RESUMO
KCNE1 is a protein of low molecular mass that is known to regulate the chromanol 293B and clofilium-sensitive K+ channel, KCNQ1, in a number of tissues. Previous work on the kidney of KCNE1 and KCNQ1 knockout mice has revealed that these animals have different renal phenotypes, suggesting that KCNE1 may not regulate KCNQ1 in the renal system. In the current study, in vivo clearance approaches and whole cell voltage-clamp recordings from isolated renal proximal tubules were used to examine the physiological role of KCNE1. Data from wild-type mice were compared to those from KCNE1 knockout mice. In clearance studies the KCNE1 knockout mice had an increased fractional excretion of Na+, Cl−, HCO3(−) and water. This profile was mimicked in wild-type mice by infusion of chromanol 293B, while chromanol was without effect in KCNE1 knockout animals. Clofilium also increased the fractional excretion of Na+, Cl− and water, but this was observed in both wild-type and knockout mice, suggesting that KCNE1 was regulating a chromanol-sensitive but clofilium-insensitive pathway. In whole cell voltage clamp recordings from proximal tubules, a chromanol-sensitive, K+-selective conductance was identified that was absent in tubules from knockout animals. The properties of this conductance were not consistent with its being mediated by KCNQ1, suggesting that KCNE1 regulates another K+ channel in the renal proximal tubule. Taken together these data suggest that KCNE1 regulates a K+-selective conductance in the renal proximal tubule that plays a relatively minor role in driving the transport of Na+, Cl− and HCO3(−).
Assuntos
Cromanos/farmacologia , Canal de Potássio KCNQ1/metabolismo , Túbulos Renais Proximais/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sulfonamidas/farmacologia , Animais , Bário/farmacologia , Bicarbonatos/metabolismo , Cloretos/metabolismo , Glucose/metabolismo , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Canal de Potássio KCNQ1/antagonistas & inibidores , Túbulos Renais Proximais/efeitos dos fármacos , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Compostos de Amônio Quaternário/farmacologia , Sódio/metabolismoRESUMO
K(+) channels play an important role in renal collecting duct cell function. The current study examined barium (Ba(2+))-sensitive whole-cell K(+) currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba(2+) in a dose- and voltage-dependent fashion, with the K (d) decreasing with hyperpolarization. The electrical distance of block by Ba(2+) was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba(2+) entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba(2+) ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K(+) channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K(+) conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K(+) handling in the principal cells of the cortical collecting duct.
Assuntos
Córtex Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Animais , Bário/farmacologia , Relação Dose-Resposta a Droga , Condutividade Elétrica , Canais Epiteliais de Sódio/metabolismo , Feminino , Expressão Gênica , Córtex Renal/citologia , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mucoproteínas/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Cloreto de Potássio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio/metabolismo , Simportadores de Cloreto de Sódio/fisiologia , Uromodulina , gama-Glutamiltransferase/metabolismoRESUMO
TWIK-1, a member of the two-pore domain K(+) channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K(+) conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (G (Quin)). G (Quin) demonstrated outward rectification and had a relatively low K(+) to Na(+) selectivity ratio. Loss of G (Quin) would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K(+) conductance and Na(+) uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that G (Quin) contributes to the resting membrane potential in the cortical collecting duct and that a fall in G (Quin) could be an adaptive response in TWIK-1 knockout ducts.
Assuntos
Túbulos Renais Coletores/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Potássio/metabolismo , Adaptação Fisiológica , Animais , Bário/metabolismo , Cátions/metabolismo , Regulação para Baixo , Feminino , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Quinidina , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The membrane protein KCNE1 has been implicated in cell volume regulation. Using a knockout mouse model, this study examined the role of KCNE1 in regulatory volume decrease (RVD) in freshly isolated renal proximal tubule cells. Cell diameter was measured using an optical technique in response to hypotonic shock and stimulation of Na(+)-alanine cotransport in cells isolated from wild-type and KCNE1 knockout mice. In HEPES buffered solutions 64% of wild-type and 56% of knockout cells demonstrated RVD. In HCO3- buffered solutions 100% of the wild-type cells showed RVD, while in the knockout cells the proportion of cells displaying RVD remained unchanged. RVD in the knockout cells was rescued by valinomycin, a K+ ionophore. In wild-type HCO3- dependent cells the K+ channel inhibitors barium and clofilium inhibited RVD. These data suggest that mouse renal proximal tubule is comprised of two cell populations. One cell population is capable of RVD in the absence of HCO3-, whereas RVD in the other cell population has an absolute requirement for HCO3-. The HCO3- dependent RVD requires the normal expression of KCNE1.
Assuntos
Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Animais , Bicarbonatos/farmacologia , Soluções Tampão , HEPES/farmacologia , Soluções Hipotônicas/farmacologia , Ionóforos/farmacologia , Íons , Túbulos Renais Proximais/efeitos dos fármacos , Camundongos , Camundongos Knockout , Potássio/metabolismo , Valinomicina/farmacologiaRESUMO
The whole-cell patch-clamp technique was used to examine nonselective conductances in single proximal tubule cells isolated from mouse kidney. Single cells were isolated in either the presence or absence of a cocktail designed to stimulate cAMP. Patches were obtained with Na+ Ringer in the bath and Cs+ Ringer in the pipette. On initially achieving the whole-cell configuration, whole-cell currents were small. In cAMP-stimulated cells, with 5 mM ATP in the pipette solution, whole-cell currents increased with time. The activated current was linear, slightly cation-selective, did not discriminate between Na+ and K+ and was inhibited by 100 microM gadolinium. These properties are consistent with the activation of a nonselective conductance, designated G(NS). Activation of G(NS) was abolished with pipette AMP-PNP, ATP plus alkaline phosphatase or in the absence of ATP. In unstimulated cells G(NS) was activated by pipette ATP together with PKA. These data support the hypothesis that G(NS) is activated by a PKA-mediated phosphorylation event. G(NS) was also activated by a hypertonic shock. However, G(NS) does not appear to be involved in regulatory volume increase (RVI), as RVI was unaffected in the presence of the G(NS) blocker gadolinium. Instead, the ATP sensitivity of G(NS) suggests that it may be regulated by the metabolic state of the renal proximal tubule cell.
Assuntos
AMP Cíclico/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/fisiologia , Mecanotransdução Celular/fisiologia , Potenciais da Membrana/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Tamanho Celular/fisiologia , Células Cultivadas , Condutividade Elétrica , Soluções Hipertônicas , Ativação do Canal Iônico/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Pressão OsmóticaRESUMO
1. Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftr(tm2cam)deltaF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT-PCR and whole-cell patch clamping. 2. Renal Na(+) clearance was similar in wild-type (1.4 +/- 0.3 microl min(-1), number of animals, N = 12) and CF mice (1.6 +/- 0.4 microl min(-1), N = 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild-type (7.0 +/- 0.8 microl min(-1), N = 8) and CF mice (9.3 +/- 1.4 microl min(-1), N = 9); no difference between genotypes was observed. 3. In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild-type (2.2 +/- 0.4 nl mm(-1) min(-1), n = 10) and CF mice (1.9 +/- 0.3 nl mm(-1) min(-1), n = 11). Addition of a forskolin-dibutyryl cAMP (db-cAMP) cocktail to the perfusate caused no significant change in Jv in either wild-type (2.6 +/- 0.7 nl mm(-1) min(-1), n = 10) or Cftr(tm2cam)deltaF508 mice (2.0 +/- 0.5 nl mm(-1) min(-1), n = 10). 4. CFTR expression was confirmed in samples of outer cortex using RT-PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin-db-cAMP using whole-cell patch clamping. 5. In conclusion, no functional deficit in proximal tubule function was found in Cftr(tm2cam)deltaF508 mice. This may be a consequence of a lack of whole-cell cAMP-dependent Cl(-) conductance in mouse proximal tubule cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Túbulos Renais Proximais/fisiopatologia , Mutação/fisiologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Técnicas de Patch-Clamp , Perfusão , Substitutos do Plasma/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio/farmacologiaRESUMO
It has been suggested that CFTR Cl(-) channels in the renal inner medullary collecting duct may be involved in mediating increased renal salt excretion during extracellular fluid volume expansion. To investigate this hypothesis, in-vivo clearance experiments were performed comparing wild-type (WT) and DeltaF508-CFTR transgenic mice (cftr (tm2Cam)). Control animals were given a 0.1-ml bolus of 0.9% saline, followed by I.V. infusion at 0.3 ml x h(-1). Volume expansion was applied by infusing a 1-ml bolus of 0.9% saline followed by infusion at 0.6 ml x h(-1). No significant differences in renal NaCl handling between WT mice ( C(Na)=1.2 +/- 0.3 microl x min(-1), C(Cl)=4.0 +/- 0.5 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=1.7 +/- 0.5 microl x min(-1), C(Cl)=4.1 +/- 0.8 microl x min(-1)) were observed under control conditions. Volume expansion resulted in large significant increases in NaCl clearance in both WT mice ( C(Na)=7.0 +/- 0.9 microl x min(-1), C(Cl)=12.0 +/- 0.6 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=7.2 +/- 1.6 microl x min(-1), C(Cl)=11.0 +/- 2.2 microl x min(-1)). However, there was no significant difference between WT and DeltaF508-CFTR mice. In conclusion, renal NaCl excretion is not significantly different under basal conditions and during saline volume expansion in DeltaF508-CFTR mice. The data suggest that CFTR is not a physiologically important mediator of volume natriuresis.
Assuntos
Volume Sanguíneo/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Anestesia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Modelos Animais de Doenças , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Natriurese/fisiologia , Cloreto de Sódio/farmacologiaRESUMO
The aims of this study were to investigate (a) if renal Na(+) handling was normal in Cftr(tm2cam) delta F508 cystic fibrosis mice, (b) whether adaptation to dietary salt depletion was preserved and (c) whether Cftr(tm2cam) delta F508 mice exhibited enhanced amiloride-sensitive Na(+) absorption. In Na(+)-replete animals (maintained on a 0.32 % NaCl diet) given a 150 mM NaCl i.v. maintenance infusion, there was no difference in fractional Na(+) excretion (FE(Na)) between wild-type (0. 42 +/- 0.06 %, n = 12) and Cftr(tm2cam) delta F508 mice (0.47 +/- 0.13 %, n = 7). Amiloride infusion significantly increased FE(Na) in both wild-type (3.14 +/- 0.83 %, n = 6) and Cftr(tm2cam) delta F508 mice (3. 47 +/- 0.63 %, n = 9), though with no significant difference between genotypes. A 14 day dietary salt restriction (animals maintained on a 0.03 % NaCl diet) and maintenance infusion with a 15 mM NaCl vehicle caused a reduction in FE(Na) to 0.14 +/- 0.05 %, n = 8 in wild-type mice and 0.14 +/- 0.04 %, n = 8 in Cftr(tm2cam) delta F508 mice. No significant difference in the ability to adapt to low salt conditions was apparent comparing the two genotypes. Treatment of salt-restricted mice with amiloride resulted in a blunted natriuresis in both wild-type mice (FE(Na) = 1.10 +/- 0.16 %, n = 7) and Cftr(tm2cam) delta F508 mice (FE(Na) = 1.97 +/- 0.29 %, n = 9). The natriuresis induced by amiloride was significantly greater in Cftr(tm2cam) delta F508 mice than in wild-type controls. In conclusion, Cftr(tm2cam) delta F508 mice exhibit normal renal salt excretion when either salt replete or salt restricted. Enhanced amiloride-sensitive FE(Na) is consistent with increased Na(+) absorption via the amiloride-sensitive sodium channel ENaC, in cystic fibrosis kidney, but this was only observed during salt restriction.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Rim/metabolismo , Sódio/sangue , Amilorida/farmacologia , Animais , Cloretos/urina , Modelos Animais de Doenças , Diuréticos/farmacologia , Canais Epiteliais de Sódio , Rim/efeitos dos fármacos , Testes de Função Renal , Masculino , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Transgênicos , Sódio/urina , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Cloreto de Sódio na Dieta/farmacologiaRESUMO
Li+ may be reabsorbed via an amiloride-sensitive mechanism in the collecting ducts of rats administered a low-Na+ diet. This was investigated by measuring the increase in fractional urinary excretion of Li+ (FELi) in response to amiloride in conscious rats at two different levels of plasma Li+ concentration and after administration of bendroflumethiazide (BFTZ), angiotensin III (ANG III), and aldosterone (Aldo). The results confirmed that amiloride increased (FELi) in rats on a low-Na+ diet (20 +/- 1 to 35 +/- 1%, means +/- SE), whereas no increase was observed in rats on a normal Na+ diet (37 +/- 1 to 38 +/- 1%). The lithiuretic effect of amiloride was 1) abolished by preadministration of BFTZ (32 +/- 1 to 33 +/- 2%) to Na(+)-deprived rats and 2) increased by ANG III (27 +/- 3 to 33 +/- 2%) and Aldo (25 +/- 2 to 37 +/- 2%) in Na(+)-replete rats. Amiloride-induced changes in FELi were independent of plasma Li+ concentration but inversely related to the fractional excretion of Na+ and the amiloride-sensitive excretion of K+. These results are compatible with the hypothesis that a low tubular Na+ concentration reduces end-tubular Na+ reabsorption and results in hyperpolarization of the apical membrane, thus favoring Li+ uptake into the cells.
Assuntos
Lítio/metabolismo , Natriurese , Sódio/metabolismo , Aldosterona/farmacologia , Amilorida/farmacologia , Angiotensina III/farmacologia , Animais , Bendroflumetiazida/farmacologia , Transporte Biológico/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Túbulos Renais/metabolismo , Natriurese/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The analysis of picomolar quantities of magnesium by electrothermal atomic absorption spectrophotometry (EAAS) was studied using a Perkin-Elmer-Zeeman 3030 spectrophotometer. The absorbance signal was not heavily dependent on the atomization temperature, but was greatly reduced when ashing temperatures in excess of 1200 degrees C were applied. The magnesium signal was significantly depressed in the presence of excess chloride in the sample matrix. However, use of NH4NO3 as a matrix modifier was sufficient to overcome this artefact. The analytical sensitivity was 0.15 absorbance units pmol-1 and the detection limit was 0.04 pmol. Using nanolitre constriction pipettes to dispense standards, the mean coefficient of variation was 5%. Measurement of magnesium handling in the rat proximal convoluted tubule revealed a significant correlation between the tubular fluid-to-plasma ultrafiltrate (TF/UF) concentration ratio for magnesium and the tubular fluid-to-plasma (TF/P) concentration ratio for [3H]inulin (r2 = 0.56, n = 17). This indicated that magnesium is concentrated during its passage along the proximal tubule. In contrast, this was not the case for sodium (r2 = 0.11, n = 16). Mean (TF/UF)Mg (1.16 +/- 0.07, n = 17) for random punctures was significantly greater than that for sodium ((TF/UF)Na = 1.02 +/- 0.02, n = 16). Despite concentration of magnesium in the lumen, significant net reabsorption of magnesium was observed along the length of the tubule (fractional reabsorption, FRMg = 19.4 +/- 3.0%, n = 17). In conclusion, EAAS provides a highly sensitive, reproducible and technically simple method for measuring picomolar quantities of magnesium in renal tubular fluid.
Assuntos
Túbulos Renais Proximais/química , Magnésio/análise , Animais , Transporte Biológico/fisiologia , Magnésio/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Espectrofotometria/métodos , Temperatura , UltrafiltraçãoRESUMO
Whole cell patch-clamp studies were performed with tissue isolated from the cystic fibrosis (CF) transgenic Cftrm1cam mouse, to determine whether anion currents in choroid plexus epithelial cells require the expression of cystic fibrosis transmembrane conductance regulator (CFTR). Inclusion of 0.25 mM adenosine 3',5'-cyclic monophosphate (cAMP) and 375 nM protein kinase A (PKA) in the pipette solution caused a significant activation of a Cl(-)-selective, inward-rectifying conductance in cells from wild-type and CF mice. The small, outward currents observed in wild-type and CF animals, however, were not activated by cAMP-PKA. There were no significant differences in the size of currents between wild-type, heterozygote, and CF cells in the presence or absence of cAMP-PKA. A second whole cell conductance was activated when cells from wild-type mice were swollen. These volume-activated currents were Cl- selective and exhibited outward rectification. They were Ca2+ independent and ATP dependent and blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The volume-activated channels were also activated in CF mutant cells, and there was no significant difference in the size of the volume-activated currents between wild-type, heterozygote, and CF cells. It is concluded that CFTR neither contributes to the whole cell conductance nor regulates the other anion conductances in choroid plexus epithelial cells.
Assuntos
Canais de Cloreto/fisiologia , Plexo Corióideo/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Animais , Canais de Cloreto/efeitos dos fármacos , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Epitélio/fisiologia , Heterozigoto , Humanos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Mutagênese , Concentração OsmolarRESUMO
1. This study used whole-cell patch clamp and RNA in situ hybridization experiments to determine whether the cAMP-activated C1- current expressed in choroid plexus epithelial cells was carried by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. 2. In patch clamp experiments, inclusion of 0.25 mM cAMP and 375 protein kinase A catalytic subunit (PKA) in the electrode solution caused activation of an inwardly rectifying current (21/23 cells). This current was C1- selective, since the current reversal potential (Erev) was -31 +/- 3 mV with equilibrium potential values for C1- (EC1) and Na+ (ENa) of -44 and 0 mV, respectively. 3. In anion substitution experiments, the relative anion permeability sequence for the inward rectifier was: I- (3.5) > HCO3-(1.5) = C1-(1.0) > Br-(0.6) > aspartate (0.2). 4. The inward rectifier was sensitive to inhibition by a range of known channel inhibitors, including: glibenclamide (100 microns), DIDS (100 and 500 microns), NPPB (100 microns) and Ba2+ (1 mM). 5. In RNA in situ hybridization experiments, using two independent rat CFTR cRNA probes, expression of CFTR could not be detected in epithelial cells from the rat choroid plexus. 6. In conclusion, the cAMP-dependent whole-cell C1- current present in choroid plexus epithelial cells from the rat has properties which are distinctly different from those of CFTR.
Assuntos
Canais de Cloreto/fisiologia , Plexo Corióideo/fisiologia , AMP Cíclico/fisiologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Bicarbonatos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Canais de Cloreto/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Eletrofisiologia , Células Epiteliais , Epitélio/metabolismo , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Hibridização In Situ , Técnicas In Vitro , Masculino , Potenciais da Membrana/fisiologia , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24-48 hr after their isolation from term placentas. Cl--selective currents were examined using K+-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mM GTPgammaS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPgammaS was found to be Ca2+-dependent since GTPgammaS failed to activate currents when included in a Ca2+-free electrode solution. In addition, similar currents could be activated by increasing the [Ca2+] of the pipette solution to 500 nM. The Ca2+-activated conductance was judged to be Cl--selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl-. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 microM. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca2+-activated Cl- conductance in human placental trophoblast are discussed.
Assuntos
Cálcio/farmacologia , Canais de Cloreto/efeitos dos fármacos , Cloretos/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Ativação do Canal Iônico/fisiologia , Trofoblastos/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Técnicas de Patch-Clamp , Gravidez , Trofoblastos/citologiaRESUMO
1. As yet there is no definitive description of the mechanism and route by which K+ reabsorption is achieved in the proximal convoluted tubule (PCT). We have assessed the contribution of convective K+ transport to net potassium ion flux (JK) by estimating the reflection coefficient of K+ (sigma K) in the proximal tubule of anaesthetized rats previously prepared for in vivo microperfusion. 2. Alterations in the luminal concentration of the impermeant solute raffinose in single-perfused (lumen only) and double-perfused (lumen and capillaries) PCTs were found to change fluid reabsorption in a predictable fashion. 3. Net potassium ion flux (JK) in single- and double-perfused tubules was significantly correlated with net fluid flux (Jv), suggesting that convective K+ transport may be a significant factor in overall K+ transport by the PCT. 4. Estimates of sigma K in single- and double-perfused tubules were very similar (0.14 +/- 0.06 and 0.13 +/- 0.05, respectively), even though K+ diffusion was not strictly controlled in the former group. The maximum effect of 'pseudo-solvent' drag in double-perfused tubules was estimated to give a sigma K of 0.40. This low value for sigma K suggests that true convection/solvent drag may be an important driving force for the reabsorption of K+ from the PCT of the rat.
Assuntos
Túbulos Renais Proximais/metabolismo , Potássio/metabolismo , Anestesia , Animais , Transporte Biológico/fisiologia , Epitélio/metabolismo , Epitélio/ultraestrutura , Inulina , Canais Iônicos/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/ultraestrutura , Masculino , Rafinose/farmacologia , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Trítio , Água/metabolismoRESUMO
The role of diffusion in transepithelial potassium flux and the importance of potassium channels in the luminal cell membrane to this process were examined by applying a luminal microperfusion technique to surface tubules in kidneys of anesthetized rats. Potassium concentration gradients were applied by altering the concentration of KCl in perfusates. To some perfusates, 2 mmol/l BaCl2 was added to block potassium channels in the luminal cell membrane. The mean applied potassium concentration gradient was highly predictive of net potassium transport in the absence of any change in fluid reabsorption, with an apparent potassium permeability of 22 x 10(-5) cm/s. Thus potassium transport in the proximal tubule may have an important diffusive component. Luminal barium significantly reduced the concentration of potassium in collected fluid under conditions of net potassium secretion, although a substantial barium-insensitive potassium permeability was also observed. However, the site of action of luminally applied barium is uncertain in proximal tubule, since barium was reabsorbed by the tubule at a rate of 13.6 pmol.mm-1.min-1. We conclude that diffusion is a significant driving force for potassium reabsorption in proximal tubule and that most diffusive potassium transport occurs via a barium-insensitive route, possibly the paracellular pathway.
Assuntos
Bário/farmacologia , Túbulos Renais Proximais/metabolismo , Potássio/metabolismo , Animais , Transporte Biológico , Difusão , Masculino , Concentração Osmolar , Perfusão , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Água/metabolismoRESUMO
The analysis of picomolar lithium, sodium, and potassium by electrothermal atomic absorption spectrophotometry was studied using a Perkin-Elmer Zeeman 3030 spectrophotometer. With ordinary pyrolytically coated graphite tubes, a number of interference effects associated with the sample matrix were observed. In particular, the lithium and potassium absorbance signal was depressed by chloride, an effect shown to be dependent on the preatomization heating. When an in situ tantalum-coated atomization surface was used, matrix interferences observed in lithium and potassium analyses were abolished, and the linear range for the potassium assay was extended. Technical difficulties encountered during sodium analysis at the primary wavelength were effectively circumvented by analysis at a less-sensitive wavelength (303.3 nm), at which tantalum coating also prevented significant chloride interference. The improved microanalyses were employed to reevaluate the handling of lithium, sodium, and potassium along the proximal convoluted tubule (PCT) of the anesthetized rat. The average tubular fluid-to-plasma concentration ratios for lithium [(TF/P)Li] and sodium [(TF/P)Na] were 1.13 +/- 0.08, n = 26, and 0.99 +/- 0.07 (n = 26), respectively. The tubular fluid-to-plasma ultrafiltrate concentration ratio for potassium [(TF/UF)K] was 1.09 +/- 0.05 (n = 13). Ratios did not change significantly with puncture site along the PCT for any of the ions. (TF/P)Li and (TF/UF)K were significantly greater than (TF/P)Na, indicating that lithium and potassium reabsorption do not directly parallel sodium reabsorption in the PCT.
Assuntos
Líquidos Corporais/química , Túbulos Renais Proximais/fisiologia , Lítio/análise , Potássio/análise , Sódio/análise , Animais , Lítio/metabolismo , Lítio/urina , Microquímica , Potássio/metabolismo , Potássio/urina , Ratos , Sensibilidade e Especificidade , Sódio/metabolismo , Sódio/urina , Espectrofotometria Atômica/métodos , TantálioRESUMO
1. The possible role of autonomic neurotransmitters in atrial natriuretic peptide secretion was investigated using spontaneously beating guinea-pig atria in vitro. Dose responses were determined for adrenaline, noradrenaline and acetylcholine and the selective alpha- and beta-adrenoceptor agonists phenylephrine and isoprenaline, respectively. Adrenoceptor effects were further studied using the selective alpha- and beta-adrenoceptor antagonists prazosin and propranolol, respectively, in conjunction with maximal adrenaline challenge. Results for rate and force of contraction and atrial natriuretic peptide secretion are expressed as a ratio (mean +/- SEM) of a 15 min treatment period (stage 2) to a corresponding pretreatment period (stage 1). 2. Adrenaline and noradrenaline caused dose-dependent increases in the rate and force of contraction and in atrial natriuretic peptide secretion with a peak secretory response at 2 x 10(-6) mol/l of 1.54 +/- 0.08 (P < 0.01) and 1.34 +/- 0.08 (P < 0.01) for adrenaline and noradrenaline, respectively. Acetylcholine decreased the rate and force of contraction, and ANP secretion was reduced to 0.47 +/- 0.06 at 3 x 10(-5) mol/l (P < 0.01). Isoprenaline increased the rate and force of contraction and atrial natriuretic peptide secretion with a peak secretory response of 1.52 +/- 0.22 at 2 x 10(-6) mol/l (P < 0.01). Phenylephrine increased the force but had no effect on the rate of contraction, and stimulated atrial natriuretic peptide secretion to 1.13 +/- 0.09 at 2 x 10(-5) mol/l (P < 0.05). After both alpha- and beta-adrenoceptor blockade, adrenaline was still able to significantly stimulate atrial natriuretic peptide secretion and positive inotropy.(ABSTRACT TRUNCATED AT 250 WORDS)