio/metabolismo , Vasos Coronarios/inervación , Vasos Coronarios/metabolismo , Inmunohistoquímica , Masculino , Arterias Mesentéricas/inervación , Arterias Mesentéricas/metabolismo , Ratas , Ratas Wistar , Arteria Renal/inervación , Arteria Renal/metabolismoRESUMEN
The present study tested two hypotheses: (1) that a receptor for extracellular Ca2+ (Ca2+ receptor [CaR]) is located in the perivascular sensory nerve system and (2) that activation of this receptor by physiological concentrations of extracellular Ca2+ results in the release of vasodilator substance that mediates Ca2+-induced relaxation. Reverse transcription-polymerase chain reaction using primers derived from rat kidney CaR cDNA sequence showed that mRNA encoding a CaR is present in dorsal root ganglia but not the mesenteric resistance artery. Western blot analysis using monoclonal anti-CaR showed that a 140-kD protein that comigrates with the parathyroid CaR is present in both the dorsal root ganglia and intact mesenteric resistance artery. Immunocytochemical analysis of whole mount preparations of mesenteric resistance arteries showed that the anti-CaR-stained perivascular nerves restricted to the adventitial layer. Biophysical analysis of mesenteric resistance arteries showed that cumulatively raising Ca2+ from 1 to 1.25 mol/L and above relaxes precontracted arteries with an ED50 value of 2.47+/-0.17 mmol/L (n=12). The relaxation is endothelium independent and is unaffected by blockade of nitric oxide synthase but is completely antagonized by acute and subacute phenolic destruction of perivascular nerves. A bioassay showed further that superfusion of Ca2+ across the adventitial surface of resistance arteries releases a diffusible vasodilator substance. Pharmacological analysis indicates that the relaxing substance is not a common sensory nerve peptide transmitter but is a phospholipase A2/cytochrome P450-derived hyperpolarizing factor that we have classified as nerve-derived hyperpolarizing factor. These data demonstrate that a CaR is expressed in the perivascular nerve network, show that raising Ca2+ from 1 to 1.25 mol/L and above causes nerve-dependent relaxation of resistance arteries, and suggest that activation of the CaR induces the release of a diffusible hyperpolarizing vasodilator. We propose that this system could serve as a molecular link between whole-animal Ca2+ balance and arterial tone.
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Proteínas de Unión al Calcio/fisiología , Calcio/farmacología , Ganglios Espinales/metabolismo , Arterias Mesentéricas/fisiología , Músculo Liso Vascular/fisiología , Neuronas Aferentes/fisiología , Vasodilatación/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Benzamidas/farmacología , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/química , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Riñón/metabolismo , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/inervación , Datos de Secuencia Molecular , Desnervación Muscular , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/inervación , Óxido Nítrico Sintasa/antagonistas & inhibidores , Piperidinas/farmacología , Quinuclidinas/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Sustancia P/análogos & derivados , Sustancia P/farmacología , Resistencia Vascular/efectos de los fármacosRESUMEN
Organ culture of mesenteric resistance arteries results in a loss of force-generating ability, which is prevented by 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We have tested the hypothesis that the culture-induced decrease in active stress is associated with altered myosin isoform expression. Rat mesenteric resistance arteries were studied immediately (fresh) or after incubation at 37 degrees C for 48 h in culture medium (control), with 300 pg/ml 1,25(OH)2D3, or with 5 microg/ml insulin. Isometric force was measured by myography; myosin heavy chain (MHC) and regulatory myosin light chain isoform (MLC) contents were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal active stress to 100 mM K+ (mN/mm2) was greater for fresh (147.8 +/- 4.9) than control (109.2 +/- 4.6, P = 0.001) or insulin (79.6 +/- 8.6, P < 0.001) but not 1,25(OH)2D3 (137.4 +/- 9.5, P = 0.197). Organ culture did not alter MLC or MHC smooth muscle (SM)-1 isoform content. MHC SM-2 content (nmol/mg protein) was greater in fresh (0.038 +/- 0.003) than control (0.026 +/- 0.003, P = 0.012) and insulin (0.027 +/- 0.002, P = 0.018) but not 1,25(OH)2D3 (0.036 +/- 0.003, P = 0.693); nonmuscle MHC (NMM) was observed in insulin. The maximal active stress response to K+ significantly correlated with SM-2 MHC isoform content (r2 = 0.483, P < 0.001). We conclude that 1) arterial organ culture alters MHC isoform content, 2) SM-2 MHC isoform content positively correlates with active stress generation, 3) 1,25(OH)2D3 maintains force-generating capacity by preventing the shift of MHC isoform expression, and 4) insulin impairs force-generating ability by lowering MHC SM-2 content and stimulating NMM expression.
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Arterias Mesentéricas/enzimología , Arterias Mesentéricas/fisiología , Miosinas/metabolismo , Resistencia Vascular , Vasoconstricción , Animales , Calcitriol/farmacología , Masculino , Arterias Mesentéricas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ratas , Ratas WistarRESUMEN
Previous studies have shown that traumatic brain injury (TBI) significantly reduces cerebral blood flow determined in vivo and reduces vascular reactivity in the pial circulation measured with cranial window preparations. We have now tested the hypothesis that TBI induces these changes by impairing intrinsic contractile activity of cerebral arteries. Anesthetized rats underwent moderate (2.2 atm) and severe (3.0 atm) midline fluid percussion TBI or sham injury following which posterior cerebral or middle cerebral arteries were isolated and isometric force generation was measured. Moderate (n = 5) and severe (n = 3) trauma had no effect on the magnitude of serotonin- or K+-induced force generation or sensitivity to serotonin in arteries isolated within 10 min of TBI. Functional disruption of the endothelium of posterior cerebral arteries isolated 10 min after moderate trauma or sham injury caused a reduction in the active tension response to serotonin that was similar in both groups. Blockade of cyclooxygenase with 5 microM indomethacin had no effect on serotonin-induced force generated by vessels with moderate trauma or in sham-treated rats. Acetylcholine induced an endothelium-dependent relaxation of posterior and middle cerebral arteries; the magnitude of the response was unaffected by moderate TBI. To determine whether prolonged in situ exposure of vessels to the traumatized cerebral milieu could reveal an alteration in intrinsic contractility, posterior cerebral arteries were isolated 30 min after TBI; again, no differences in the tension or relaxation responses were observed. It is concluded that midline fluid percussion TBI did not affect contraction or relaxation of proximal middle or posterior cerebral arteries in rats.
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Lesiones Encefálicas/fisiopatología , Arterias Cerebrales/fisiopatología , Endotelio Vascular/fisiopatología , Contracción Isométrica , Acetilcolina/farmacología , Animales , Arterias Cerebrales/efectos de los fármacos , Arterias Cerebrales/fisiología , Circulación Cerebrovascular , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Técnicas In Vitro , Indometacina/farmacología , Contracción Isométrica/efectos de los fármacos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/fisiopatología , Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Serotonina/farmacologíaRESUMEN
OBJECTIVE: The effects of agonists and guanosine 5'-triphosphate binding proteins (G proteins) on contractile properties were investigated in rat longitudinal myometrial tissues in late gestation and during delivery. STUDY DESIGN: The effect of carbachol was examined on the intracellular Ca++ concentration in intact thin muscle strips from pregnant rat myometrium. In addition, the action of carbachol with guanosine 5'-triphosphate was examined on the Ca(++)-induced contractions in beta-escin-treated skinned strips (membrane-permeable conditions and chemical clamping of intracellular Ca++ concentrations). The effects of guanosine 5'-0-(gamma-thiotriphosphate) (a nonhydrolyzable analog of guanosine 5'-triphosphate), prostaglandin F2 alpha with guanosine 5'-triphosphate, prostaglandin E2 with guanosine 5'-triphosphate, and okadaic acid (a phosphatase inhibitor) were also examined in skinned strips. RESULTS: In intact longitudinal rat myometrium at late gestation the maximum contractions induced by carbachol were larger than the maximum contractions induced by high K+ (118 mmol/L), whereas increases in intracellular Ca++ concentration produced by both agents were similar. In beta-escin-treated skinned myometrial strips from late gestation, 0.3 mumol/L Ca++ evoked contractions. Carbachol (10 mumol/L) plus guanosine 5'-triphosphate (10 mumol/L) enhanced the 0.3 mumol/L Ca(++)-induced contractions of skinned strips; the increase was antagonized by 1 mmol/L guanosine 5'-0-(beta-thiodiphosphate). Guanosine 5'-0-(gamma-thiotriphosphate) (0.1 to 100 mumol/L), prostaglandin F2 alpha (10 mumol/L) plus guanosine 5'-triphosphate (10 mumol/L), prostaglandin E2 (10 mumol/L) plus guanosine 5'-triphosphate (10 mumol/L), and okadaic acid (1 nmol/L) also augmented 0.3 mumol/L Ca++ contractions in skinned strips. The increases of 0.3 mumol/L Ca(++)-induced contractility by the agonists with guanosine 5'-triphosphate or guanosine 5'-0-(gamma-thiotriphosphate) were similar between late gestation and delivery. CONCLUSION: These results suggest that agonists such as carbachol, prostaglandin F2 alpha, and prostaglandin E2 enhance the Ca(++)-induced contraction of myometrium at late gestation through G protein-mediated mechanisms. The agonist/G protein-mediated Ca(++)-sensitizing effects on contractile elements produce additional contractile force with the same amount of intracellular calcium, thus providing expelling forces for delivery of the fetuses.
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Calcio/metabolismo , Carbacol/farmacología , Guanosina Trifosfato/farmacología , Agonistas Muscarínicos/farmacología , Miometrio/efectos de los fármacos , Potasio/farmacología , Contracción Uterina/efectos de los fármacos , Animales , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/agonistas , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Miometrio/metabolismo , Miometrio/fisiología , Ácido Ocadaico , Embarazo , Prostaglandinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The mechanism by which 1 alpha,25-dihydroxycholecalciferol [1,25(OH)2D3] enhances smooth muscle force generation was examined. Rats were injected on three mornings with 1,25(OH)2D3 (35 ng/100 g) or vehicle, and on the fourth morning mesenteric resistance arteries were isolated and used for simultaneous measurement of intracellular Ca2+ and force or myosin light chain phosphorylation. 1,25(OH)2D3 did not affect media thickness or wall-to-lumen ratio, but it increased basal intracellular Ca2+ (vehicle = 49.2 +/- 2.2 nM vs. 1,25(OH)2D3 = 65.9 +/- 4.0 nM, P < 0.05, n = 24-26 rats). 1,25(OH)2D3 enhanced the active stress and intracellular Ca2+ responses to increasing doses of norepinephrine, and the increases were normalized by verapamil (10 microM). In a second group of animals, 1,25(OH)2D3 significantly increased both basal intracellular Ca2+ and light chain phosphorylation and the active stress and Ca2+ mobilization responses to norepinephrine (10 microM). The hormone did not affect peak or steady-state light chain phosphorylation. Myofilament Ca2+ sensitivity, determined during stimulation with 2 microM norepinephrine, was depressed in vessels isolated from rats treated with 1,25(OH)2D3 [vehicle Ca2+ 50% effective dosé (ED50) = 82.7 +/- 3.8 nM vs. 1,25(OH)2D3 = 104.8 +/- 4.9 nM, P = 0.002]. We conclude that 1,25(OH)2D3 enhances resistance artery force generation by altering smooth muscle Ca2+ homeostasis, with effects on basal and verapamil-sensitive, agonist-induced Ca2+ mobilization.