RESUMO
We studied ntimicrobial activity of L. plantarum strain against different pathogens: Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus. It was shown that supernatant of 48-h L. plantarum culture in liquid nutrient medium exhibits inhibitory activity against gram-positive and gram-negative pathogenic microorganisms. Supernatant of 24-h culture exhibited lower activity, while supernatant of 72-h culture produced no inhibitory effect. Boiling and proteinase K treatment did not affect activity of the preparation, i.e. antimicrobial activity of the supernatant was not associated with protein or peptide component. These data were confirmed by the results observed after ultrafiltration of the preparation: the growth of E. coli, P. aeruginosa, and S. aureus was inhibited by the low-molecular-weight fraction, but not high-molecular-weight fraction of the supernatant. On the other hand, the high-molecular-weight fraction suppressed the growth of streptococcus by 3 times. We hypothesized that L. plantarum supernatant obtained in our experiments contained at least two antimicrobial components with different molecular weights.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Lactobacillus plantarum/citologia , Lactobacillus plantarum/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/patogenicidade , Técnicas Bacteriológicas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Escherichia coli , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Staphylococcus aureus , Streptococcus pyogenesRESUMO
OBJECTIVE: The purpose of the present investigation was to compare calcium sensitivity of contractile machinery in aorta and portal vein smooth muscle cells (SMC) in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive Okamoto rats (SHR), and to shed light upon the mechanisms of possible differences. DESIGN: Investigations into calcium sensitivity of SMC myofilaments can only be made on skinned muscular strips. METHODS: The vascular strips were made hyperpermeable by detergent skinning with saponin. The isometric calcium-induced contractions of SMC were recorded using a force displacement transducer coupled to a physiograph. RESULTS: It was shown that the pCa-tension (negative logarithm of calcium concentration versus tension) relationship for aorta and portal vein SMC in SHR shifted to the left in comparison with WKY rats. Putative protein kinase C inhibitors 1-(S-isoquionolinyl-sulfonyll)-2-methylpiperasine (H-7) and polymyxin B shifted the pCa-tension relationship more significantly to the right in the SMC of SHR than in WKY rats. It has also been shown that H-7 and polymyxin B sharply reduced the maximum tension developed by SMC in SHR whilst causing a non-significant decrease in maximum tension of SMC from WKY rats. These results are consistent with higher protein kinase C activity in SMC of SHR. CONCLUSION: These results indicate that the increase in calcium sensitivity of vascular SMC contractile machinery in SHR may be linked with the increase in their protein kinase C activity.
Assuntos
Cálcio/farmacologia , Hipertensão/genética , Contração Muscular/fisiologia , Proteínas Musculares/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Proteína Quinase C/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Animais , Hipertensão/fisiopatologia , Isoquinolinas/farmacologia , Músculo Liso Vascular/fisiologia , Piperazinas/farmacologia , Polimixina B/farmacologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
OBJECTIVE: The present study was designed to examine, using isolated preparations of thoracic aorta, the effects of artificial phosphatidylcholine vesicles (liposomes) on vascular endothelium-dependent responses in spontaneously hypertensive rats (SHR) compared with those in Wistar-Kyoto (WKY) normotensive rats. DESIGN: Phosphatidylcholine liposomes, which possess the ability to repair the plasma membrane of living cells, were used in these experiments. METHODS: Liposomes were prepared from egg phosphatidylcholine. A suspension of lipid was subjected to ultrasound treatment at 20 degrees C at a frequency of 44 kHz for 45 s. The contraction of vascular smooth muscle was recorded using a force-displacement transducer coupled with a physiograph. RESULTS: It was shown that aortic smooth muscle from SHR demonstrated a loss of endothelium-dependent relaxation in acetylcholine (10(-6) mol/l) compared with WKY rat aortic smooth muscle. Liposomes in a concentration of 100-125 micrograms/ml restored these endothelium-dependent responses more effectively than L-arginine (10(-5) to 10(-4) mol/l), which is known to be a precursor of endothelium-derived relaxing factor (EDRF). This effect was not observed in denuded aortic rings, and liposomes lost their ability of repairing endothelium-dependent vascular relaxant responses in the presence of methylene blue (5 x 10(-5) mol/l), which inhibits soluble guanylyl cyclase activation by nitric oxide (NO), and N omega-nitro-L-arginine (L-NNA, 5 x 10(-5) to 10(-4) mol/l), a potent and selective inhibitor of NO synthase. CONCLUSION: The present results suggest that the loss of vascular endothelium-dependent responses in SHR may be, at least partly, due to endothelial cells membrane damage, and that the phosphatidylcholine liposomes can repair the function of endothelial cells and restore synthesis or release, or both, of EDRF in hypertension.
Assuntos
Acetilcolina/farmacologia , Endotélio Vascular/fisiologia , Hipertensão/fisiopatologia , Lipossomos/farmacologia , Fosfatidilcolinas/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica/fisiopatologia , Técnicas In Vitro , Óxido Nítrico/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca2+]i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca2+]i and contractile force were done. All vascular rings were precontracted either using a high-K+-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC50 for GTN under K+ stimulation of VSM comprised (2.48 +/- 1.6) x 10(-5) M, whereas the mean EC50 under PE stimulation was (3.05 +/- 2.3) x 10(-4) M (p < 0.05, n = 9). The EC50 for SNP under K+ stimulation of VSM comprised (1.09 +/- 0.47) x 10(-7) M, whereas the EC(50) under PE stimulation was (8.01 +/- 2.4) x 10(-6) M (p < 0.05, n = 9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca2+]i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca2+]i. The main peculiarity of SNP action under K+ stimulation as compared to PE stimulation was the transient decrease in [Ca2+]i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K+ depolarization than that seen under PE stimulation, indicating that Ca2+ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca2+ entry through receptor- operated calcium channels or intracellular Ca2+ release, or both.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Doadores de Óxido Nítrico/administração & dosagem , Animais , Canais de Cálcio/efeitos dos fármacos , Relação Dose-Resposta a Droga , Soluções Isotônicas/administração & dosagem , Masculino , Modelos Animais , Modelos Cardiovasculares , Contração Miocárdica/efeitos dos fármacos , Nitroglicerina/administração & dosagem , Nitroprussiato/administração & dosagem , Fenilefrina/administração & dosagem , Canais de Potássio/administração & dosagem , Ratos , Ratos Wistar , Estimulação Química , Grau de Desobstrução Vascular/efeitos dos fármacos , Vasoconstritores/administração & dosagem , Vasodilatadores/administração & dosagemRESUMO
The protective action of passive saline filled ("empty") phosphatidylcholine liposomes (PCL) on endothelial function was examined in thoracic aortas obtained from gamma irradiated (6 Gy) Chinchilla rabbits, and then verified in experiments on non-anesthetized and anesthetized rats. Acetylcholine (ACh)-induced vascular relaxant responses in isolated vascular tissues rats were used as the test of endothelial integrity and its functional ability. It was shown that when added to the bath solution (100 microg/ml), PCL effectively restored endothelium-dependent ACh relaxations of isolated vascular rings damaged resulting from gamma-irradiation but had no effect on endothelium-independent vascular responses to therapeutic nitric oxide (NO) donors. The liposomes were also without protective effect when injected to the rabbits intraperitoneally (30 mg/kg) 1 hour before irradiation. In contrast, PCL, being injected at the same dose 1 hour after radiation impact, promote normalization of both endothelium-dependent vascular responses to ACh and nitric oxide (NO) donors. PCL restored also the sensitivity of vascular tissues to authentic NO (aqueous NO solution) that was surprisingly increased after irradiation, and normalized relationship between ACh-stimulated NO release and relaxant response amplitudes in irradiated aortas. Experiments on non-anesthetized and anesthetized rats demonstrated that irradiation led to significant elevation in the level of arterial blood pressure without any changes in cardiac contractility. PCL administration (25 mg/kg, i.v.) effectively normalized an increased arterial blood pressure in irradiated animals. In conclusion, it appears that PCL due to its ability to normalize NO-dependent vascular tone control mechanisms might be worthwhile therapeutic approach in case of ionizing irradiation accident. These result support the concept that the depression of endothelium-dependent vascular responses after irradiation may be result of decreased NO bioavailability due to its conversion to less potent vasodilators during irradiation-induced oxidative attack.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/efeitos da radiação , Raios gama , Fosfatidilcolinas/administração & dosagem , Cloreto de Sódio/farmacologia , Acetilcolina/farmacologia , Adulto , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Aorta Torácica/efeitos da radiação , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/efeitos da radiação , Endotélio Vascular/fisiologia , Humanos , Técnicas In Vitro , Lipossomos , Pessoa de Meia-Idade , Doadores de Óxido Nítrico/farmacologia , Coelhos , Ratos , Ratos Endogâmicos WKY , Recuperação de Função Fisiológica , Cloreto de Sódio/química , Vasoconstrição , Vasodilatadores/farmacologiaRESUMO
The effect of platelet-activating factor (PAF) on the development of isometric tension by saponin-skinned coronary artery was studied. PAF caused two types of contractions of coronary smooth muscle cells (SMC): (i) rapid, transient (phasic) contractions of SMC were induced by inositol 1,4,5-trisphosphate dependent Ca2+ release from sarcoplasmic reticulum (SR); (ii) slow sustained (tonic) contractions were induced by increase in Ca(2+)-sensitivity of the contractile apparatus of SMC by protein kinase C activation. The present results support the hypothesis that, in SMC of coronary artery, PAF receptors are located not only on the plasma membrane, but also on the SR membranes.
Assuntos
Vasos Coronários/fisiologia , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Fator de Ativação de Plaquetas/farmacologia , Animais , Cálcio/farmacologia , Cálcio/fisiologia , Vasos Coronários/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Saponinas , Retículo Sarcoplasmático/fisiologia , SuínosRESUMO
The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30µM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5mg/kg) 30min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine, known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously (70mg/kg). We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hipóxia/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Tionas/farmacologia , Fosfolipases Tipo C/metabolismo , Aminoquinolinas/farmacologia , Animais , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/farmacologia , Indóis/farmacologia , Injeções Intravenosas , Masculino , Norbornanos , Fosforilcolina/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Artéria Pulmonar/metabolismo , Ratos , Ratos Wistar , Tiocarbamatos , Tionas/administração & dosagem , Fosfolipases Tipo C/antagonistas & inibidores , Vasoconstrição/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity. EXPERIMENTAL APPROACH: Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries. KEY RESULTS: Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers. CONCLUSIONS AND IMPLICATIONS: We conclude that in DM enhanced myofilament Ca(2+) sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.
Assuntos
Artérias/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Quinase C/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Artérias/fisiopatologia , Benzofenantridinas/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Fenilefrina/farmacologia , Ratos , Ratos Sprague-Dawley , EstreptozocinaRESUMO
The cellular mechanisms underlying smooth muscle hypoxic relaxation were examined in experiments on isolated chemically skinned muscle strips of rat portal vein. Low PO2 (3.9 kPa) shifted the pCa-tension relation to the right by 0.29 +/- 0.01 pCa units as compared to control curves (PO2, 18.8 kPa). Thus the Ca2+ sensitivity of the filaments had decreased. Addition of cyclic AMP (30 mumol l-1) with theophylline (5 mmol l-1) to the buffer solution produced a similar shift to low PO2. Low PO2 also decreased the maximal force to 40-50% of control. Inclusion of sodium fluoride (10 mmol l-1) and aluminium chloride (10 mumol l-1) in a bath solution caused partial (25-30%) relaxation of skinned smooth muscle preconstricted with 10 mumol l-1 Ca2+. The rate and amplitude of smooth muscle relaxation at low PO2 were significantly decreased by tolbutamide (5 mmol l-1), which is known to inhibit cyclic AMP-dependent protein kinases. We suggest that PO2 changes can alter myofilament responsiveness to Ca2+ and this effect may be related to cyclic AMP-dependent phosphorylation of myosin light chain kinase, its inactivation and subsequent uncoupling between Ca2+ and contractile machinery in smooth muscle.
Assuntos
Citoesqueleto de Actina/fisiologia , Compostos de Alumínio , Cálcio/farmacologia , Hipóxia/fisiopatologia , Músculo Liso Vascular/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Alumínio/farmacologia , Cloreto de Alumínio , Animais , Cloretos/farmacologia , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Fluoreto de Sódio/farmacologia , Teofilina/farmacologia , Tolbutamida/farmacologiaRESUMO
The goal of the present study was to evaluate the role of protein kinase C (PKC) in the depression of endothelium-dependent vacular response in spontaneously hypertensive Okamoto rats (SHR). Aortae from SHR demonstrated a decreased relaxant response to acetylcholine (Ach) as compared to aortae from normotensive Wistar-Kyoto (WKY) rats, while papaverine lowered the force of aorta to a similar degree in both strains of rats. PKC inhibitors, H-7 (5 x 10(-6) M) and chelerythrine chloride (10(-6) M), produced a greater decrease in the force developed by the aortae from SHR vs. WKY rats both in intact and chemically permeabilized tissues. In SHR aortae PKC inhibitors enhanced relaxation to Ach to a greater extent as compared to WKY aortae. Furthermore, in the presence of PKC inhibitors, the constrictor responses of SHR aortae to Ach were transformed into relaxant responses, and the concentration-response curve to Ach was shifted to the left. The sensitivity of aortae from SHR to authentic nitric oxide (NO) was lowere compared to WKY rats. EC50s for authentic NO in SHR and WKY rat aortae were different: -2.9 +/- 0.15 x 10(-6) M and 4.58 +/- 0.1 x 10(-7) M (n = 15, p < 0. 001), respectively. Bioassay experiments using SHR aortae showed that the addition of chelerythrine (10(-6) M) to the detector superfusate caused relaxation during treatment of the donor segment with Ach, indicating that the sensitivity of the aortae to NO had been restored. When SHR detector ring was substituted for denuded aortae from WKY rats and PKC inhibitors were not added to the detector superfusate, the relaxation of the detector aortae was also close to the normal Ach-induced relaxation. WKY aortae demonstrated a positive relationship between Ach-stimulated NO release and relaxant response amplitudes (correlation coefficient r = 0.905, p < 0.001, n = 10). In contrast, there was a significant negative correlation in SHR aortae (r = -0.712, p < 0.05, n = 10). Detection of NO release by chemiluminescence showed no significant difference in NO release in SHR and WKY aortae. Taken together, these data suggest that the blunted endothelium-dependent relaxations seen in SHR aortae are mainly due to a decreased sensitivity of vascular smooth muscle to EDRF/NO resulting from an increased PKC activity.