RESUMO
BACKGROUND: Some cannabinoids, a family of compounds derived from Cannabis sativa (marijuana), have previously shown vasodilator effects in several studies, a feature that makes them suitable for the generation of a potential treatment for hypertension. The mechanism underlying this vasodilator effect in arteries is still controversial. In this report, we explored how the synthetic cannabinoids ACPA (CB1-selective agonist) and JWH-133 (CB2-selective agonist) regulate the vascular tone of rat superior mesenteric arteries. METHODS: To screen the expression of CB1 (Cannabinoid receptor 1) and CB2 (Cannabinoid receptor 2) receptors in arterial rings or isolated smooth muscle cells obtained from the artery, immunocytochemistry, immunohistochemistry, and confocal microscopy were performed. In addition, the effects on vascular tone induced by the two cannabinoids were tested in isometric tension experiments in rings obtained from superior mesenteric arteries. The participation of voltage and calcium-activated potassium channel of big conductance (BKCa) and the role of nitric oxide (NO) release on the vascular effects induced by ACPA and JWH-133 were tested. RESULTS: CB1 and CB2 receptors were highly expressed in the rat superior mesenteric artery, in both smooth muscle and endothelium. The vasodilation effect shown by ACPA was endothelium-dependent through a mechanism involving CB1 receptors, BKCa channel activation, and NO release; meanwhile, the vasodilator effect of JWH-133 was induced by the activation of CB2 receptors located in smooth muscle and by a CB2 receptor-independent mechanism inducing NO release. CONCLUSIONS: CB1 and CB2 receptor activation in superior mesenteric artery causes vasorelaxation by mechanisms involving BKCa channels and NO release.
Assuntos
Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/agonistas , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/metabolismo , Microscopia Confocal , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40 ± 2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca²âº were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca²âº at various concentrations. Maximum contractility (Emax) in the shock group increased with NE (from 0.179 ± 0.038 to 0.440 ± 0.177 g/mg, P<0.05) and Ca²âº (from 0.515 ± 0.043 to 0.646 ± 0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca²âº at various concentrations in the shock+drainage group (from 0.744 ± 0.187 to 0.570 ± 0.143 g/mg in Emax for NE and from 0.729 ± 0.037 to 0.645 ± 0.056 g/mg in Emax for Ca²âº, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.
Assuntos
Cálcio/metabolismo , Linfa/fisiologia , Artéria Mesentérica Superior/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Quinase de Cadeia Leve de Miosina/fisiologia , Choque Hemorrágico/fisiopatologia , Animais , Masculino , Artéria Mesentérica Superior/metabolismo , Contração Muscular , Músculo Liso Vascular/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/análise , Distribuição Aleatória , Ratos Wistar , Choque Hemorrágico/enzimologiaRESUMO
Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40±2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca2+ were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca2+ at various concentrations. Maximum contractility (Emax) in the shock group increased with NE (from 0.179±0.038 to 0.440±0.177 g/mg, P<0.05) and Ca2+ (from 0.515±0.043 to 0.646±0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca2+ at various concentrations in the shock+drainage group (from 0.744±0.187 to 0.570±0.143 g/mg in Emax for NE and from 0.729±0.037 to 0.645±0.056 g/mg in Emax for Ca2+, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.
Assuntos
Animais , Masculino , Cálcio/metabolismo , Linfa/fisiologia , Artéria Mesentérica Superior/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Quinase de Cadeia Leve de Miosina/fisiologia , Choque Hemorrágico/fisiopatologia , Contração Muscular , Artéria Mesentérica Superior/metabolismo , Músculo Liso Vascular/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/análise , Distribuição Aleatória , Ratos Wistar , Choque Hemorrágico/enzimologiaRESUMO
PURPOSE: To investigate the role of protein kinase G (PKG) in blocking post-shock mesenteric lymph (PSML) return ameliorating the calcium sensitivity in hemorrhagic shock rats. METHODS: Male Wistar rats were randomly divided into sham, shock, shock+ligation (shock plus mesenteric lymph duct ligation (MLDL)), shock+drainage (shock plus PSML drainage) groups. After shock (hypotension 40 mm Hg) for three hours or corresponding times, the superior mesenteric artery (SMA) was taken out for detecting the PKG and phospho PKG (p-PKG) contents, and the vascular rings of SMA were prepared for assaying the calcium sensitivity using an isolated organ perfusion system. RESULTS: The PKG and p-PKG contents of SMA in shock group were significantly increased than that of sham group, and MLDL or PSML drainage reducing the levels of PKG and p-PKG. Meanwhile, the vascular calcium sensitivity in shock group was significantly lower than that of sham group, MLDL or PSML drainage enhanced the calcium sensitivity. After incubating with PKG regulators in shock+ligation and shock+drainage groups, the PKG agonist 8Br-cGMP reduced the contractility of vascular rings to gradient calcium ions and Emax and the PKG inhibitor agonist KT5823 elevated the calcium sensitivity significantly. CONCLUSION: Protein kinase G plays an important role in post-shock mesenteric lymph blockage improving vascular calcium sensitivity.
Assuntos
Cálcio/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Artéria Mesentérica Superior/metabolismo , Choque Hemorrágico/metabolismo , Animais , Western Blotting , Cálcio/análise , Proteínas Quinases Dependentes de GMP Cíclico/análise , Ensaio de Imunoadsorção Enzimática , Masculino , Artéria Mesentérica Superior/fisiopatologia , Contração Muscular , Distribuição Aleatória , Ratos , Ratos Wistar , Choque Hemorrágico/fisiopatologiaRESUMO
PURPOSE: To investigate the role of protein kinase G (PKG) in blocking post-shock mesenteric lymph (PSML) return ameliorating the calcium sensitivity in hemorrhagic shock rats. METHODS: Male Wistar rats were randomly divided into sham, shock, shock+ligation (shock plus mesenteric lymph duct ligation (MLDL)), shock+drainage (shock plus PSML drainage) groups. After shock (hypotension 40mmHg) for three hours or corresponding times, the superior mesenteric artery (SMA) was taken out for detecting the PKG and phospho PKG (p-PKG) contents, and the vascular rings of SMA were prepared for assaying the calcium sensitivity using an isolated organ perfusion system. RESULTS: The PKG and p-PKG contents of SMA in shock group were significantly increased than that of sham group, and MLDL or PSML drainage reducing the levels of PKG and p-PKG. Meanwhile, the vascular calcium sensitivity in shock group was significantly lower than that of sham group, MLDL or PSML drainage enhanced the calcium sensitivity. After incubating with PKG regulators in shock+ligation and shock+drainage groups, the PKG agonist 8Br-cGMP reduced the contractility of vascular rings to gradient calcium ions and Emax and the PKG inhibitor agonist KT5823 elevated the calcium sensitivity significantly. CONCLUSION: Protein kinase G plays an important role in post-shock mesenteric lymph blockage improving vascular calcium sensitivity.
Assuntos
Animais , Masculino , Ratos , Cálcio/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Artéria Mesentérica Superior/metabolismo , Choque Hemorrágico/metabolismo , Western Blotting , Cálcio/análise , Proteínas Quinases Dependentes de GMP Cíclico/análise , Ensaio de Imunoadsorção Enzimática , Contração Muscular , Artéria Mesentérica Superior/fisiopatologia , Distribuição Aleatória , Ratos Wistar , Choque Hemorrágico/fisiopatologiaRESUMO
PURPOSE: We investigated the effects of ischemia/reperfusion in the intestine (I/R-i) on purine receptor P2X2-immunoreactive (IR) neurons of the rat ileum. METHODS: The superior mesenteric artery was occluded for 45 min with an atraumatic vascular clamp and animals were sacrificed 4 h later. Neurons of the myenteric and submucosal plexuses were evaluated for immunoreactivity against the P2X2 receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT), calbindin, and calretinin. RESULTS: Following I/R-i, we observed a decrease in P2X2 receptor immunoreactivity in the cytoplasm and surface membranes of neurons of the myenteric and submucosal plexuses. These studies also revealed an absence of calbindin-positive neurons in the I/R-i group. In addition, the colocalization of the P2X2 receptor with NOS, ChAT, and calretinin immunoreactivity in the myenteric plexus was decreased following I/R-i. Likewise, the colocalization between P2X2 and calretinin in neurons of the submucosal plexus was also reduced. In the I/R-i group, there was a 55.8% decrease in the density of neurons immunoreactive (IR) for the P2X2 receptor, a 26.4% reduction in NOS-IR neuron, a 25% reduction in ChAT-IR neuron, and a 47% reduction in calretinin-IR neuron. The density of P2X2 receptor and calretinin-IR neurons also decreased in the submucosal plexus of the I/R-i group. In the myenteric plexus, P2X2-IR, NOS-IR, ChAT-IR and calretinin-IR neurons were reduced in size by 50%, 49.7%, 42%, and 33%, respectively, in the I/R-i group; in the submucosal plexus, P2X2-IR and calretinin-IR neurons were reduced in size by 56% and 72.6%, respectively. CONCLUSIONS: These data demonstrate that ischemia/reperfusion of the intestine affects the expression of the P2X2 receptor in neurons of the myenteric and submucosal plexus, as well as density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X2-IR enteric neurons that could result in alterations in intestinal motility.
Assuntos
Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal , Íleo/inervação , Íleo/fisiopatologia , Receptores Purinérgicos P2X2/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Calbindina 2 , Calbindinas , Colina O-Acetiltransferase/metabolismo , Íleo/metabolismo , Masculino , Artéria Mesentérica Superior/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
This study investigated the cardiovascular responses to the essential oil of Aniba canelilla (EOAC) and its main constituent 1-nitro-2-phenylethane (NP) in spontaneously hypertensive rats (SHRs). In anesthetized SHRs, intravenous (i.v.) bolus injections of EOAC (1-20 mg/kg) or NP (1-10 mg/kg) elicited dose-dependent hypotensive and bradycardiac effects, which were characterized in two periods (phases 1 and 2). The first rapid component (phase 1) evoked by EOAC and NP both at 10 mg/kg was absent after left ventricle injection, fully abolished by bilateral vagotomy and perineural treatment of both cervical vagus nerves with capsaicin (250 µg/mL) while remained unaltered by i.v. pretreatment with capsazepine (1 mg/kg) or ondansetron (30 µg/kg). In conscious SHRs, NP (5 and 10 mg/kg, i.v.) evoked rapid hypotensive and bradycardiac effects (phase 1) that were fully abolished by methylatropine (1 mg/kg, i.v.) pretreatment. In rat endothelium-containing mesenteric preparations, increasing concentrations (0.1-1000 µg/mL) of EOAC and NP relaxed the phenylephrine-induced contraction in a concentration-dependent manner. It is concluded that NP induces a vago-vagal bradycardiac and depressor reflex (phase 1) that apparently results from the stimulation of vagal pulmonary rather than cardiac C-fiber afferents. This effect does not appear to involve activation of either vanilloid TPRV(1) or 5-HT(3) receptors located on vagal sensory nerves. The phase 2 hypotensive response to i.v. NP seems to result, at least in part, from its direct vasodilatory effect on the peripheral smooth muscle. All in vivo and in vitro effects of EOAC are mostly attributed to the actions of its main constituent NP.
Assuntos
Derivados de Benzeno/farmacologia , Hipertensão/tratamento farmacológico , Lauraceae/química , Óleos Voláteis/farmacologia , Animais , Derivados de Benzeno/administração & dosagem , Derivados de Benzeno/isolamento & purificação , Bradicardia/induzido quimicamente , Relação Dose-Resposta a Droga , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/metabolismo , Óleos Voláteis/administração & dosagem , Óleos Voláteis/isolamento & purificação , Ratos , Ratos Endogâmicos SHR , Reflexo/efeitos dos fármacos , Nervo Vago/efeitos dos fármacos , Nervo Vago/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
This study was performed to investigate the mechanisms involved in the vasorelaxation induced by mesoionic 2-(4-chlorophenyl)-3-methyl-4-(4-methoxyphenyl)-1;3-thiazolium-5-thyolate (CMMTT), a newly synthesized mesoionic compound, in rat superior mesenteric arteries. In phenylephrine (10 microM)-pre-contracted mesenteric rings, CMMTT (10(-14) - 10(-6) M) induced a concentration-dependent relaxation [pD(2) = 10.26 +/- 0.05, E(max) = 80.8 +/- 5.8%], and this effect was almost abolished after either removal of the vascular endothelium [E(max) = 17.7 +/- 4.2%, P<0.001], removal of the vascular endothelium plus100 microM N(omega)-nitro-L-arginine methyl esther (L-NAME) [E(max) = 21.0 +/- 2.0 %, P<0.001], or after pre-treatment of the rings with 100 microM L-NAME [E(max) = 13.3 +/- 2.4%, P<0.001] or 10 microM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) [E(max) = 13.6 +/- 4.8%, P<0.001]. However, endothelium-dependent relaxation induced by CMMTT was not significantly modified after 1 microM indomethacin plus 1 nM atropine [pD(2) = 11.12 +/- 0.08, E(max) = 73.8 +/- 5.15%] or 100 nM charybdotoxin (ChTX) plus 100 nM apamin [pD(2) = 10.89 +/- 0.08, E(max) = 58.91 +/- 9.8%]. In mesenteric rings, CMMTT (10(-6) M) was able to increase nitric oxide (NO)(x) levels, and this effect was abolished after removal of the vascular endothelium. In conclusion, the present study, using combined functional and biochemical approaches, demonstrated that CMMTT induced a significant vasorelaxant effect, almost completely mediated by the endothelium, likely via NO release and activation of the NO-cGMP pathway.
Assuntos
Endotélio Vascular/fisiologia , Óxido Nítrico/fisiologia , Tiazóis/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia , Animais , Fatores Biológicos/fisiologia , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Artéria Mesentérica Superior/efeitos dos fármacos , Artéria Mesentérica Superior/metabolismo , Artéria Mesentérica Superior/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Oxidiazóis/farmacologia , Fenilefrina/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Vasoconstritores/farmacologiaRESUMO
Previous studies have established the hepatoprotective, gastroprotective, hypolipidemic and hypoglycemic effects of kolaviron (KV), a biflavonoid complex from Garcinia kola seeds. In this study, we investigated the mechanisms involved in the vasorelaxant effects of KV in isolated superior mesenteric arteries from normotensive rats. KV (1, 10, 30, 100, 300, 500 and 1,000 microg/ml) concentration-dependently inhibited the contractions induced by phenylephrine (PHE) (10 microM) and KCl (80 mM) in both endothelium-intact (E(max) = 58.3 +/- 1.7% and 51.4 +/- 1.3%, respectively) and -denuded rings (E(max) = 59.3 +/- 5.5% and 64.3 +/- 2.4%, respectively). Furthermore, KV reduced CaCl(2)-induced contraction in Ca(2+)-free medium containing KCl 60 mM, thus acting as a Ca(2+)-antagonist. In addition, KV inhibited the transient contraction by PHE in Ca(2+)-free medium containing EGTA, suggesting a possible action on the release of intracellular Ca(2+) via the inositol-1,4,5-triphosphate (IP(3)) pathway. KV is not a specific alpha-adrenoceptor blocker, since it also caused a concentration-dependent inhibition of contractile responses to KCl, suggesting that KV also blocks the L-type Ca(2+)-channel. As a Ca(2+) antagonist, KV (100 microg/ml) potentiates the relaxant effects of nifedipine in denuded rings (E(max) = 97.6 +/- 1.2%; control = 75.1 +/- 3.0%, P<0.05). Also, the vasorelaxation induced by KV was significantly inhibited after pre-treatment of the denuded rings with 4-aminopyridine (4-AP) 1 mM, a selective blocker of voltage-dependent K(+) (K(v)) channels and, tetraethylammonium (TEA) 1 mM or charybdotoxin (ChTX) 0.1 microM, non-selective blockers of large and intermediate conductance Ca(2+)-activated K(+) (BK(Ca)) channels. In contrast, neither glibenclamide (10 microM), BaCl2 (1 mM) nor apamin (0.1 microM), blockers of K(ATP), K(IR) and SK(Ca) channels, respectively affected the KV-induced vasorelaxation. In conclusion, our results provide functional evidence that the vasorelaxant effects by KV involve extracellular Ca(2+) influx blockade, inhibition of intracellular Ca(2+) release and the opening of K(+) channels sensitive to 4-AP and ChTX with a resultant membrane hyperpolarization/ repolarization.
Assuntos
Flavonoides/farmacologia , Garcinia kola , Artéria Mesentérica Superior/metabolismo , Sementes , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , 4-Aminopiridina/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Charibdotoxina/farmacologia , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Endotélio Vascular/metabolismo , Flavonoides/química , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Neurotoxinas/farmacologia , Técnicas de Cultura de Órgãos , Fenilefrina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Cloreto de Potássio/metabolismo , Ratos , Ratos Wistar , Vasoconstritores/farmacologiaRESUMO
Reperfusion of ischemic vascular beds may lead to recruitment and activation of leukocytes, release of mediators of the inflammatory process and further injury to the affected vascular bed and to remote sites. Neutrophils appear to play a major role in the pathophysiology of reperfusion injury. Amongst inflammatory mediators shown to activate neutrophils and induce their recruitment in vivo, much interest has been placed on the role of leukotriene (LT)B(4). Here, we have assessed the effects of the BLT receptor antagonist (+)-1-(3S, 4R)-[3-(4-phenyl-benzyl)-4-hydroxy-chroman-7-yl]-cyclopentane carboxylic acid (CP 105,696) in a model of neutrophil-dependent ischemia and reperfusion injury in the rat. The superior mesenteric artery was isolated and ischemia was induced by its total occlusion for 30 min. After 30 min of reperfusion, injury was assessed by evaluating the extravasation of Evans blue, an index of vascular permeability, and the levels of myeloperoxidase, an index of neutrophil accumulation, in the intestine, mesentery and lung. The neutrophil-dependence of the local (intestine and mesentery) and remote (lung) injury was confirmed by using fucoidin, a selectin blocker, and WT-3, an anti-CD18 monoclonal antibody. Post-ischemic treatment with CP 105,696 dose-dependently inhibited vascular permeability and neutrophil accumulation in the intestine and mesentery. CP 105,696 also blocked the vascular permeability changes, but not neutrophil accumulation, in the lungs after reperfusion injury. Virtually identical results were obtained with another BLT receptor antagonist, 1-(5-ethyl-2-hydroxy-4-(6-methyl-6-(1H-tetrazol-5-yl)-heptoxy++ +)-phenyl )ethanone (LY255283). Our results suggest that post-ischemic treatment with BLT receptor antagonists may inhibit local and remote ischemia and reperfusion injury by blocking both the accumulation and/or activation of neutrophils.