RESUMO
This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions , AMP Cíclico/biossíntese , Túbulos Renais Proximais/efeitos dos fármacos , Ouabaína/farmacologia , Animais , Toxina da Cólera/farmacologia , Colforsina/farmacologia , AMP Cíclico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Técnicas In Vitro , Túbulos Renais Proximais/metabolismo , SuínosRESUMO
Angiotensin-(1-7) (Ang-(1-7)) modulates the Na+-ATPase, but not the Na+,K+-ATPase activity present in pig kidney proximal tubules. The Na+-ATPase, insensitive to ouabain, but sensitive to furosemide, is stimulated by Ang-(1-7) (68% by 10(-9) M), in a dose-dependent manner. This effect is due to an increase in Vmax, while the apparent affinity of the enzyme for Na+ is not modified. Saralasin, a general angiotensin receptor antagonist, abolishes the stimulation, demonstrating that the Ang-(1-7) effect is mediated by receptor. The Ang-(1-7) stimulatory effect is not changed by either PD 123319, an AT2 receptor antagonist, or A779, an Ang-(1-7) receptor antagonist. On the other hand, increasing the concentration of the AT1 receptor antagonist losartan from 10(-11) to 10(-9) M, reverses the Ang(1-7) stimulation completely. A further increase to 10(-3) M losartan reverses the Na+-ATPase activity to a level similar to that obtained with Ang-(1-7) (10(-9) M) alone. The stimulatory effect of Ang-(1-7) at 10(-9) M is similar to the effect of angiotensin II (AG II) alone. However, when the two peptides are both present, Na+-ATPase activity is restored to control values. These data suggest that Ang-(1-7) selectively modulates the Na+-ATPase activity present in basolateral membranes of kidney proximal tubules through a losartan-sensitive receptor. This receptor is probably different from the receptor involved in the stimulation of the Na+-ATPase activity by angiotensin II.
Assuntos
Adenosina Trifosfatases/metabolismo , Angiotensina I/farmacologia , Proteínas de Transporte de Cátions , Túbulos Renais Proximais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais Proximais/enzimologia , Natriurese , Ouabaína , Piridinas/farmacologia , Receptores de Angiotensina/agonistas , ATPase Trocadora de Sódio-Potássio/metabolismo , SuínosRESUMO
Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.
Assuntos
Adenosina Trifosfatases/metabolismo , Angiotensina II/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Proteína Quinase C/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Técnicas In Vitro , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , SuínosRESUMO
Angiotensin II (AG II) stimulates the ouabain-insensitive, furosemide- sensitive Na+-ATPase present in the basolateral membrane of pig renal proximal tubules in a dose dependent manner. Maximum effect was obtained with 10-8 M AG II, which corresponded to an activity 134% higher than control. Half of the maximum effect was observed between 10-11 M and 10-10 M, corresponding to physiological hormone levels. Saralasin, an AG II peptide analogue receptor antagonist, abolished the phenomenon, demonstrating that AG II interacts with specific sites in pig proximal tubules. The AG II stimulatory effect was also prevented by dithiothreitol (DTT), a reducing compound, and by 10 nM losartan, a non-peptide antagonist highly specific for AT1 receptors, characterizing AG II binding to AT1 receptors. GTPgammaS, a non-hydrolysable GTP analogue, increased by 159% the enzyme activity as compared to the control values. The simultaneous addition of 10-5 M GTPgammaS and 10-8 M AG II did not have additive effects. Furthermore, the stimulatory action of AG II was completely abolished by 0.1 microM GDPbetaS, a non-hydrolysable GDP analogue. Two microgram ml-1 pertussis toxin, an inhibitor of Gi-protein, did not modulate the AG II stimulatory effect. On the other hand, the Na+-ATPase activity was enhanced 100% in the presence of cholera toxin and 85% in the presence of both AG II and cholera toxin. Taken together, these data suggest that AG II activates the Na+-ATPase activity through AT1 receptors coupled to a pertussis-insensitive and cholera-sensitive G-protein.
Assuntos
Adenosina Trifosfatases/metabolismo , Angiotensina II/farmacologia , Proteínas de Transporte de Cátions , Proteínas de Ligação ao GTP/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Ativação Enzimática , Túbulos Renais Proximais/metabolismo , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/metabolismo , SuínosRESUMO
This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from renal proximal tubule basolateral membranes (BLM) by protein kinase C (PKC). Two PKC isoforms were identified in BLM, one of 75 kDa and the other of 135 kDa. The former correlates with the PKC isoforms described in the literature but the latter seems to be a novel isoform, not yet identified. Both PKC isoforms of BLM are functional since a protein kinase C activator, TPA, increased the total hydroxylamine-resistant 32P(i) incorporation from [gamma-32P]ATP into the BLM. In parallel, TPA stimulated the Na(+)-ATPase activity from BLM in a dose-dependent manner, the effect being reversed by the PKC inhibitor sphingosine. The stimulatory effect of TPA on Na(+)-ATPase involved an increase in the V(max) (from 13.4+/-0.6 nmol P(i) mg(-1) min(-1) to 25.2+/-1.4 nmol P(i) mg(-1) min(-1), in the presence of TPA, P<0.05) but did not change the apparent affinity for Na(+) (K(0.5)=14.5+/-2.1 mM in control and 10.0+/-2.1 mM in the presence of TPA, P>0.07). PKC involvement was further confirmed by stimulation of the Na(+)-ATPase activity by the catalytic subunit of PKC (PKC-M). Finally, the phosphorylation of an approx. 100 kDa protein in the BLM (the suggested molecular mass of Na(+)-ATPase [1]) was induced by TPA. Taken together, these findings indicate that PKCs resident in BLM stimulate Na(+)-ATPase activity which could represent an important mechanism of regulation of proximal tubule Na(+) reabsorption.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions , Túbulos Renais Proximais/metabolismo , Proteína Quinase C/metabolismo , Animais , Fracionamento Celular , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Isoenzimas/análise , Túbulos Renais Proximais/enzimologia , Cinética , Fosforilação , Proteína Quinase C/análise , Transdução de Sinais , Sódio/metabolismo , SuínosRESUMO
In previous papers we showed that Ang II increases the proximal tubule Na+-ATPase activity through AT1/PKC pathway [L.B. Rangel, C. Caruso-Neves, L.S. Lara, A.G. Lopes, Angiotensin II stimulates renal proximal tubule Na+-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316, L.B.A. Rangel, A.G. Lopes, L.S. Lara, C. Caruso-Neves, Angiotensin II stimulates renal proximal tubule Na+)-ATPase activity through the activation of protein kinase C. Biochim. Biophys. Acta 1564 (2002) 310-316]. In the present paper, we study the involvement of PI-PLCbeta on the stimulatory effect of angiotensin II (Ang II) on the proximal tubule Na+-ATPase activity. Western blotting assays, using a polyclonal antibody for PI-PLCbeta, show a single band of about 150 KDa, which correspond to PI-PLCbeta isoforms. Ang II induces a rapid decrease in PIP2 levels, a PI-PLCbeta substrate, being the maximal effect observed after 30 s incubation. This effect of Ang II is completely abolished by 5 x 10(-8) M U73122, a specific inhibitor of PI-PLCbeta. In this way, the effect of 10(-8) M Ang II on the proximal tubule basolateral membrane (BLM) Na+-ATPase activity is completely abolished by 5 x 10(-8) M U73122. The increase in diacylglycerol (DAG) concentration, an product of PI-PLCbeta, from 0.1 to 10 nM raises the Na+-ATPase activity from 6.1+/-0.2 to 13.1+/-1.8 nmol Pi mg(-1) min(-1). This effect is similar and non-additive to that observed with Ang II. Furthermore, the stimulatory effect of 10 nM DAG is completely reversed by 10(-8) M calphostin C (Calph C), an inhibitor of PKC. Taken together these data indicate that Ang II stimulates the Na+-ATPase activity of proximal tubule BLM through a PI-PLCbeta/PKC pathway.
Assuntos
Adenosina Trifosfatases/metabolismo , Angiotensina II/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Isoenzimas/metabolismo , Túbulos Renais Proximais/enzimologia , Fosfatidilinositol Diacilglicerol-Liase/metabolismo , Animais , Diglicerídeos/metabolismo , Estrenos/metabolismo , Isoenzimas/antagonistas & inibidores , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol Diacilglicerol-Liase/antagonistas & inibidores , Pirrolidinonas/metabolismo , SuínosRESUMO
For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption.
Assuntos
Angiotensina II/fisiologia , Angiotensina I/fisiologia , Espaço Extracelular/enzimologia , Túbulos Renais Proximais/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/urina , Animais , Espaço Extracelular/fisiologia , Receptores de Angiotensina/fisiologiaRESUMO
For several years it was believed that angiotensin II (Ang II) alone mediated the effects of the renin-angiotensin system. However, it has been observed that other peptides of this system, such as angiotensin-(1-7) (Ang-(1-7)), present biological activity. The effect of Ang II and Ang-(1-7) on renal sodium excretion has been associated, at least in part, with modulation of proximal tubule sodium reabsorption. In the present review, we discuss the evidence for the involvement of Na+-ATPase, called the second sodium pump, as a target for the actions of these compounds in the regulation of proximal tubule sodium reabsorption
Assuntos
Animais , Angiotensina II/fisiologia , Angiotensina I/fisiologia , Espaço Extracelular/enzimologia , Túbulos Renais Proximais/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/urina , Espaço Extracelular/fisiologia , Receptores de Angiotensina/fisiologiaRESUMO
O teste de fluorescencia pode ser usado para identificar bacilos diftericos toxinogenicos ou nao, alem de evitar esforco adicional, no processo de identificacao de microrganismos corineformes, nao fluorescentes, semelhantes ao C. diphtheriae. O teste da pirazina-carboxilamidase, uma vez que pode ser realizado em duas horas, mostra-se tambem de grande valor para o diagnostico laboratorial
Assuntos
Corynebacterium diphtheriae , ImunofluorescênciaRESUMO
Descrevemos o isolamento do C. diphtheriae em uma regiäo endêmica da leishmaniose cutâneo-mucosa por Leishmania braziliensis braziliensis. Materiais das lesöes cutâneas foram obtidos de dezesseis pacientes. Nove deles foram portadores do bacilo (56%). Os microrganismos suspeitos foram identificados pelos testes de fluorescência sob luz ultravioleta, teste de virulência "in vitro" (por imunodifusäo radial simples), pesquisa da enzima pirazina-carboxilamidase (Pyz) e por imunofluorescência direta e indireta