RESUMEN
PURPOSE: This study was performed to clarify the possible mechanism behind the ocular hypotensive effect of unoprostone isopropyl (Rescula; Novartis Ophthalmics AG, Basel, Switzerland), a new docosanoid that has been shown to reduce intraocular pressure (IOP) in patients with ocular hypertension or primary open-angle glaucoma. To gain insight into the possible mode of action, the effects of unoprostone on ciliary muscle (CM) and trabecular meshwork (TM) contractility, intracellular calcium levels, and membrane channels were investigated. METHODS: The effects of unoprostone (M1 metabolite = free acid, 10(-5) M) and endothelin (ET)-1 (10(-9) M) on bovine TM (BTM) and ciliary muscle (CM) strips were investigated, by using a custom-made force-length transducer system. The effects of unoprostone and ET-1 (5 x 10(-8) M) on intracellular Ca(2+) mobilization in cultured human TM (HTM) were measured using fura-2AM as a fluorescent probe. Patch-clamp experiments were performed on HTM and BTM cells to investigate the unoprostone-dependent modulation of membrane currents. RESULTS: In isolated TM and CM strips, unoprostone almost completely inhibited ET-induced contractions (TM: 2.9% +/- 4.3% vs. 19.6% +/- 5.7%, P < 0.05, n = 6; CM: 1.4% +/- 1.6% vs. 30.1% +/- 5.3%, P < 0.01, n = 6; 100% = maximal carbachol-induced (10(-6) M) contraction). However, neither carbachol-induced contraction nor baseline tension was affected by unoprostone. Furthermore, unoprostone had no effect on baseline intracellular calcium levels (baseline: 126 +/- 45 nM versus unoprostone: 132 +/- 42 nM, n = 8) in HTM cells. The endothelin-induced increase (679 +/- 102 nM), however, was almost completely (P < 0.01) blocked by unoprostone (178 +/- 40 nM). In patch-clamp recordings, unoprostone could be shown to double the amplitude of outward current (HTM: 200% +/- 33%; n = 6; BTM: 179% +/- 20%; n = 8). This effect was blocked by the specific inhibitor of maxi-K channels, iberiotoxin. CONCLUSIONS: This study presents evidence for direct interaction of unoprostone with the contractility of the TM and CM. This compound may lower IOP by affecting aqueous outflow, most probably conventional outflow pathways (i.e., TM) through inhibition of ET-dependent mechanisms. In addition, unoprostone interacts with the maxi-K channel. Although primarily Ca(2+)-sensitive signal-transduction pathways seem to be involved, effects of unoprostone on Ca(2+)-independent pathways and uveoscleral outflow cannot be excluded.
Asunto(s)
Antihipertensivos/farmacología , Dinoprost/análogos & derivados , Dinoprost/farmacología , Malla Trabecular/efectos de los fármacos , Malla Trabecular/fisiología , Adulto , Anciano , Animales , Calcio/metabolismo , Carbacol/farmacología , Bovinos , Células Cultivadas , Agonistas Colinérgicos/farmacología , Cuerpo Ciliar/efectos de los fármacos , Cuerpo Ciliar/fisiología , Conductividad Eléctrica , Endotelinas/farmacología , Humanos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Persona de Mediana Edad , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Concentración Osmolar , Malla Trabecular/citologíaRESUMEN
The murine gastric mucosa possesses very high secretory type phospholipase A2 activity. Northern and Western blots indicated that the pancreatic-type, sPLA2-IB represents the predominant form of sPLA2 enzymes present in the gastric mucosa. Both sPLA2-IB mRNA and protein in the gastric mucosa exceeded levels found in the pancreas, and in contrast to the pancreatic enzyme it was present primarily in the active state. The sPLA2-IB gene is not expressed in the murine small intestine and colon. Infection by the gastritis-inducing bacteria, Helicobacterfelis (H. felis) dramatically and time dependently decreased the PLA2 activity in the glandular stomach of the mouse strain, C57BL/6, sensitive to the organism, which appeared to be related to a decrease in the percentage of sPLA2-IB present in the active form. This bacterial-induced reduction in PLA2 activity was not observed in BALB/c mice that fail to develop gastritis in response to H. felis infection. C57BL/6 mice do not, while BALB/c mice express, the PLA2-II enzyme. The H. felis-induced reduction in sPLA2-IB activity may weaken the gastric barrier by reducing the local concentration of arachidonic and linoleic acid, liberated from membrane phospholipids, the major precursors of 'cytoprotective' prostaglandins. Data presented here suggest that both sPLA2-IB and sPLA2-II enzymes may contribute to the gastric response to Helicobacter infection.
Asunto(s)
Mucosa Gástrica/enzimología , Infecciones por Helicobacter/enzimología , Fosfolipasas A/biosíntesis , Fosfolipasas A/metabolismo , Animales , Northern Blotting , Western Blotting , Femenino , Fosfolipasas A2 Grupo II , Infecciones por Helicobacter/genética , Íleon/enzimología , Intestinos/enzimología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosfolipasas A/genética , Fosfolipasas A2 , ARN Mensajero/biosíntesis , Especificidad de la Especie , Factores de TiempoRESUMEN
We measured the activities of total Na+, K+-ATPase (Na, K-ATPase), its alpha1 and alpha2/alpha3 isoforms and the angiotensin-converting enzyme (ACE) in the microvascular and neural compartments of the retina, and/or retinal pigment epithelium (RPE) of streptozotocin (STZ)-diabetic rats. The effect of captopril, an ACE inhibitor on Na, K-ATPase activities was also determined and correlated to morphological changes. Insulin-dependent diabetes mellitus was induced by a single intraperitoneal injection of STZ (60 mg/kg) in male Long-Evans rats. ACE activity was inhibited by captopril (10 mg/kg given in the drinking water) for 1 month. Na, K-ATPase activity was measured spectrophotometrically or by a radioassay (32P-labeled ATP). The activity of ACE was determined by a radioassay using tritiated benzoyl-gly-gly-gly as substrate. Both the alpha1 and alpha2/alpha3 isoforms of Na, K-ATPase were present in the microvascular and neural compartments of retinas, whereas only one isoform, the alpha2/alpha3, was found in the RPE. In 2-month diabetic rats, the activity of the alpha2/alpha3 isoform was reduced in both the microvascular and neural compartments of retinas, while the activity of the alpha1 isoform was reduced only in the neural isolates. ACE activity was significantly decreased in the retinal neural compartment and unaltered in the microvascular compartment from 2-month diabetic rats. In 5-month diabetic rats, Na, K-ATPase activity was moderately but not significantly reduced in RPE preparations. Ultrastructural studies revealed a significant deepening of basal infoldings in the RPE and a noticeable increase in the size of the extracellular space between the basal infoldings of 5-month diabetic animals. Captopril stimulated Na, K-ATPase activity in the neural retina, but not in the RPE. Diabetes-induced morphological changes in the RPE were not improved by captopril. An enlargement of intercellular space between the RPE cells was a frequent finding in the treated group. In conclusion, captopril stimulated Na, K-ATPase activity in the neural retina of diabetic rats. This stimulation seems to be beneficial to the neural retina. ACE inhibition, however, did not improve RPE morphological changes. Although the clinical significance of increased intercellular spacing between RPE cells in treated animals is not clearly established, we speculate that it might contribute to an increased alteration of their barrier function. Additional studies are necessary to assess both the desirable and adverse effects of captopril and other ACE inhibitors in the retinas of diabetic patients.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Captopril/farmacología , Diabetes Mellitus Experimental/enzimología , Epitelio Pigmentado Ocular/enzimología , Retina/enzimología , Animales , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/enzimología , Retinopatía Diabética/patología , Retinopatía Diabética/fisiopatología , Masculino , Peptidil-Dipeptidasa A/metabolismo , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/ultraestructura , Ratas , Ratas Long-Evans , Retina/efectos de los fármacos , Retina/ultraestructura , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
BACKGROUND: Helicobacter pylori has become recognized as a fundamental pathogen in the development of gastritis and peptic ulcer disease. Bismuth compounds in combination with antibiotics are widely used to treat H. pylori associated peptic ulcer disease. METHODS: In this study we measured and analysed the inhibitory effect of ranitidine bismuth citrate (RBC, Pylorid, Tritec) on the activity and kinetics of phospholipase A2 (PLA2) (E.C.3.1.1.4) of commercial cobra (Naja naja) venom and H. pylori (French press lysates) using L-alpha-dipalmitoyl-(2[1-14C]palmitoyl)-phosphatidylcholine as substrate. RESULTS: Our data suggest that RBC might exert a dose-dependent uncompetitive inhibition on PLA2 activity of both H. pylori and Naja naja venom. the inhibitory effect of RBC on the PLA2 activity cannot be abolished by the optimal concentration of calcium (10 mM), indicating its mechanism to be unrelated to the displacement of calcium from the activation site of the enzyme. CONCLUSION: Our results suggest that one of the mechanisms by which bismuth compounds are therapeutically effective in the treatment of H. pylori associated gastritis is by inhibiting the activity of the degradative PLA2 enzyme secreted by H. pylori. As a consequence of the inhibitory action of RBC on PLA2 of the bacteria, the extracellular and/or intracellular phospholipid components of the gastric mucosal barrier are preserved.
Asunto(s)
Antiulcerosos/farmacología , Bismuto/farmacología , Venenos Elapídicos/enzimología , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Antagonistas de los Receptores H2 de la Histamina/farmacología , Fosfolipasas A/efectos de los fármacos , Ranitidina/análogos & derivados , Animales , Antiulcerosos/uso terapéutico , Bismuto/uso terapéutico , Relación Dosis-Respuesta a Droga , Gastritis/tratamiento farmacológico , Gastritis/microbiología , Infecciones por Helicobacter/tratamiento farmacológico , Antagonistas de los Receptores H2 de la Histamina/uso terapéutico , Humanos , Fosfolipasas A2 , Ranitidina/farmacología , Ranitidina/uso terapéuticoRESUMEN
Helicobacter pylori infection has been linked to the development of gastritis which can then progress to a number of disease entities including peptic ulcer disease and gastric cancer. Since the pathogenic mechanism by which the bacteria causes gastritis is unresolved, we employed a model system, the H. felis-infected mouse to investigate the temporal relationship between bacterially-induced alterations in the hydrophobic phospholipid barrier of the stomach and the development of gastritis. In the present study, C57BL/6 mice were inoculated with 10(9) CFU of H. felis and the changes in gastric wet weight, histology, surface hydrophobicity, phospholipid/phosphatidylcholine concentration, phospholipase A2 activity, and the pH of collected gastric juice were measured 0.5-2 months postinoculation. In related experiments, we investigated the effects of treating H. felis infected mice with antibiotic/ bismuth therapy on the above gastric properties. It was determined that both gastric surface hydrophobicity and phospholipid composition were significantly attenuated as early as 2-4 weeks postinfection, preceding signs of mucosal inflammation and glandular atrophy as indicated by increases in gastric wet weight, pH and a disappearance in parietal cells. These early H. felis-induced changes in gastric surface hydrophobicity and phospholipid concentration were reversed by antibiotic/bismuth therapy. Based on these results we conclude that H. felis infection induces an early transformation of the stomach from a hydrophobic to an acid-sensitive hydrophilic state that may trigger the subsequent development of gastritis.
Asunto(s)
Gastritis/microbiología , Infecciones por Helicobacter/metabolismo , Fosfolípidos/análisis , Animales , Bismuto/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/tratamiento farmacológico , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Fosfatidilcolinas/análisis , Fosfolipasas A/análisis , Fosfolipasas A2 , Estómago/patologíaRESUMEN
PURPOSE: To determine the involvement of rat kallikrein-binding protein (RKBP) in the development of diabetic retinopathy. METHODS: Diabetes was induced by streptozotocin (STZ) (55 mg/kg body weight in 0.05 M citrate buffer, pH 4.5) in male Sprague-Dawley rats (150 to 175 g, 6 weeks old) as confirmed by hyperglycemia and reduced body weight. Retinas were dissected from animals at 1, 2, and 4 months of diabetes. The functional activity of RKBP in retinal homogenates was determined by its complex formation with tissue kallikrein. Immunoreactive RKBP levels were measured by enzyme-linked immunosorbent assay. The RKBP messenger RNA (mRNA) levels in the retina were measured by Northern blot analysis using the RKBP complementary DNA probe. The activity of total Na+,K(+)-ATPase was determined by a radioassay. Total protein concentration was determined by a protein assay. RESULTS: The kallikrein-binding activity was reduced in the retinas of STZ-diabetic rats at 1 (59%), 2 (50%), and 4 (38%) months of diabetes compared to those of age-matched control subjects. Levels of immunoreactive RKBP were significantly lower in the diabetic animals at each time point examined compared to those of control subjects. At 1 and 2 months of diabetes, RKBP levels (nanogram/milligram protein) were decreased significantly to 6.9 +/- 0.7 (n = 8) and 10.6 +/- 1.0 (n = 8), respectively, compared to those of age-matched control subjects (14.1 +/- 0.7, n = 8, P < 0.001, and 14.1 +/- 1.2, n = 8, P < 0.01). At 4 months of diabetes, retinal RKBP levels were lower in both control and diabetic groups, but RKBP levels in diabetic groups were significantly lower (5.8 +/- 0.6, n = 8) than those of the age-matched control subjects (8.4 +/- 0.9, n = 8, P < 0.01). Similarly, Northern blot analysis showed that RKBP mRNA levels were reduced in the retina of each group of STZ-diabetic rats, suggesting that the decrease in RKBP occurred at the level of transcription. CONCLUSIONS: The results show that STZ-induced diabetic rats have decreased retinal RKBP; moreover, this suggests that RKBP may contribute to diabetic retinopathy.
Asunto(s)
Proteínas Portadoras/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Calicreínas/metabolismo , Retina/metabolismo , Serpinas/metabolismo , Animales , Northern Blotting , Proteínas Portadoras/genética , Ensayo de Inmunoadsorción Enzimática , Calicreínas/genética , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Serpinas/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , EstreptozocinaRESUMEN
PURPOSE: To investigate whether serum and/or retinal angiotensin-converting enzyme (ACE) activity might correlate with the decrease in sodium potassium adenosine triphosphatase (Na,K-ATPase) activity in the retina of experimentally diabetic rats. METHODS: Insulin-dependent diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) in male Sprague-Dawley rats. Male Zucker fatty diabetic (ZDF/Gmifa) rats were used as models of non-insulin-dependent diabetes mellitus. ACE activity in the serum and retina of diabetic rats (1 through 5 months) and age-matched control animals was measured by radioimmunoassay using benzoyl-gly-gly-gly as substrate. The activity of total Na,K-ATPase was determined spectrophotometrically. The alpha 1 and alpha 3 isozymes of Na,K-ATPase were distinguished pharmacologically by their differential sensitivity to ouabain and were measured in the retina. RESULTS: Serum ACE activity was significantly increased in rats with STZ-induced diabetes at 3 weeks through 4 months of diabetes (28% to 32%) but was significantly decreased in ZDF rats after 2 to 5 months of diabetes (-9% to -16%). The activity of ACE in retinas obtained from the same groups of STZ and ZDF rats was significantly reduced at all time points examined in both models (-43% and -55%, respectively). The effect of angiotensin II (AngII) on the activity of Na,K-ATPase in retinas from normal rats was also studied in vitro. AngII significantly lowered the activities of total Na,K-ATPase (-16%) and its alpha 1 and alpha 3 isozymes. The inhibitory effect of AngII was abolished completely by losartan (0.1 microM), a specific antagonist of the AT1 receptor-subtype of AngII, and by nordihydroguaiaretic acid (50 microM), which at this concentration inhibits the lipoxygenase and cytochrome P-450-dependent pathways of arachidonic acid metabolism. The inhibitory effect of AngII on the Na,K-ATPase activity was not altered significantly by NG-iminoethyl ornithine (10 microM), an irreversible nitric oxide synthase inhibitor. CONCLUSIONS: The authors suggest that systemic ACE probably is not involved in the mechanisms responsible for the reduced activity of Na,K-ATPase in diabetes. Although AngII inhibits retinal Na,K-ATPase by a mechanism possibly involving arachidonic acid metabolites, it is unlikely that AngII contributes to the decreased Na,K-ATPase activity because of its reduced formation by retinal ACE in diabetes. The possible importance of reduced retinal ACE activity in diabetes warrants further investigation.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Peptidil-Dipeptidasa A/metabolismo , Retina/enzimología , Angiotensina II/antagonistas & inhibidores , Angiotensina II/farmacología , Animales , Antihipertensivos/farmacología , Compuestos de Bifenilo/farmacología , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Imidazoles/farmacología , Isoenzimas/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Losartán , Masculino , Masoprocol/farmacología , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Retina/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tetrazoles/farmacología , Vasoconstrictores/antagonistas & inhibidores , Vasoconstrictores/farmacologíaRESUMEN
PURPOSE: To examine the effect of captopril, an angiotensin-converting enzyme (ACE) inhibitor, on the activity of retinal sodium-potassium ATPase (Na,K-ATPase) and the activity of ACE in the serum and retina of streptozotocin (STZ)-induced diabetic rats. METHODS: Experimental diabetes was induced in male Long-Evans rats by a single intraperitoneal injection of STZ (55 mg/kg body weight). Some groups of normal and diabetic animals were treated with captopril (10 mg/kg per day) added to the drinking water for either a week or a month. After 2 and 4 months of diabetes, the specific activity of retinal total Na,K-ATPase was determined. The components of the activity of Na,K-ATPase caused by the alpha 1 and alpha 3 isoforms were pharmacologically separated by their different sensitivity to ouabain. The activity of ACE in the serum and retina was measured by radioassay using benzoyl-gly-gly-gly as substrate (10(5) cpm, 5 mM). RESULTS: The total Na,K-ATPase activity was decreased significantly after 2 (16%, P < 0.02) and 4 months (15%, P < 0.02) of diabetes. At both time points examined, the activities of the alpha 1-low-ouabain-affinity isoform and the alpha 3-high-ouabain-affinity isoform of retinal Na,K-ATPase were significantly reduced compared to those of age-matched controls (alpha 1, 9% to 14%, P < 0.05; alpha 3, 14% to 19%, P < 0.05 and P < 0.02 respectively). After 1 month of captopril administration, the activities of both Na,K-ATPase isoforms were at control level in 2-month diabetic rats, whereas they were restored only partially in 4-month diabetic rats. In age-matched normal animals, 1 month of captopril treatment did not alter the specific activities of either Na,K-ATPase isoform. One week or 1 month of captopril administration to diabetic rats did not change the activities of retinal Na,K-ATPase isoforms. Serum ACE activity was elevated significantly in both groups of untreated STZ rats (55% and 40%, respectively). One month of captopril administration further increased the ACE levels in 2- and 4-month diabetic rats (101% and 94%, respectively) and also enhanced significantly the serum ACE activity in normal animals (131%) versus the basal values. In contrast, retinal ACE activity was decreased significantly in both groups of untreated STZ rats (approximately 37%). Captopril exerted a significant inhibitory effect on the retinal ACE activity in 2- and 4-month diabetic rats (37% and 31%, respectively) compared to untreated diabetic animals as well as in normal rats (29%). CONCLUSIONS: These data suggest that stimulation of retinal Na,K-ATPase activity in diabetes is most likely one of the mechanisms through which captopril can improve retinal complications. The effect of captopril seems to be related to local effects in the retina. Whether the inhibition of retinal ACE is part of the mechanism of action of captopril requires further study.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Captopril/farmacología , Diabetes Mellitus Experimental/enzimología , Retinopatía Diabética/enzimología , Retina/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Antibacterianos , Glucemia/análisis , Diabetes Mellitus Tipo 1/enzimología , Inhibidores Enzimáticos/farmacología , Isoenzimas/metabolismo , Masculino , Ouabaína/farmacología , Peptidil-Dipeptidasa A/metabolismo , Distribución Aleatoria , Ratas , Retina/efectos de los fármacos , EstreptozocinaRESUMEN
The temporal pattern of changes in the specific activities of retinal Na+, K(+)-ATPase (Na, K-ATPase) and Mg(2+)-ATPase (Mg-ATPase) were determined at several time intervals following the onset of diabetes in streptozotocin-induced diabetic (STZ: at 1, 2, 4 and 6 months) Long-Evans hooded rats, spontaneously diabetic Zucker diabetic fatty (ZDF: at 1, 2 and 4 months) rats and their age-matched controls. These animals were utilized as models for insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), respectively. Na, K-ATPase specific activity, using 10(3) M ouabain, was decreased (-6% to -14%) at all time points after the appearance of hyperglycemia in the ZDF rat, but was reduced only after 4 and 6 months in the STZ rat (-8% and -14%, respectively). In contrast, Mg-ATPase activity was significantly increased (13%) after 4 months in the ZDF rat and after 6 months in the STZ rat (8%). The concentration-dependent inhibitory effects of ouabain (10(-9) to 10(-3) M) on the activity of Na, K-ATPase in diabetic rats and age-matched controls was used to assess the time-dependent effects of diabetes on the alpha 3-high ouabain affinity or the alpha 1-low ouabain affinity retinal Na, K-ATPase isozymes. The retinal Na, K-ATPase activity for the alpha 3 isozyme was significantly lower at all times examined for the ZDF (-5% to -26%) and STZ-induced diabetic rats (-8% to -14%). This was reflected in the markedly decreased half-maximal inhibitory concentrations (IC50) of ouabain for the alpha 3 isozyme. For example, after four months of diabetes, the mean +/- SEM IC50 values were 12 +/- 3 nM in the STZ rats and 48 +/- 6 nM in the age-matched controls and 19 +/- 3 nM in the ZDF rats and 30 +/- 4 nM in the age-matched controls. In contrast, the activity of the alpha 1 isozyme was slightly, but significantly, decreased at 2 and 4 months in the ZDF rats (-4% to -7%) and after 4 and 6 months in the STZ-induced diabetic rats (-3% to -9%) while the IC50 values were unchanged. Moreover, the Hill coefficient for the alpha 3 isozyme was decreased in both diabetic groups while it was unchanged for the alpha 1 isozyme.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Retina/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Retinopatía Diabética/enzimología , Hiperglucemia/enzimología , Isoenzimas/metabolismo , Masculino , Ouabaína/farmacología , Ratas , Ratas Zucker , Retina/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Estreptozocina , Factores de TiempoRESUMEN
In this study we measured phospholipase A (PLA) and C (PLC) activity of media filtrates and French Press lysates of the gastritis-inducing bacteria Helicobacter pylori. We report here that both H. pylori lysates and filtrates contain PLA1, PLA2, and C enzymes, which readily hydrolyze a radiolabeled dipalmitoylphosphatidylcholine (DPPC) and phosphorylcholine substrates, respectively. The specific activity of both PLA and C enzymes were greatest in the 6.5-7.0 and 8.4-8.8 pH ranges, respectively. Colloidal bismuth subcitrate (CBS) induced a dose-dependent inhibition of PLA2 and C activity of both H. pylori lysates and filtrates. This inhibitory effect of CBS on PLA2 was antagonized in a dose-dependent fashion by the addition of CaCl2 to the incubation mixture, suggesting that calcium and bismuth may be competing for the same site on the enzyme. In contrast, the ability of bismuth salts to inhibit PLC activity of H. pylori lysates was not antagonized by CaCl2. Employing a biophysical assay system for surface wettability, it was determined that H. pylori lysates had the capacity to remove a synthetic phospholipid monolayer off a glass in a dose-dependent fashion. This ability of the bacterial lysates to catalyze the transformation of a hydrophobic surface to a wettable state was significantly attenuated in the presence of bismuth salts. Our experimental results are, therefore, consistent with the possibility that H. pylori colonization compromises the stomach's barrier to acid by eroding a phospholipid lining, possibly a monolayer, on the surface of the gastric mucus gel and that this process is blocked in response to bismuth therapy.
Asunto(s)
Antibacterianos/farmacología , Helicobacter pylori/enzimología , Compuestos Organometálicos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , Bismuto/farmacología , Úlcera Duodenal/microbiología , Infecciones por Helicobacter/microbiología , Humanos , Concentración de Iones de Hidrógeno , Fosfolipasas A1 , Fosfolipasas A2 , Úlcera Gástrica/microbiologíaRESUMEN
The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.
Asunto(s)
Músculo Liso/enzimología , Neprilisina/metabolismo , Útero/enzimología , Animales , Fraccionamiento Celular , Electroforesis en Gel de Poliacrilamida , Femenino , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Cinética , Neprilisina/análisis , Neprilisina/antagonistas & inhibidores , Oxitocina/metabolismo , Embarazo , Ratas , Especificidad por Sustrato , Contracción Uterina/fisiologíaRESUMEN
The role of the neuropeptide galanin in the regulation of anterior pituitary function was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells. Galanin (50-200 ng; 15-60 pmol) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of plasma growth hormone (GH) concentrations, whereas galanin increased prolactin (PRL) and decreased thyroid-stimulating hormone (TSH) levels only at the highest dose (60 pmol) tested. Intravenous galanin failed to alter PRL and TSH levels in these rats. In contrast with the results with intraventricular injection of the peptide, intravenous injection of 30 or 300 pmol of galanin produced small, brief, dose-related increases in plasma GH. The response to the 300-pmol dose was less than that induced by a factor-of-20-lower intraventricular dose, which establishes a central action of galanin. Galanin in concentrations ranging from 1 nM to 1 microM failed to alter significantly GH, PRL, or TSH release from dispersed anterior pituitary cells. It also failed to alter GH secretion in response to 100 nM GH-releasing hormone; however, at this dose galanin did potentiate the effect of 100 nM TSH-releasing hormone on TSH and PRL release. Thus, the effects of third-ventricular injection of the peptide are mediated by the hypothalamus. To determine the physiological significance of galanin in control of pituitary hormone release, highly specific antiserum against galanin was injected intraventricularly. Third-ventricular injection of 3 microliter of galanin antiserum resulted in a dramatic decrease in plasma GH values as compared with those of normal rabbit serum-injected controls within 15 min, which persisted until the end of the experiment (5 hr postinjection). Galanin antiserum did not decrease plasma PRL or TSH levels at any time period after its third-ventricular injection; however, a transient increase of plasma TSH levels occurred after 30 and 60 min in comparison with TSH levels in normal rabbit serum-injected controls. Since there was no effect of the antiserum on plasma PRL and only a transient elevation in TSH, galanin may not be physiologically significant enough during resting conditions to alter PRL and TSH release in the male rat. The results of the experiments with galanin antiserum indicate that endogenous galanin has a tonic action within the hypothalamus to stimulate GH release. The rapidity of onset of the effects of galanin and the antiserum directed against it suggest that it acts to stimulate release of GH-releasing hormone from periventricular structures, which then stimulates the release of GH.
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Sistema Hipotálamo-Hipofisario/fisiología , Péptidos/fisiología , Adenohipófisis/fisiología , Animales , Galanina , Hormona del Crecimiento/sangre , Masculino , Prolactina/sangre , Ratas , Ratas Endogámicas , Tirotropina/sangreRESUMEN
Prostacyclin (PGI2) or its stable metabolite, 6-keto-PGF1 alpha (1-5 micrograms) in 2.5 microliter 0.05 M phosphate buffer (pH 7.4), was injected into the third ventricle (3 V) of ovariectomized (OVX), freely moving rats. Control animals received 2.5 microliter of buffer. In the initial experiments a control blood sample was taken and then the PGI2 was injected and frequent samples taken thereafter. With this protocol injection of 2 micrograms of PGI2 produced a significant decrease in mean plasma LH only at 60 min after its injection (p less than .05), while the higher dose (5 micrograms) decreased plasma LH concentrations at 30 and 60 min (p less than .01 and p less than .001, respectively). In subsequent experiments, blood was removed from indwelling external jugular vein cannulae every 5-6 min during 2 hours and plasma LH and PRL levels were determined by radioimmunoassay. LH pulses were monitored and several parameters of LH pulsation were calculated during the hour before and after injection of phosphate buffer, PGI2 or 6-keto-PGF1 alpha. Intraventricular injection of phosphate buffer failed to modify the characteristic pulsatile release of LH and did not alter plasma PRL levels. The amplitude of LH pulses was significantly reduced by PGI2 and the inhibitory effect was dose-related. Even a dose of 1 microgram produced a significant reduction in pulse height and the response was graded with maximal reduction occurring with the 5 microgram dose which essentially abolished the LH pulses. Following the microinjection of 6-keto-PGF1 alpha, no significant changes were observed in plasma LH values and the pulses of the hormone. Five micrograms PGI2 considerably elevated plasma PRL values during the 20-25 min following its 3V injection, whereas the same dose of 6-keto-PGF1 alpha produced only a very slight stimulatory effect. Since PGI2 had no effect to alter LH release by cultured pituitary cells in vitro, it is concluded that PGI2 can act on structures near the 3V to inhibit pulsatile release of LHRH.
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Ventrículos Cerebrales/fisiología , Epoprostenol/farmacología , Hormona Luteinizante/metabolismo , Ovariectomía , Prolactina/metabolismo , 6-Cetoprostaglandina F1 alfa/administración & dosificación , 6-Cetoprostaglandina F1 alfa/farmacología , Animales , Ventrículos Cerebrales/efectos de los fármacos , Epoprostenol/administración & dosificación , Femenino , Inyecciones Intraventriculares , Cinética , Hormona Luteinizante/sangre , Prolactina/sangre , Ratas , Ratas EndogámicasRESUMEN
The presence of FMRFamide (Phe-Met-Arg-Phe-amide)-like immunoreactivity in neuronal elements in the hypothalamus suggested a role for this in the hypothalamic control of the anterior pituitary function. We report here the action of FMRFamide on growth hormone release following intracerebroventricular administration to rats. The injection of 200 ng (313.8 picomoles) of FMRFamide (in 2 ul) produced a significantly increased plasma GH 15 min after injection. The GH-increasing effect of 400-800 ng (627-1255 picomoles) of FMRFamide was already developed after 5 min and lasted up to 30 min. No change was detected in the plasma FSH, LH and prolactin levels at any time during the experimental period. The intravenous administration of 10, 30 or 100 ug of FMRFamide had no effect on the plasma GH level. We conclude that FMRFamide can act at low doses to increase GH release through the inhibition of somatostatin release or the stimulation of GRF. We could not exclude a direct site of action in the pituitaries.
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Hormona del Crecimiento/metabolismo , Neuropéptidos/farmacología , Adenohipófisis/metabolismo , Animales , FMRFamida , Femenino , Hipotálamo/efectos de los fármacos , Inyecciones Intraventriculares , Ovariectomía , Ratas , Ratas Endogámicas , Estimulación QuímicaRESUMEN
The effects of different doses of human pancreatic polypeptide (HPP) injected into the third ventricle was studied on plasma follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl) and somatotropin (GH) in freely moving ovariectomized rats. Two hundred ng of HPP produced a significant decrease in plasma LH at 15, 30, and 60 min following microinjection. The LH-lowering effect of 400 and 800 ng of HPP developed at 5 min and persisted throughout the experiment. The strongest inhibition was observed at 15 and 30 min. No change in plasma FSH was detected at any time during the experimental period. Two hundred and 400 ng of HPP failed to influence the plasma Prl, while 800 ng resulted in a moderate but significant decrease in plasma Prl levels at 15 and 30 min following injection. Intraventricular microinjection of 400 ng of HPP decreased the GH level at 15 min and 800 ng caused a more pronounced decrease which was significant at 15, 30, and 60 min after the injection. The study suggested that HPP, either from the periphery if it can pass the blood brain barriers or produced in the brain, can influence pituitary function.
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Gonadotropinas Hipofisarias/metabolismo , Hormona del Crecimiento/metabolismo , Polipéptido Pancreático/farmacología , Adenohipófisis/metabolismo , Animales , Femenino , Hormona Folículo Estimulante/metabolismo , Humanos , Hormona Luteinizante/metabolismo , Prolactina/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Galanin is a 29 amino acid peptide that was isolated and characterized from porcine intestinal extracts. The presence of galanin-like immunoreactivity in neuronal elements in the hypothalamus and median eminence suggested a role for it in the hypothalamic control of anterior pituitary function. A hypothalamic site of action of galanin to stimulate growth hormone (GH) release is suggested by our observation that doses as low as 50 picomoles when infused into the third cerebroventricle of conscious, unrestrained ovariectomized rats resulted in significantly elevated plasma levels of GH. This effect was specific for GH and was dose-related. The failure of galanin to alter GH release from dispersed, cultured anterior pituitary cells in vitro further suggests that endogenous galanin plays a neuromodulatory role at the level of the median eminence, possibly affecting the release of known GH-releasing and/or inhibiting factors.
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Hormona del Crecimiento/metabolismo , Sistema Hipotálamo-Hipofisario/fisiología , Péptidos/farmacología , Adenohipófisis/metabolismo , Animales , Ventrículos Cerebrales/efectos de los fármacos , Ventrículos Cerebrales/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Galanina , Hormona del Crecimiento/sangre , Inyecciones Intraventriculares , Ovariectomía , Péptidos/administración & dosificación , Adenohipófisis/efectos de los fármacos , Prolactina/sangre , Ratas , Ratas EndogámicasRESUMEN
Several members of the secretin family of hormones have been demonstrated to alter anterior pituitary hormone secretion. Here we report the action of gastric inhibitory polypeptide (GIP) on gonadotropin and somatotropin release. Intraventricular injection of 1 microgram (0.2 nmole) GIP (2.5 microliters) produced a significant decrease in plasma FSH at 30 (p less than 0.02) and 60 min after its injection (p less than 0.01). The FSH-lowering effect of a higher dose of 5 micrograms (1 nmole) of GIP was already developed at 15 min (p less than 0.01) and was prolonged until the end of the experiment (60 min, p less than 0.05). No change in plasma LH was detected at any time during the experimental period. If 5 micrograms of estradiol-benzoate were given SC 48 hr prior to experiment, the initial values of FSH and LH were markedly decreased. In these animals GIP failed to influence plasma FSH and LH. When dispersed anterior pituitary cells from OVX rats were cultured overnight and incubated in vitro with GIP, the peptide was found to induce both FSH and LH release. Highly significant release occurred with the lowest dose tested of 10(-7) M and there was a dose-response effect for both hormones. The slope of the dose-response curve was similar for both FSH and LH release. GIP was less potent than LHRH which produced a greater stimulation of both FSH and LH release at a dose of 10(-9) M than did 10(-7) M GIP. The two peptides had an additive effect on the release of both FSH and LH.(ABSTRACT TRUNCATED AT 250 WORDS)
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Polipéptido Inhibidor Gástrico/farmacología , Hormonas Gastrointestinales/farmacología , Adenohipófisis/efectos de los fármacos , Hormonas Adenohipofisarias/metabolismo , Animales , Castración , Estradiol/farmacología , Femenino , Polipéptido Inhibidor Gástrico/administración & dosificación , Inyecciones Intraventriculares , Adenohipófisis/metabolismo , Ratas , Ratas Endogámicas , Tasa de Secreción/efectos de los fármacosRESUMEN
Prostacyclin (PGI2) (1-5 micrograms in 3 microliters 0.05 M Tris/HCl buffer, pH 7.5) and its stable metabolite, 6-oxo-PGF1 alpha, were microinjected into the third ventricle of ovariectomized rats, and plasma FSH, GH, PRL, and TSH levels were measured by RIA. Control animals received 3 microliters buffer. Injection of 5 micrograms PGI2 dramatically elevated plasma PRL values (4- to 5-fold) at 5 and 15 min, whereas the same dose of 6-oxo-PGF1 alpha produced a significant but smaller (2-fold) stimulatory effect. A delayed increase (1.5-fold) in plasma GH occurred after intraventricular PGI2 at 30 and 60 min. 6-Oxo-PGF1 alpha failed to alter GH levels. There were no alterations in plasma FSH and TSH after intraventricular injection of PGI2. Dispersed, overnight cultured cells from anterior pituitaries of ovariectomized rats were tested with 10(-4)-10(-7) M PGI2 and its metabolite. After 15 min of incubation, 3 X 10(-5) PGI2 produced a highly significant elevation in GH release (P less than 0.001), whereas there was no alteration in PRL levels. Only pharmacological doses of 6-oxo-PGF1 alpha (10(-4) M) stimulated GH release. There was no alteration in PRL release by the cultured cells even in the presence of 10(-4) PGI2. These results suggest that PGI2 stimulates PRL release by a hypothalamic action either to increase the release of PRL-releasing factor, or to decrease release of PRL-inhibiting factor, or by both mechanisms. The delayed stimulatory effect of PGI2 on the release of GH may be exerted via an effect on the anterior lobe itself, since PGI2 was effective in stimulating GH release by the incubated pituitary cells.
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6-Cetoprostaglandina F1 alfa/farmacología , Epoprostenol/farmacología , Hormona del Crecimiento/metabolismo , Hipotálamo/metabolismo , Adenohipófisis/metabolismo , Prolactina/metabolismo , 6-Cetoprostaglandina F1 alfa/administración & dosificación , Animales , Células Cultivadas , Epoprostenol/administración & dosificación , Femenino , Hormona Folículo Estimulante/sangre , Hormona del Crecimiento/sangre , Inyecciones Intraventriculares , Prolactina/sangre , Ratas , Ratas Endogámicas , Tirotropina/sangreRESUMEN
The synthesis of prostacyclin and prostaglandins was examined in isolated blood-free brain capillaries of guinea-pigs and rats using 1-14C-arachidonic acid as a precursor. The main prostaglandins synthesized by guinea-pig microvessels were prostaglandin D2 and prostaglandin E2. Substantially less prostaglandin F2 alpha or the prostacyclin stable metabolite, 6-oxo-prostaglandin F1 alpha was synthesized. Rat capillary prostaglandin distribution differed substantially from that of the guinea-pigs although the principle prostaglandin was also PGD2. Total prostaglandin conversion was greater in guinea-pig capillaries than in the rat. Norepinephrine stimulated the prostaglandin forming capacity of blood free cerebral microvasculature of guinea-pigs. Prostacyclin and prostaglandins could be involved in the activity dependent regulation of regional cerebral blood flow and permeability.
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Capilares/metabolismo , Corteza Cerebral/irrigación sanguínea , Epoprostenol/biosíntesis , Prostaglandinas/biosíntesis , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Capilares/efectos de los fármacos , Femenino , Glutatión/farmacología , Cobayas , Masculino , Norepinefrina/farmacología , Ratas , Especificidad de la EspecieRESUMEN
A high amount of histamine was found in capillaries isolated by subcellular fractionation from the brain. In view of the important effects of histamine on vascular permeability in peripheral vessels, it was thought that histamine had a similar function in the cerebral vasculature. Intracarotic histamine infusion resulted in an enhanced pinocytosis of endothelial cells and te oedematous swelling of the astrocytic end-feet system. [3H]-Histamine, injected in the cerebral ventricles, accumulated in the capillary wall. Histamine and cimetidine activated hydroxyfatty acid and prostaglandin D2 synthesis in isolated brain capillaries. The possible function of the capillary histamine in the regulation of permeability of microvessels is discussed.