RESUMO
Endogenous carbon monoxide (CO) contributes to vasodilator responses of cerebral microvessels in newborn pigs. We investigated the expression, intracellular localization, and activity of heme oxygenase (HO), the key enzyme in CO production, in quiescent cerebral microvascular endothelial cells (CMVEC) from newborn pigs. HO-1 and HO-2 isoforms were detected by RT-PCR, immunoblotting, and immunofluorescence. HO-1 and HO-2 are membrane-bound proteins that have a strong preference for the nuclear envelope and perinuclear area of the cytoplasm. Betamethasone (10(-6) to 10(-4) M for 48 h) was associated with upregulation of HO-2 protein by approximately 50% and inhibition of Cox-2 but did not alter HO-1 or endothelial nitric oxide synthase expression in CMVEC. In vivo betamethasone treatment of newborn pigs (0.2 and 5.0 mg/kg im for 48 h) upregulated HO-2 in cerebral microvessels by 30-60%. HO activity as (14)CO production from [(14)C]glycine-labeled endogenous heme was inhibited by chromium mesoporphyrin (10(-6) to 10(-4) M). L-Glutamate (0.3-1.0 mM) stimulated HO activity 1.5-fold. High-affinity specific binding sites for L-[(3)H]glutamate suggestive of the glutamate receptors were detected in CMVEC. Altogether, these data suggest that, in cerebral circulation of newborn pigs, endothelium-derived CO may contribute to basal vascular tone and to responses that involve glutamate receptor activation.
Assuntos
Córtex Cerebral/irrigação sanguínea , Endotélio Vascular/enzimologia , Ácido Glutâmico/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Animais , Animais Recém-Nascidos , Betametasona/farmacologia , Monóxido de Carbono/metabolismo , Fracionamento Celular , Córtex Cerebral/fisiologia , Ciclo-Oxigenase 2 , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática , Glucocorticoides/farmacologia , Ácido Glutâmico/química , Heme Oxigenase (Desciclizante)/genética , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Ensaio Radioligante , SuínosRESUMO
We investigated the relationships among expression, activity, and spatial organization of cyclooxygenase (COX-1 and COX-2) in endothelial cells from porcine and human cerebral microvessels and from human umbilical vein. In quiescent cells, COX-1 was detected in the perinuclear zone and the cytoplasm, while COX-2 was mainly a nuclear resident possibly connected with the nuclear matrix. COX-2 immunogold labeling was situated in the nuclear envelope, at the nuclear pores, and in connection with the perichromatin regions of the nucleus, considered to be the sites of active transcription. In human endothelial cells transcriptionally activated by interleukin (IL)-1beta, the nucleus remained a major COX-2 localization site during the first 12 h of stimulation, when COX-2 expression was maximally induced. The continuous rise in prostanoid synthesis at 17-23 h of stimulation was associated with COX-2 relocation from the nucleus to the nuclear envelope and the cytoplasm. IL-1beta did not affect COX-1 expression, activity, and localization. COX-2 nuclear localization sites and trafficking between the nucleus and the cytoplasm in endothelial cells may indicate a novel function of COX-2 in regulating gene expression.
Assuntos
Núcleo Celular/enzimologia , Endotélio Vascular/enzimologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Capilares/enzimologia , Núcleo Celular/química , Células Cultivadas , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/ultraestrutura , Humanos , Immunoblotting , Interleucina-1/farmacologia , Isoenzimas/genética , Proteínas de Membrana , Microscopia de Fluorescência , Fosfolipases A/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandinas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Telencéfalo/irrigação sanguínea , Veias Umbilicais/citologia , Fator de von Willebrand/imunologiaRESUMO
Sodium channels using cAMP as a second messenger play a role in the regulation of cerebral circulation and metabolism. Cerebrospinal fluid (CSF) cAMP levels have been shown to correlate with the degree and duration of hypoxic injury and outcome and to be an indicator of cerebral vascular reactivity. We hypothesize that sodium channel inhibition either before or at termination of experimental asphyxia will attenuate cerebrovascular alterations and maintain CSF cAMP levels. Three groups of piglets with closed cranial windows were studied: asphyxia or group 1 (n = 5) and two treatment groups. Pigs were treated with 50 mg/kg of sodium channel blocker before asphyxia (group 2, n = 6) and after the termination of asphyxia and start of reventilation (group 3, n = 6). Asphyxia was sustained over 60 min by ventilating piglets with 10% O2 gas mixture and decreasing minute ventilation followed by 60 min of reventilation with room air. Every 10 min, pial arterial diameters were measured, and CSF samples were collected for cAMP determination. Vascular reactivity to topically applied isoproterenol (10(-4) M) was evaluated 60 min after recovery. During asphyxia, cAMP levels in group 2 peaked and declined at a later time with mean values remaining significantly higher than those of groups 1 and 3. During reventilation, CSF cAMP concentrations were highest in group 3 and lowest in group 1. Pial arteriolar dilation occurred during asphyxia in all three groups but to a lesser degree in the pretreated group compared with groups 1 and 3. Pial arteriolar reactivity to isoproterenol postasphyxia was preserved in both groups 2 and 3. In summary, in newborn pigs, pretreatment with sodium channel blocker resulted in higher CSF cAMP levels and a lesser degree of pial arteriolar dilation during prolonged asphyxia. Pretreatment or treatment at reventilation restored vascular tone and reactivity.
Assuntos
Asfixia/fisiopatologia , Vasos Sanguíneos/fisiopatologia , Encéfalo/irrigação sanguínea , Tono Muscular , Músculo Liso Vascular/fisiopatologia , Bloqueadores dos Canais de Sódio , Animais , AMP Cíclico/líquido cefalorraquidiano , Feminino , Masculino , SuínosRESUMO
Endothelium-derived prostanoids are predominant vasorelaxant factors in the cerebral circulation of newborn pigs in vivo, whereas in older pigs nitric oxide (NO)-mediated responses also contribute to the regulation of cerebral vascular tone. We compared the expression and activities of NO synthase and cyclooxygenase in the cerebral microcirculation of newborn and adult pigs. In adult animals, expression and activity of endothelial NO synthase in cerebral microvessels and in cultured cerebral endothelial cells is two- to threefold higher than in newborn pigs; acetylcholine and bradykinin cause a greater increase in NO production in adult pigs. Expression and activity of cyclooxygenase in cerebral microvascular endothelial cells is similar in newborn and adult pigs; acetylcholine and bradykinin stimulated dilator prostanoid production to the same degree in both age groups. Endothelial prostanoid synthesis in cerebral microvessels and cultured endothelial cells was inhibited 30-70% by NS-398, reflecting a large contribution of COX-2 in both newborn and adult animals. These data indicate that in the cerebral circulation of pigs, NO synthase is age-dependently upregulated, whereas endothelial cyclooxygenase is not altered during postnatal development.
Assuntos
Envelhecimento , Encéfalo/irrigação sanguínea , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Vasodilatadores/sangue , Acetilcolina/farmacologia , Animais , Animais Recém-Nascidos , Bradicinina/farmacologia , Células Cultivadas , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Dinoprostona/biossíntese , Isoenzimas/metabolismo , Microcirculação/enzimologia , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/biossíntese , SuínosRESUMO
The mechanism through which iloprost permits cerebral vasodilation induced by specific stimuli is incompletely understood. Previous study suggests there might be interplay between the adenylyl cyclase and phospholipase C (PLC) systems. Coupling of the prostacyclin receptor with the PLC pathway system was investigated. Iloprost, a stable prostacyclin analog, was used as a prostacyclin receptor agonist. We investigated the effects of iloprost (10-12-10-6 M) on inositol 1,4,5-trisphosphate (IP3) production by piglet cerebrovascular smooth muscle cells in primary culture. Iloprost caused concentration- and time-dependent increases in IP3 production in control cells and in cells pretreated with LiCl (to prevent further IP3 metabolism). Iloprost treatment (10-12 M) of cerebrovascular smooth muscle cells, in the absence and presence of 20 mM LiCl, resulted in 2-fold and 4-fold increases in the formation of IP3, respectively. In contrast, 10-10 M to 10-6 M iloprost, either in the presence or absence of LiCl, induced moderate or no increase in IP3 formation. Iloprost (10-10-10-12 M) strongly stimulated diacylglycerol (DAG) generation, whereas higher concentrations (10-8 M) did not induce an increase. In conclusion, the results suggest that prostacyclin receptors on cerebromicrovascular smooth muscle can couple to PLC, generating the second messengers, IP3 and DAG.
Assuntos
Circulação Cerebrovascular , Endotelina-1/farmacologia , Iloprosta/farmacologia , Microcirculação , Músculo Liso Vascular/metabolismo , Receptores de Prostaglandina/agonistas , Fosfolipases Tipo C/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Diglicerídeos/metabolismo , Ativação Enzimática , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Cloreto de Lítio/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Receptores de Epoprostenol , SuínosRESUMO
Nigericin decreases intracellular pH (pH(i)) and stimulates prostanoid (PG) synthesis in endothelial cells from cerebral microvessels of newborn pigs. Nigericin-induced PG production was abolished by protein tyrosine kinase (PTK) inhibitors and amplified by phorbol 12-myristate 13-acetate (PMA) or protein tyrosine phosphatase (PTP) inhibitors. Nigericin-induced PG production in PMA-primed cells was potentiated by PTP inhibitors and abrogated by PTK inhibitors. Phospholipase A(2) (PLA(2)) activity was stimulated by nigericin in a phosphorylation-dependent manner. Nigericin's effects on PG production and PLA(2) activity were reproduced by ionomycin, which activates cytosolic PLA(2) (cPLA(2)). cPLA(2) was immunodetected in endothelial cell lysates. We found no evidence that nigericin's effects are mediated via mitogen-activated protein (MAP) kinase [extracellularly regulated kinase 1 (ERK1) and ERK2] activation: although nigericin stimulated detergent-soluble MAP kinase, its effects were not amplified by PMA or PTP inhibitors. Phosphorylation-dependent stimulation of PG synthesis was also observed when pH(i) was decreased by sodium propionate or a high level of CO(2). Altogether, our data indicate that nigericin and decreased pH(i) stimulate PG synthesis by a protein phosphorylation-dependent mechanism involving cross talk between pathways mediated by PTK and PTP and by protein kinase C; cPLA(2) appears to be a key enzyme affected by nigericin and decreased pH(i).
Assuntos
Circulação Cerebrovascular , Endotélio Vascular/metabolismo , Nigericina/farmacologia , Prostaglandinas/biossíntese , Animais , Cálcio/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Ionomicina/farmacologia , Microcirculação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Concentração Osmolar , Fosfolipases A/fisiologia , Fosforilação , Prostaglandina-Endoperóxido Sintases/metabolismo , SuínosRESUMO
OBJECTIVE: In piglets prolonged asphyxia resulted in decreased cerebrospinal fluid (CSF) 3;,5;-cyclic adenosine monophosphate (cAMP) during recovery; this was associated with reduced pial arteriolar responses to stimuli that use cAMP as a second messenger. We hypothesized that asphyxia in human neonates results in decreased CSF cAMP and that low CSF cAMP is associated with abnormal outcome. DESIGN: We studied 27 infants with evidence of hypoxic-ischemic insult; 19 were term (group 1) and 8 were preterm (group 2). The normal values of CSF cAMP were determined from 75 infants with no asphyxia; 44 were term (group 3) and 31 were preterm (group 4). CSF cAMP was measured by using radioimmunoassay procedures. RESULTS: CSF cAMP levels in infants with asphyxia (groups 1 and 2) were 12 +/- 9. 5 and 7.9 +/- 7.1 pmol/mL, respectively, significantly lower than those of groups 3 and 4 (control infants), that is, 21.1 +/- 8.7 and 27.1 +/- 9.2 pmol/mL, respectively (P <.0001). Among infants with asphyxia, 3 died and 10 had abnormal neurologic outcome. Univariate analysis showed that abnormal outcomes were significantly related to CSF cAMP levels, phenobarbital use, and multi-organ failure. However, only CSF cAMP was retained in the model by stepwise logistic regression. CSF cAMP of 10.0 pmol/mL discriminated between those with normal and those with abnormal neurologic outcome. Low CSF cAMP concentration was associated with abnormal long-term outcome, estimated odds ratio of 12.4 (95% CI, 2.1-109.3; P <.006), and sensitivity, specificity, and positive and negative predictive values of 85%, 69%, 73%, and 80%, respectively. CONCLUSION: CSF cAMP concentrations were decreased in infants with asphyxia. Low CSF cAMP levels were associated with poor neurologic outcome.
Assuntos
Asfixia Neonatal/líquido cefalorraquidiano , AMP Cíclico/líquido cefalorraquidiano , Hipóxia Encefálica/líquido cefalorraquidiano , Índice de Apgar , Peso ao Nascer , População Negra , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Valor Preditivo dos Testes , Prognóstico , Radioimunoensaio , Valores de Referência , População BrancaRESUMO
The role of tyrosine phosphorylation was investigated using protein tyrosine phosphatase inhibitors in newborn pigs equipped with a cranial window in vivo. We tested the hypothesis that cyclooxygenase and nitric oxide (NO) synthase are physiological targets for tyrosine phosphorylation in cerebral circulation. Phenylarsine oxide dilated pial arterioles and increased prostacyclin and prostaglandin E2 in cortical periarachnoid fluid; these responses were inhibited by indomethacin. Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NNA) inhibited the vasodilation to phenylarsine oxide; the effects of NO synthase inhibitors and indomethacin were additive. Cyclooxygenase-mediated vascular responses were assessed using topical application of arachidonic acid. Phenylarsine oxide and sodium orthovanadata potentiated vasodilation and prostanoid synthesis in response to arachidonic acid. Nomega-nitro-L-arginine methyl ester and Nomega-nitrol-arginine did not affect vasodilation or prostanoid production in response to arachidonic acid, indicating no cross talk between cyclooxygenase and NO synthase. These data indicate that cyclooxygenase and NO synthase are physiological targets for tyrosine phosphorylation in the cerebral circulation of newborn pigs.
Assuntos
Animais Recém-Nascidos/fisiologia , Circulação Cerebrovascular/fisiologia , Tirosina/metabolismo , Sistema Vasomotor/fisiologia , Animais , Arsenicais/farmacologia , Arteríolas/fisiologia , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Fosforilação , Pia-Máter/irrigação sanguínea , Prostaglandina-Endoperóxido Sintases/metabolismo , Prostaglandinas/farmacologia , Suínos , Vanadatos/farmacologia , Vasodilatadores/farmacologiaRESUMO
Endothelium-derived cyclooxygenase (COX) products regulate cerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebral microvessels. We investigated the role of protein phosphorylation in the regulation of COX activity. The protein tyrosine phosphatase (PTP) inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acid rapidly stimulated COX activity, whereas the protein tyrosine kinase inhibitors, genistein and tyrphostins, inhibited it. Protein synthesis inhibitors did not reverse the stimulation of COX activity evoked by PTP inhibitors. Similar changes were observed in other vascular cells from newborn pigs that also express COX-1 and COX-2 (cerebral microvascular smooth muscle cells and aortic endothelial cells) but not in human umbilical vein endothelial cells or Swiss 3T3 fibroblasts that express COX-1 only. Tyrosine-phosphorylated proteins were immunodetected in endothelial cell lysates. COX-2 immunoprecipitated from 32P-loaded endothelial cells incorporated 32P that was increased by PTP inhibitors. COX-2, but not COX-1, was detected in endothelial fractions immunoprecipitated with anti-phosphotyrosine. These data indicate that tyrosine phosphorylation posttranslationally regulates COX activity in newborn pig vascular cells and that COX-2 is a substrate for phosphorylation.
Assuntos
Córtex Cerebral/irrigação sanguínea , Endotélio Vascular/enzimologia , Isoenzimas/metabolismo , Músculo Liso Vascular/enzimologia , Fosfotirosina/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Tirfostinas , 6-Cetoprostaglandina F1 alfa/farmacologia , Animais , Animais Recém-Nascidos , Aorta , Células Cultivadas , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/farmacologia , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/biossíntese , Proteínas de Membrana , Microcirculação , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Nitrilas/farmacologia , Nitrobenzenos/farmacologia , Fenóis/farmacologia , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteínas Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Suínos , Vanadatos/farmacologiaRESUMO
This study investigated the effects of intraventricular/periventricular blood on cerebral cAMP production and cortical cerebrovascular reactivity. Under halothane and N2O anesthesia, 3 mL of either autologous blood or artificial cerebrospinal fluid (CSF) were injected into the left caudate nucleus; volume was adequate to result in extrusion of fluid or blood into the lateral ventricles of 1-2-d-old piglets. Twenty-four hours later, a closed cranial window was implanted over the left parietal cortex. Pial arteriolar responses to vasodilator and vasoconstrictor stimuli were monitored. Before the application of vasoactive agents, cortical periarachioid CSF was collected for cAMP measurement. Pial arteriolar responses to topical application of endothelin-1 (10(-9) and 10(-8) M) and to leukotriene C4 (10(-10) and 10(-9) M) were similar between the two groups. However, pial arteriolar responses to topical application of cAMP-mediated vasodilators, prostaglandin E2 (10(-6) and 10(-5) M), and histamine (10(-6) and 10(-5) M), respectively, were markedly reduced in the blood group when compared with the artificial CSF (control) group. Mean CSF cAMP level in the blood group was significantly lower than the control group (199 +/- 31 versus 1092 +/- 238 fmol/mL, p = 0.0006). We conclude that in newborn pigs intraventricular/periventricular blood results in a marked reduction of CSF cAMP concentration and attenuation of the cerebrovascular responses to cAMP-mediated vasodilators on the cortical surface remote from the site of blood or hematoma.
Assuntos
Animais Recém-Nascidos/fisiologia , Ventrículos Cerebrais , Circulação Cerebrovascular/fisiologia , AMP Cíclico/biossíntese , Animais , Artérias , Coagulação Sanguínea/fisiologia , Fenômenos Fisiológicos Sanguíneos , Pressão Sanguínea/fisiologia , Volume Sanguíneo/fisiologia , Dióxido de Carbono/análise , Dióxido de Carbono/sangue , Ventrículos Cerebrais/irrigação sanguínea , Líquido Cefalorraquidiano/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Dinoprostona/farmacologia , Endotelina-1/farmacologia , Hemorragia/fisiopatologia , Histamina/farmacologia , Concentração de Íons de Hidrogênio , Leucotrienos/farmacologia , Oxigênio/análise , Oxigênio/sangue , Pressão Parcial , Suínos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Cyclooxygenase (COX) isoform expression, intracellular localization, and function in endothelial cells from the newborn pig cerebral microvessels were investigated using COX-1- and COX-2-specific antibodies and the COX-2 inhibitor NS-398. Cerebral microvessels, microvascular endothelium, and cultured endothelial cells constitutively express both COX-1 and COX-2. NS-398 inhibits 70-90% of endothelial prostanoid production. Endothelial cells grown in noncontact coculture with smooth muscle cells for 24-48 h demonstrate a stable induction of COX-2 protein and an NS-398-sensitive increase in prostanoid synthesis. The induction of endothelial COX in mixed cell coculture is accompanied by intracellular redistribution of COX-2. In cocultured endothelial cells, COX-2 is observed in the nucleus, nuclear envelope, and cytoplasm, compared with the mainly intranuclear localization of COX-2 in cells cultured separately. No changes were observed in COX-1 protein, localized in endothelial cell cytoplasm and the nuclear envelope. These results indicate that smooth muscle cells may modify endothelial function by upregulating COX-2, which is a major functional COX isoform in cerebral microvascular endothelial cells.
Assuntos
Circulação Cerebrovascular , Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica , Isoenzimas/biossíntese , Microcirculação , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Animais Recém-Nascidos , Ácido Araquidônico/farmacologia , Encéfalo/irrigação sanguínea , Núcleo Celular/enzimologia , Células Cultivadas , Técnicas de Cocultura , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Citosol/enzimologia , Dexametasona/farmacologia , Dinoprostona/farmacologia , Endotélio Vascular/citologia , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Ionomicina/farmacologia , Cinética , Nitrobenzenos/farmacologia , Prostaglandinas/metabolismo , Sulfonamidas/farmacologia , SuínosRESUMO
Cerebrovascular reactivity is preserved after acute severe asphyxia/reventilation in piglets. We hypothesize that prolonged, partial asphyxia with hypotension causes loss of cerebrovascular reactivity and altered cerebral hemodynamics during recovery. We investigated the changes in cerebrospinal fluid cAMP and cGMP, pial arteriolar diameters and flow, and cerebral blood flow during 1 h of asphyxia and 1 h of recovery. During asphyxia, blood pressure decreased from 10 +/- 0.7 to 4.7 +/- 0.3 kPa and increased during recovery to 6 +/- 0.7 kPa. cAMP increased 3-fold by 20 min of asphyxia, returning to baseline at 40 min of asphyxia. During recovery, cAMP increased 2-fold initially, followed by a decrease to 50% below baseline. cGMP increased after 20 min of asphyxia, with maximum levels observed at 40 min; reventilation resulted in a transient increase in cGMP. Pial arteriolar diameters increased at the onset of asphyxia, then decreased toward baseline; during recovery, a similar pattern occurred. Blood flow to the cerebrum (microspheres) decreased during asphyxia and remained very low during recovery. Pial arteriolar flow but not pial arteriolar diameters followed the changes in cortical cerebral blood flow (i.e. virtually no flow during recovery). During recovery, pial arteriolar reactivity to isoproterenol and histamine decreased significantly. We conclude that 60 min of asphyxic-hypotensive insult results in alterations of cerebral cAMP metabolism which may compromise cellular communications during recovery. Prolonged asphyxia induces "no-reflow" during recovery, even when partial pressures of arterial CO2 and O2 have returned to baseline values, and blood pressure is within the autoregulatory range.
Assuntos
Asfixia Neonatal/fisiopatologia , Encéfalo/irrigação sanguínea , Circulação Cerebrovascular , AMP Cíclico/líquido cefalorraquidiano , GMP Cíclico/líquido cefalorraquidiano , Hemodinâmica , Animais , Animais Recém-Nascidos , Asfixia Neonatal/metabolismo , Pressão Sanguínea , Córtex Cerebral/irrigação sanguínea , Feminino , Humanos , Concentração de Íons de Hidrogênio , Recém-Nascido , Masculino , Especificidade de Órgãos , Pia-Máter/irrigação sanguínea , Fluxo Sanguíneo Regional , Suínos , Fatores de TempoRESUMO
The hypothesis that endothelium-dependent components contribute to the cerebromicrovascular dilation to hypoxia in the newborn pig was addressed. Piglets anesthetized with ketamine-acepromazine and maintained on alpha-chloralose were equipped with closed cranial windows. Injury to the endothelium of pial arterioles was produced by light activation of fluorescein dye. Light/dye injury reduced the pial arteriolar dilation to hypoxia (5 min, arterial PO2 approximately 30 mmHg) from 57 +/- 9 to 19 +/- 5%. Light/dye injury abolished the pial arteriolar dilation to hypercapnia but did not affect dilation to sodium nitroprusside. The pial arteriolar dilation to hypoxia was not affected by tetrodotoxin, N(omega)-nitro-L-arginine, glibenclamide, iberiotoxin, charybdotoxin, tetraethylammonium, or 8-phenyltheophylline. Hypoxia caused increases in the cerebral cortical production of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. Cerebral vasodilation to hypoxia was inhibited by 5,8,11,14-eicosatetraynoic acid but was not greatly affected by cyclooxygenase or lipoxygenase inhibitors. In contrast, the cytochrome P-450 epoxygenase inhibitor miconazol decreased cerebral vasodilation to hypoxia from 45 +/- 5 to 17 +/- 4%. Therefore, the vascular endothelium appears to participate in cerebral microvascular dilation to hypoxia in newborn pigs. The mechanism may include cytochrome P-450 epoxygenase metabolites of arachidonic acid.
Assuntos
Circulação Cerebrovascular/fisiologia , Hipóxia Encefálica/fisiopatologia , Microcirculação/fisiologia , Vasodilatação , Ácido 5,8,11,14-Eicosatetrainoico/farmacologia , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Arteríolas/fisiopatologia , Dióxido de Carbono/sangue , Dióxido de Carbono/farmacologia , Hipóxia Encefálica/metabolismo , Isoproterenol/farmacologia , Masoprocol/farmacologia , Microcirculação/efeitos dos fármacos , N-Metilaspartato/farmacologia , Nitroprussiato/farmacologia , Oxigênio/sangue , Pressão Parcial , Pia-Máter/irrigação sanguínea , Suínos , Tetrodotoxina/farmacologia , Teofilina/análogos & derivados , Teofilina/farmacologiaRESUMO
Experiments addressed the hypothesis that dilator prostanoids contribute to maintenance of low cerebral microvascular tone during hypoxia in the newborn. Anesthetized newborn pigs equipped with closed cranial windows were used to measure responses of pial arterioles (approximately 60 microns) to treatments. Hypoxia (Pao2 approximately equal to 25 mmHg) caused dilation of pial arterioles (approximately 50% increase in diameter). Hypoxia (5 min) caused an increase in cortical cerebrospinal fluid 6-ketoprostaglandin F1 alpha concentration from 907 +/- 171 (normoxia) to 1,408 +/- 213 pg/ml (hypoxia). Pretreatment with indomethacin (5 mg/kg) did not affect pial arteriolar dilation to hypoxia. Conversely, indomethacin treatment during hypoxia caused a rapid decrease in arteriolar diameter to nearly the normoxia diameter within 3 min, returning to the original hypoxia diameter by 10 min. Ibuprofen treatment (30 mg/kg) had no effect on pial arteriolar diameter during normoxia or hypoxia, and pretreatment did not alter dilation to hypoxia. However, pretreatment with ibuprofen abolished the constrictor effect of indomethacin given during hypoxia. These data suggest that the primary mechanism by which hypoxia produces cerebral vasodilation does not involve prostanoids, but prostanoids can contribute to cerebral vasodilation in response to hypoxia.
Assuntos
Circulação Cerebrovascular , Hipóxia/fisiopatologia , Prostaglandinas/fisiologia , Vasodilatação , 6-Cetoprostaglandina F1 alfa/líquido cefalorraquidiano , Animais , Animais Recém-Nascidos/fisiologia , Arteríolas/efeitos dos fármacos , Arteríolas/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Hipercapnia/metabolismo , Hipóxia/metabolismo , Ibuprofeno/farmacologia , Indometacina/farmacologia , Pia-Máter/irrigação sanguínea , Suínos , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
The involvement of protein kinase C (PKC) and protein tyrosine kinase (PTK) in hypercapnia-induced cerebral vasodilation in newborn pigs was investigated with closed cranial windows using the PKC stimulator phorbol 12-myristate 13-acetate (PMA), and the PTK inhibitors, genistein and herbimycin A. The influence of prostaglandin I2 was eliminated using the prostaglandin cyclooxygenase inhibitor, indomethacin. Changes in pial arteriolar diameters in response to hypercapnia [partial pressure of arterial CO2 approximately 9.3 kPa (70 torr)] were analyzed. Genistein (40 micrograms/mL), herbimycin A (10 microM), or PMA (1 microM) did not affect cerebral vasodilation to hypercapnia when applied topically. Indomethacin (5 mg/kg i.v.) treatment blocked the dilation to hypercapnia and attenuated hypercapnia-induced increase in cortical cAMP. Genistein and herbimycin A restored the response to hypercapnia to indomethacin-treated piglets. PMA also restored the pial arteriolar dilation and the cAMP response to hypercapnia to indomethacin-treated piglets. One-hour exposure to 10 microM PMA, to down-regulate PKC, blocked vasodilation to hypercapnia but did not inhibit vasodilation to sodium nitroprusside. After prolonged (2 h) topical exposure of indomethacin-treated piglets to 10 microM PMA, neither genistein nor iloprost could restore dilation to hypercapnia. These results indicate that PKC stimulation and/or PTK inhibition may permit hypercapnia-induced vasodilation. These data further suggest that PKC is downstream from PTK in the regulatory pathway. Because previous data showed prostaglandin I2 at subdilator concentrations can also return dilation to hypercapnia to piglets treated with indomethacin, prostaglandin I2 could provide its permissive input by activating PKC and/or inhibiting PTK.
Assuntos
Anti-Hipertensivos/farmacologia , Epoprostenol/farmacologia , Pia-Máter/irrigação sanguínea , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/metabolismo , Vasodilatação/fisiologia , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Benzoquinonas , Regulação para Baixo , Genisteína , Hipercapnia/fisiopatologia , Isoflavonas/farmacologia , Lactamas Macrocíclicas , Pia-Máter/efeitos dos fármacos , Quinonas/farmacologia , Rifabutina/análogos & derivados , Suínos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In the newborn pig cerebral circulation, arteriolar dilation in response to hypercapnia requires the presence of intact endothelium and is accompanied by an indomethacin-sensitive increase in cortical adenosine 3',5'-cyclic monophosphate (cAMP). The effects of short-term hypercapnia on production of dilator prostanoids and cAMP were investigated using newborn pig cerebral microvascular smooth muscle cells and endothelial cells cultured both separately and in noncontact coculture. Microvascular smooth muscle cells respond to hypercapnia (pH 7.00 +/- 0.05 PCO2 75 +/- 3 mmHg) by a 1.3- to 1.7-fold increase in basal cAMP production that is not affected by indomethacin, whereas hypercapnia and 80 mM sodium propionate do not affect iloprost-stimulated cAMP production. Microvascular endothelial cells cultured on Millicel inserts respond to hypercapnia by a two- to fourfold increase in prostacyclin (as 6-keto-prostaglandin F1 alpha) and prostaglandin E2 production in both luminal and abluminal compartments. For noncontact coculture, Millicel inserts with endothelial cells (as hypercapnia-sensitive producers of prostanoids) were installed into cell culture dishes with aspirin-pretreated smooth muscle cells (as targets for endothelium-derived dilator prostanoids). Exposure of noncontact microvascular cell cocultures to hypercapnia results in a three- to fourfold stimulation of prostanoid and cAMP production. Therefore, short-term hypercapnia increases cAMP production by microvascular smooth muscle cells via 1) a direct (prostanoid independent) mechanism and 2) an endothelial-dependent pathway that involves prostanoids. Endothelium-produced prostanoid signals are necessary for a full increase in cAMP production by cerebral microvascular smooth muscle cells in response to hypercapnia.
Assuntos
Circulação Cerebrovascular , AMP Cíclico/biossíntese , Hipercapnia/metabolismo , Músculo Liso Vascular/metabolismo , Prostaglandinas/biossíntese , Animais , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Microcirculação , Músculo Liso Vascular/citologia , Propionatos/farmacologia , Suínos , Vasodilatadores/farmacologiaRESUMO
Inflammatory cytokines may affect cerebral circulation under pathological conditions. Responses of cerebral pial arterioles to one such cytokine, interleukin (IL)-1 beta and its inhibitor [soluble IL-1 receptor (sIL-1R)] were examined in anesthetized newborn pigs using closed cranial windows. Levels of prostanoids and cyclic nucleotides in periarachnoid cerebral spinal fluid (CSF) were measured. To examine the structure-activity relationship of the parent IL-1 beta molecule, two IL-1 beta fragments with amino acid sequences of 187-204 [IL-1 beta-(187-204)] and 208-240 [IL-1 beta-(208-240)] were tested for their effects on pial arterioles. Diameter changes of pial arterioles were sequentially recorded every 5 min for 30 min after topical application of IL-1 peptides. CSF was sampled at the end of the 30 min. IL-1 beta dose dependently induced arteriolar dilation and increased prostaglandin E2 (PGE2), 6-keto-PGF1 alpha adenosine 3',5'-cyclic monophosphate (cAMP), and guanosine 3',5'-cyclic monophosphate (cGMP). Intravenous indomethacin blocked the IL-1 beta-induced vasodilation, the increased prostanoids, and the increased cAMP, but not the increased cGMP. Neither heat-inactivated IL-1 beta nor IL-1 beta vehicle affected arteriolar diameter or CSF levels of prostanoids. The sIL-1R blocked the IL-1 beta-induced vasodilation and the increased CSF prostanoids. IL-1 beta-(208-240) also induced pial arteriolar dilation; however, its vasodilatory potency was 1,000 times less than that of the whole IL-1 beta molecule. IL-1 beta-(187-204) did not induce pial arteriolar dilation even when its dose was increased to the level of IL-1 beta-(208-240). These results suggest that IL-1 beta, through the activation of membrane-bound IL-1 beta receptors, induces pial arteriolar dilation via mechanisms that involve prostanoids and cyclic nucleotides. The results also indicate that the 208-240 amino acid sequence of IL-1 beta has a sequence-specific physiological function.
Assuntos
Circulação Cerebrovascular/efeitos dos fármacos , Interleucina-1/farmacologia , Fragmentos de Peptídeos/farmacologia , Pia-Máter/irrigação sanguínea , Vasodilatação , 6-Cetoprostaglandina F1 alfa/antagonistas & inibidores , 6-Cetoprostaglandina F1 alfa/líquido cefalorraquidiano , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Dinoprostona/antagonistas & inibidores , Dinoprostona/líquido cefalorraquidiano , Indometacina/farmacologia , Receptores de Interleucina-1/fisiologia , Solubilidade , SuínosRESUMO
The present study in piglets was designed to examine cerebrovascular effects of tumor necrosis factor-alpha (TNF alpha) and potential mechanisms involved. Anesthetized new-born pigs with closed cranial windows implanted were used. Effects of nitric oxide synthase (NOS) inhibitors, NG-nitro-L-arginine (L-NNA) and aminoguanidine, and a prostaglandin H synthase inhibitor, indomethacin, on pial arteriolar responses to TNF alpha were determined. In addition, cortical cerebrospinal fluid (CSF) prostanoids (PGE2 and 6-keto-PGF1 alpha) and cyclic nucleotides (cAMP and cGMP) were examined as indices of local cerebral production. Diameters of pial arterioles were recorded every 5 min for 30 min following topical infusion of TNF alpha under the window. CSF was sampled at the end of the 30-min recordings. TNF alpha (10(-8) and 10(-7) M) caused dilation of pial arterioles and increased CSF prostanoids and cyclic nucleotides. Indo-methacin blocked TNF alpha-induced vasodilation and the increase of prostanoids and cAMP, but not of cGMP. L-NNA and amino-guanidine blocked TNF alpha-induced vasodilation. Both inhibitors attenuated TNF alpha-induced prostanoid increase. Aminoguanidine blocked TNF alpha-induced increased cGMP and attenuated the increase in cAMP. These results are consistent with the hypothesis that TNF alpha increases cAMP via prostanoid synthesis. They also suggest that TNF alpha increases cGMP through nitric oxide synthesis, which, in addition, may promote production of prostanoids and, thus, cAMP.
Assuntos
Artérias Cerebrais/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Animais Recém-Nascidos , AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Suínos , Fatores de Tempo , Vasodilatação/efeitos dos fármacosRESUMO
Cerebral vascular reactivity to prostaglandin E2 was investigated in newborn pigs using closed cranial windows. Exogenous prostaglandin E2 is a dilator of pial arterioles that elevates cyclic AMP in cortical cerebrospinal fluid. Pial arterioles are less sensitive to prostaglandin E2 than to the prostacyclin receptor agonist iloprost, but their maximal responses to the dilator prostanoids are similar. The cerebrovascular effects of prostaglandin E2 and iloprost are not additive. Pretreatment with either iloprost or prostaglandin E2 decreases pial arteriolar responsiveness to iloprost without affecting responses to isoproterenol. The homologous desensitization of pial arterioles suggests that auto- and cross-tachyphylaxis in vascular effects of iloprost and prostaglandin E2 occur at the receptor level. Indomethacin, which selectively inhibits prostacyclin receptor-mediated responses in cerebral vascular smooth muscle, greatly reduces the vascular responses to prostaglandin E2. These results suggest that vasodilator effects of prostaglandin E2 in the newborn cerebral circulation are mediated via prostacyclin receptors coupled to adenylyl cyclase.
Assuntos
Animais Recém-Nascidos/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Dinoprostona/farmacologia , Vasodilatadores/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Aspirina/farmacologia , Córtex Cerebral/metabolismo , AMP Cíclico/líquido cefalorraquidiano , Dinoprostona/antagonistas & inibidores , Iloprosta/farmacologia , Indometacina/farmacologia , Receptores de Epoprostenol , Receptores de Prostaglandina/agonistas , SuínosRESUMO
Effects of the lipid mediator lysophosphatidic acid (LPA) were studied on the cerebral circulation of newborn pigs using closed cranial windows. Topical application of synthetic LPA caused dose-dependent vasoconstriction and inhibited vasodilation to hypercapnia and isoproterenol. These vasodilators elicited a rise in the adenosine 3',5'-cyclic monophosphate (cAMP) content of the cerebrospinal fluid, which was inhibited dose dependently by LPA. Pertussis toxin (1 microgram/ml) completely abolished LPA-induced vasoconstriction and the altered vascular reactivity, and LPA no longer decreased cAMP. Electrophysiological recording of currents evoked by LPA-like lipids in Xenopus oocytes showed that cerebrospinal fluid is normally devoid of LPA-like factors. In contrast, the amount of LPA-like factors generated 4 days after intrathecal injection of autologous blood was in the range of 1-10 microM LPA equivalents. The data indicate that LPA-like bioactive mediators were generated in an intracranial hematoma model and that these phospholipids might play a role in the pathophysiology of altered vascular reactivity often found in posthemorrhagic conditions and could also contribute to the development of posthemorrhagic vasoconstriction.