RESUMO
Diabetes mellitus and hypertension are diseases that are strongly correlated. A major factor in this correlation is the renin-angiotensin system (RAS), with the peptide angiotensin II being a key component. This study analyzed the impact of Angiotensin Type 1 receptor (AT1R) and Angiotension Type 2 receptor (AT2R) in atrial function. MAIN METHODS: To perform the experiments, Wistar Kyoto rats (WKY), diabetic streptozotocin-induced WKY rats and spontaneously hypertensive rats (SHR) were used, and stimulation of cardiovascular function was done by means of the following drugs: angiotensin II, novokinin and the antagonists losartan and PD123177. We also measured the systolic blood pressure (SBP). RESULTS: An increase in AT1R function was observed in diabetic and hypertensive rats (18% in right atria [RA] and 11% in left atria [LA]). We also observed an increase in calcium release from the endoplasmic reticulum in right atria of diabetic rats (31%) and in right atria of hypertensive rats (35%). On the other hand, a decreased response of AT2R in diabetic and hypertensive rats was observed, this decreased response was greater in hypertensive rats (RA, 10%; LA, 12%). These results have demonstrated a dysfunction of the RAS that may contribute to the common dysfunctions of the cardiovascular system in diabetic and hypertensive rats.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Átrios do Coração/fisiopatologia , Contração Muscular , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Pressão Sanguínea , Diabetes Mellitus Experimental/metabolismo , Ratos , Ratos Endogâmicos SHRRESUMO
Background: Blockage of the Na+/Ca2+ exchanger (NCX) is used to determine the role of NCX in arrhythmogenesis. Trisulfated heparin disaccharide (TD) and Low Molecular Weight Heparins (LMWHs) can directly interact with the NCX and accelerate its activity. Objective: In this work, we investigated the antiarrhythmic effect of heparin oligosaccharides related to the NCX activity. Methods: The effects of heparin oligosaccharides were tested on the NCX current (patch clamping) and intracellular calcium transient in rat cardiomyocytes. The effects of heparin oligosaccharides were further investigated in arrhythmia induced in isolated rat atria and rats in vivo. Results: The intracellular Ca2+ concentration decreases upon treatment with either enoxaparin or ardeparin. These drugs abolished arrhythmia induction in isolated atria. The NCX antagonist KB-R7943 abolished the enoxaparin or ardeparin antiarrhythmic effects in isolated atria. In the in vivo measurements, injection of TD 15 min both before coronary occlusion or immediately after reperfusion, significantly prevented the occurrence of reperfusion-induced arrhythmias (ventricular arrhythmia and total AV block) and reduced the lethality rate. The patch clamping experiments showed that, mechanistically, TD increases the forward mode NCX current. Conclusion: Together, the data shows that heparin oligosaccharides may constitute a new class of antiarrhythmic drug that acts by accelerating the forward mode NCX under calcium overload.
RESUMO
Comorbidity of diabetes and hypertension is frequent. Here, we have performed a comparative study in three animal models namely, normotensive Wistar Kyoto (WKY) rats, streptozotocin-induced diabetic rats (STZ), and spontaneously hypertensive rats (SHR). With respect WKY rats, we have found the following alterations in adrenal chromaffin cells from STZ and SHR rats: (1) diminished Ca2+ currents; (2) augmented [Ca2+]c elevations and catecholamine release in cells stimulated with angiotensin II or high K+; (3) unchanged expression of angiotensin II receptors AT1 and AT2; (4) higher density of secretory vesicles at subplasmalemmal sites; (5) mitochondria with lower cristae density that were partially depolarized; and (6) lower whole cell ATP content. These alterations may have their origin in (i) an augmented capacity of the endoplasmic reticulum [Ca2+] store likely due to (ii) impaired mitochondrial Ca2+ uptake; (iii) augmented high-[Ca2+]c microdomains at subplasmalemmal sites secondary to augmented calcium-induce calcium release and to inositol tris-phosphate receptor mediated enhanced Ca2+ mobilization from the endoplasmic reticulum; and (iv) augmented vesicle pool. These alterations seem to be common to the two models of human hypertension here explored, STZ diabetic rats and SHR hypertensive rats.
Assuntos
Sinalização do Cálcio , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Células Cromafins/patologia , Diabetes Mellitus Experimental/patologia , Mitocôndrias/patologia , Animais , Contagem de Células , Masculino , Ratos , Ratos Endogâmicos SHR , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
It is known that red wine has cardioprotective properties. However, its influence is unknown about purinergic system. Therefore, we study the influence of the treatment with red wine or ethanol in purinergic neurotransmission. We used Wistar Kyoto rats (WKY), diabetic streptozotocin-induced WKY and spontaneously hypertensive rats (SHR), treated with red wine (12.5%) or ethanol (12.5%). The cardiovascular function stimulated with purinergic agonists and systolic blood pressure (SBP) was assessed. In atria of diabetics and SHRs, the P1 receptor response was decreased, unlike the P2 receptor response was increased. Likewise, in aorta the affinity to adenosine (ADO) was decreased from SHRs and diabetics. Furthermore, the P2X function was increased just SHRs. All these alterations were improved after treatment with red wine, resulting in reduction of SBP from diabetics and SHRs, but not when treated with ethanol. This study has important implications, because it is shown that consumption of red wine can improve cardiovascular system by purinergic neurotransmission.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Hipertensão/tratamento farmacológico , Receptores Purinérgicos/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Vitis , Vinho , Adenosina/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Etanol/farmacologia , Hipertensão/etiologia , Hipertensão/metabolismo , Masculino , Preparações de Plantas/farmacologia , Preparações de Plantas/uso terapêutico , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
In the normotensive rat atrium, adenosine-5'-triphosphate and uridine-5'-triphosphate exert a biphasic effect consisting of an initial negative inotropic effect (NIE) followed by a subsequent positive inotropic effect (PIE). We comparatively studied these responses in normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Compared with NWRs, the NIE responses in the atria were lower and the PIE responses were higher in SHRs. The P1 purinoceptor antagonist, D 8-cyclopentyl-1,3-dipropylxanthine, partially blocked the NIE responses of both ATP and UTP and mildly enhanced the PIE responses in both NWRs and SHRs. Furthermore, the P2 purinoceptor blockers suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium salt induced a pronounced block of the PIE responses in both atria types. The PIE responses to ATP were inhibited more efficiently by nifedipine. These responses were depressed by ryanodine and, to a lesser extent, carbonyl cyanide 3-chlorophenylhydrazone in SHR atria compared with NWR atria. The higher responses in SHR rats suggest the existence of an augmented endoplasmic reticulum Ca(2+) store and faster mitochondrial Ca(2+) cycling in SHR atria compared with NWR atria. These data support the hypothesis that a dysfunction of purinergic neurotransmission and enhanced sympathetic activity are contributing factors in the pathogenesis of hypertension.
Assuntos
Átrios do Coração , Hipertensão/fisiopatologia , Contração Miocárdica/fisiologia , Receptores Purinérgicos P1/fisiologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Mitocôndrias/metabolismo , Nifedipino/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Antagonistas do Receptor Purinérgico P2/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Rianodina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Diabetes augments the risk of hypertension. Although several factors have been implicated in the development of such hypertensive state, we designed this study to investigate blood pressure development, the activity of angiotensin-converting enzyme (ACE) in blood as well as sympathetic neurotransmission in the vas deferens of diabetic rats. We used streptozotocin (STZ)-induced diabetic rats (60 mg/kg) in order to evaluate the systolic blood pressure (SBP), ACE activity and peripheral sympathetic neurotransmission. We observed the following changes of parameters: increase of SBP, decrease of heart rate, augmentation of plasma ACE activity, enhancement of phasic and tonic vas deferens contractions elicited by electrical stimulation at 5 Hz, increase of maximal response to noradrenaline (NA) and decrease of adenosine triphosphate (ATP)-elicited contraction of vasa deferentia. The results reveal that in the development of hypertension in diabetic rats, augmentation of circulating ACE activity precedes the sympathetic dysfunction. Additionally, it seems that the purinergic and noradrenergic neurotransmission is compromised.
Assuntos
Diabetes Mellitus Experimental/complicações , Hipertensão/etiologia , Músculo Liso/inervação , Peptidil Dipeptidase A/sangue , Sistema Nervoso Simpático/fisiopatologia , Ducto Deferente/inervação , Animais , Pressão Sanguínea , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Frequência Cardíaca , Hipertensão/sangue , Hipertensão/enzimologia , Hipertensão/fisiopatologia , Masculino , Contração Muscular , Fármacos Neuromusculares/farmacologia , Ratos , Estreptozocina , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Transmissão Sináptica , Sístole , Fatores de Tempo , Regulação para CimaRESUMO
Enhanced activity of the sympatho-adrenal axis and augmented circulating catecholamines has been implicated in the development of hypertension. Release of catecholamine from stimulated adrenal medulla chromaffin cells has been shown to be higher and longer in spontaneously hypertensive rats (SHRs), compared with normotensive Wistar rats (NWRs). Whether differences in the functional expression of voltage-dependent calcium channels (VDCCs) of the L-, N-, or P/Q subtypes may contribute to such distinct secretory behaviour, is unknown. We therefore approached here this study in voltage-clamped NWR and SHR chromaffin cells, using 10mM Ba(2+) as charge carrier (IBa) and selective blockers of each channel type. We found that compared with NWR cells, SHR chromaffin cells exhibited the following differences: (1) 30% diminution of the IBa fraction carried by L channels; (2) a doubling of the IBa fraction carried by P/Q channels; (3) more visible current modulation by ATP that could be linked to a 10-fold higher mRNA levels for purinergic receptors of the P2Y2 subtype; and (3) a higher contribution of PQ channels to the transients of the cytosolic calcium concentrations ([Ca(2+)]c) generated by K(+), compared with L channels. These results may contribute to the better understanding of the greater calcium signalling and exocytotic responses of SHR compared with NWR chromaffin cells, found in three previous reports from our laboratories.
Assuntos
Medula Suprarrenal/fisiologia , Canais de Cálcio/fisiologia , Células Cromafins/fisiologia , Hipertensão/metabolismo , Trifosfato de Adenosina/farmacologia , Medula Suprarrenal/citologia , Medula Suprarrenal/efeitos dos fármacos , Animais , Bário/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Potássio/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Receptores Purinérgicos P2Y2/genéticaRESUMO
Previous studies conducted in our laboratory indicated that administration of amphetamine, fluoxetine or sibutramine affects the sympathetic nervous system of the rat vas deferens. Therefore, our goal was to verify the role of calcium in vasa deferentia from young rats pretreated with a single dose of these drugs. Young 40-day-old male Wistar rats were pretreated with amphetamine 3 mg/kg, fluoxetine 10 mg/kg or sibutramine 6 mg/kg for 4 h before the experiments. CaCl(2) (10 mM) was used to induce contraction through time-effect curves in calcium-free solution to measure phasic and tonic components. We also evaluated the calcium-induced fluorescence of vas deferens cut into thin slices. In rats pretreated with amphetamine, we found an increase of the tonic contraction component which was reduced by verapamil. The phasic and tonic responses were increased in the group treated with fluoxetine, but only the tonic response was more sensitive to the antagonism by verapamil. The group treated with sibutramine showed an increase of phasic response whereas the tonic component was decreased. In this group an increase of the affinity for verapamil antagonism was found. In the calcium fluorescence study it was observed that the group treated with amphetamine, fluoxetine or sibutramine showed higher basal Ca(2+) fluorescence after stimulus with KCl (70 mM), noradrenaline (10(-4)M) or acetylcholine (10(-4)M). In all pretreated groups the calcium fluorescence was diminished by nifedipine 10(-7)M. Therefore, the pretreatment with amphetamine, fluoxetine or sibutramine seems to affect the calcium contractility and homeostasis in young rat vas deferens.
Assuntos
Cálcio/metabolismo , Fármacos do Sistema Nervoso Central/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Contração Muscular/efeitos dos fármacos , Ducto Deferente/citologia , Ducto Deferente/fisiologia , Anfetamina/farmacologia , Animais , Ciclobutanos/farmacologia , Fluoxetina/farmacologia , Homeostase/efeitos dos fármacos , Técnicas In Vitro , Masculino , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ratos , Ratos Wistar , Fatores de Tempo , Ducto Deferente/efeitos dos fármacosRESUMO
Cardiac dysfunctions are described in diabetes. However, the role of purinergic neurotransmission in diabetes-related cardiovascular diseases is unknown. The purpose of this study was to evaluate the purinergic neurotransmission in isolated atria from streptozotocin-induced diabetic rats. The animals were grouped as control and diabetic with 30 days (D30) and 60 days (D60) after streptozotocin-induced diabetes. The isolated left and right atria were used in functional experiments. The effects of adenosine triphosphate, uridine diphosphate, and adenosine were evaluated on atrial inotropism and chronotropism. The antagonists 8-cyclopentyl-1,3-dipropylxanthine and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate were also used, as blockers of P1 and P2 receptors, respectively. A negative inotropic effect followed by a positive inotropic effect was induced by adenosine triphosphate in isolated atria. This negative inotropic effect was decreased by 25% in left atria of D30. Additionally, the apparent affinity for adenosine was diminished in left atria of D30, suggesting changes in P1 receptor function. No changes were found in the right atria of D30 stimulated by adenosine. The left atria and right atria stimulated by uridine diphosphate showed an increased inotropic effect of 92% and 17%, respectively. No changes were observed in left and right atria of D30 stimulated by uridine diphosphate. Our data showed the involvement of purinergic neurotransmission in diabetes-related cardiovascular changes.
Assuntos
Complicações do Diabetes/fisiopatologia , Diabetes Mellitus Experimental/fisiopatologia , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Masculino , Agonistas Purinérgicos/farmacologia , Ratos , Ratos Wistar , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Estreptozocina , Fatores de Tempo , Difosfato de Uridina/farmacologiaRESUMO
We have recently synthesized a new series of hybrid compounds having the moieties of tacrine, a potent inhibitor of brain and peripheral acetylcholinesterase (AChE), and nimodipine, a blocker of L-type voltage-dependent calcium channels (VDCCs). These compounds were designed to target AChE and L calcium channels in the brain, as potential therapeutic agents in Alzheimer's disease. We performed the present study to determine the main peripheral side effects of two of these compounds, ITH12117 and ITH12118. We have here shown that in rat vas deferens these compounds inhibited AChE with a potency about 1000-fold lower than that of physostigmine or tacrine. Furthermore, the hybrid compounds enhanced contractions evoked by acetylcholine, with a potency about 100-fold lower than that of physostigmine or tacrine. Additionally, contractions induced by Ca2+ on depolarized vas deferens were blocked by nimodipine with greater efficacy, compared with ITH12117 and ITH12118. Compound ITH12118 (1 µM) caused a pronounced inhibition of the tonic (but not phasic) contraction elicited by electrical field stimulation. Furthermore, the same dose of nimodipine and ITH12118 blocked by 75% cytosolic Ca2+ elevations produced by acetylcholine, noradrenaline, or ATP. As a matter of comparison, we showed that rat brain cortex AChE was inhibited by ITH12118 with a potency 10 to 20-fold higher than that for vas deferens. This study shows that ITH12118 could be a paradigmatic multitarget compound having selective brain effects with smaller peripheral side effects. This may help to orient the search of new neuroprotective compounds with potential therapeutic application in Alzheimer's disease.
Assuntos
Acetilcolinesterase/metabolismo , Cálcio/metabolismo , Inibidores da Colinesterase/farmacologia , Contração Muscular/efeitos dos fármacos , Tacrina/farmacologia , Ducto Deferente/metabolismo , Ducto Deferente/fisiologia , Acetilcolina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Butirilcolinesterase/metabolismo , Cálcio/farmacologia , Córtex Cerebral/enzimologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Estimulação Elétrica , Fura-2/farmacologia , Humanos , Técnicas In Vitro , Masculino , Nimodipina/farmacologia , Norepinefrina/farmacologia , Fisostigmina/farmacologia , Ratos , Ratos Wistar , Ducto Deferente/citologia , Ducto Deferente/efeitos dos fármacosRESUMO
Pronounced differences in the kinetics of single-vesicle catecholamine release from adrenal chromaffin cells stimulated with acetylcholine or high potassium (K(+)) have been recently found between normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Such differences could be explained on the basis of distinct mechanisms of calcium (Ca(2+)) handling by chromaffin cells of NWRs and SHRs. We have explored here this hypothesis in adrenal medullary slices loaded with calcium fluorescent probes to measure the changes in Ca(2+) concentration in the cytosol ([Ca(2+)](c)), endoplasmic reticulum ([Ca(2+)](er)), and mitochondria ([Ca(2+)](m)). We found the following differences on calcium handling in SHRs, as compared with NWR: (i) higher basal [Ca(2+)](c) and basal [Ca(2+)](m); (ii) greater [Ca(2+)](c) elevations elicited by acetylcholine and K(+), with faster activation but slower inactivation; (iii) greater [Ca(2+)](c) elevations elicited by CRT (a mixture of caffeine, ryanodine, and thapsigargin) and by the mitochondrial protonophore FCCP (carbonylcyanide p-(trifluoromethoxy) phenylhydrazone). The higher basal [Ca(2+)](c) and [Ca(2+)](m) suggest an enhanced mitochondrial Ca(2+) uptake, and the greater [Ca(2+)](c) elevations produced by FCCP indicates a higher mitochondrial Ca(2+) release into the cytosol. This alteration of intracellular Ca(2+) movements could explain the greater quantal catecholamine release responses seen in SHRs, as compared with NWRs in previous studies. Furthermore, enhanced mitochondrial Ca(2+) cycling may be the basis for the dysfunction of mitochondrial bioenergetics, reported to be present in hypertensive states.
Assuntos
Medula Suprarrenal/citologia , Medula Suprarrenal/patologia , Cálcio/metabolismo , Citosol/metabolismo , Hipertensão/metabolismo , Hipertensão/patologia , Mitocôndrias/metabolismo , Acetilcolina/farmacologia , Medula Suprarrenal/efeitos dos fármacos , Medula Suprarrenal/metabolismo , Animais , Cafeína/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Células Cromafins/patologia , Citosol/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fura-2/metabolismo , Técnicas In Vitro , Masculino , Movimento/efeitos dos fármacos , Potássio/farmacologia , Ratos , Ratos Endogâmicos SHR , Rianodina/farmacologia , Tapsigargina/farmacologiaRESUMO
PURPOSE: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy. The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. METHODS: New Zealand rabbits (n = 10) adipose panicle (2-3 cm) were removed by lipectomy, fragmented and washed with PBS and enzymatically dissociated with trypsin/EDTA. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. RESULTS: The fat total cells (CTF) were 1.62 x10(6) cells/mL and presented 98% of viability. These cells were taken for cultivation and after 20 days were counted 2.88 x10(6) cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10(6) cells/mL MSC. CONCLUSION: The lipectomy of adipose panicule is a very satisfactory method to extract stem cells from fat, quantitatively and qualitatively.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/cirurgia , Animais , Separação Celular , Lipectomia , Modelos Animais , CoelhosRESUMO
PURPOSE: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy. The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. METHODS: New Zealand rabbits (n= 10) adipose panicle (2-3 cm) were removed by lipectomy, fragmented and washed with PBS and enzymatically dissociated with trypsin/EDTA. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. RESULTS: The fat total cells (CTF) were 1.62 x10(6) cells/mL and presented 98 percent of viability. These cells were taken for cultivation and after 20 days were counted 2.88 x10(6) cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10(6) cells/mL MSC. CONCLUSION: The lipectomy of adipose panicule is a very satisfactory method to extract stem cells from fat, quantitatively and qualitatively.
OBJETIVO: Apresentar um modelo experimental de análise qualitativa e quantitativa de células tronco mesênquimais proveniente da gordura de coelhos obtido por lipectomia. A gordura poderia ser uma grande fonte de obtenção de células tronco mesenquimais, criando condições para a reparação de tecidos lesados. MÉTODOS: Foram removidos os panículos adiposos (2-3 cm) da região cervical de Coelhos Nova Zelândia (n = 10) por lipectomia. Os panículos foram fragmentados e lavados com PBS e, posteriormente, dissociados enzimaticamente com tripsina / EDTA. As células extraídas do panículo adiposo foram incubadas em meio de cultura DMEM e após 20 dias, foi realizada uma análise quantitativa da adesão de primeira e segunda passagem das células mesênquimais em garrafas de cultura. RESULTADOS: Foram extraídas 1,62 x106 cel/ mL células totais de gordura (CTG) with 98 por cento de viabilidade. Essas células foram levadas para o cultivo e após 20 dias, foi realizada a primeira passagem (1pd) sendo quantificadas 2,88 x10(6) cel/mL células tronco mesênquimais (CTM). Na segunda passagem (2pd) foi obtido 4,28 x10(6) cel/mL CTM. CONCLUSÃO: A lipectomia do paniculo adiposo é um método muito satisfatório para extrair células tronco a partir de gordura, quantitativamente e qualitativamente.
Assuntos
Animais , Coelhos , Tecido Adiposo/citologia , Células-Tronco Mesenquimais , Tecido Adiposo/cirurgia , Separação Celular , Lipectomia , Modelos AnimaisRESUMO
Here, we present the first study on the effects of compounds that interfere with calcium (Ca(2+)) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal catecholamine release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K(+) pulses triggered spike bursts of secretion by Ca(2+) entry through Ca(2+) channels. ER Ca(2+) release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K(+), CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca(2+) handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K(+) were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca(2+) entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.
Assuntos
Catecolaminas/metabolismo , Células Cromafins/metabolismo , Retículo Endoplasmático/metabolismo , Hipertensão/metabolismo , Mitocôndrias/metabolismo , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Separação Celular , Inibidores Enzimáticos/farmacologia , Potássio/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Rianodina/farmacologia , Tapsigargina/farmacologia , Desacopladores/farmacologiaRESUMO
In a previous study performed in the intact adrenal gland (Lim et al., 2002), stimulation with acetylcholine (ACh) or high K(+) concentrations (K(+)) produced greater catecholamine release in spontaneously hypertensive rats (SHR), as compared with normotensive animals. In this study, the time course of secretion was in the range of minutes. Hence, we do not know whether enhanced release is due to greater quantal content and/or distinct kinetics in SHRs and control animals. To get insight into the mechanism involved in such enhanced catecholamine secretory responses, we performed a single-vesicle release study in primary cultures of adrenal chromaffin cells, recorded with amperometry. Cells were stimulated with 2-s pulses of 1 mM ACh or 70 mM K(+). The secretory responses to ACh or K(+) pulses in SHR cells as compared with control cells had the following characteristics: 1) double number of secretory events, 2) 4-fold augmentation of total secretion, 3) cumulative secretion that saturated slowly, 4) 3-fold higher complex events with two to four superimposed spikes that may be explained by faster spike kinetics, 5) about 2- to 3-fold higher event frequency at earlier post stimulation periods, and 6) 2- to 5-fold higher quantal content of simple spikes. We conclude that SHR cells have faster and larger catecholamine release responses, explained by more vesicles ready to undergo exocytosis and greater quantal content of vesicles. This could have relevance to further understand the pathogenic mechanisms involved in the development of high blood pressure, as well as in the identification of new drug targets to treat hypertension.