Ultra scale-down device to predict dewatering levels of solids recovered in a continuous scroll decanter centrifuge.

During centrifugation operation, the major challenge in the recovery of extracellular proteins is the removal of the maximum liquid entrapped within the spaces between the settled solids-dewatering level. The ability of the scroll decanter centrifuge (SDC) to process continuously large amounts of feed material with high concentration of solids without the need for resuspension of feeds, and also to achieve relatively high dewatering, could be of great benefit for future use in the biopharmaceutical industry. However, for reliable prediction of dewatering in such a centrifuge, tests using the same kind of equipment at pilot-scale are required, which are time consuming and costly. To alleviate the need of pilot-scale trials, a novel USD device, with reduced amounts of feed (2 mL) and to be used in the laboratory, was developed to predict the dewatering levels of a SDC. To verify USD device, dewatering levels achieved were plotted against equivalent compression (Gtcomp ) and decanting (Gtdec ) times, obtained from scroll rates and feed flow rates operated at pilot-scale, respectively. The USD device was able to successfully match dewatering trends of the pilot-scale as a function of both Gtcomp and Gtdec , particularly for high cell density feeds, hence accounting for all key variables that influenced dewatering in a SDC. In addition, it accurately mimicked the maximum dewatering performance of the pilot-scale equipment. Therefore the USD device has the potential to be a useful tool at early stages of process development to gather performance data in the laboratory thus minimizing lengthy and costly runs with pilot-scale SDC.

Prediction and verification of centrifugal dewatering of P. pastoris fermentation cultures using an ultra scale-down approach.

Recent years have seen a dramatic rise in fermentation broth cell densities and a shift to extracellular product expression in microbial cells. As a result, dewatering characteristics during cell separation is of importance, as any liquor trapped in the sediment results in loss of product, and thus a decrease in product recovery. In this study, an ultra scale-down (USD) approach was developed to enable the rapid assessment of dewatering performance of pilot-scale centrifuges with intermittent solids discharge. The results were then verified at scale for two types of pilot-scale centrifuges: a tubular bowl equipment and a disk-stack centrifuge. Initial experiments showed that employing a laboratory-scale centrifugal mimic based on using a comparable feed concentration to that of the pilot-scale centrifuge, does not successfully predict the dewatering performance at scale (P-value <0.05). However, successful prediction of dewatering levels was achieved using the USD method (P-value ≥0.05), based on using a feed concentration at small-scale that mimicked the same height of solids as that in the pilot-scale centrifuge. Initial experiments used Baker's yeast feed suspensions followed by fresh Pichia pastoris fermentation cultures. This work presents a simple and novel USD approach to predict dewatering levels in two types of pilot-scale centrifuges using small quantities of feedstock (<50 mL). It is a useful tool to determine optimal conditions under which the pilot-scale centrifuge needs to be operated, reducing the need for repeated pilot-scale runs during early stages of process development.

Prostaglandin E2 modulates proximal tubule Na+-ATPase activity: cooperative effect between protein kinase A and protein kinase C.

Previous data showed that prostaglandin E2 (PGE2) mediates the inhibitory effect of bradykinin (BK) on proximal tubule (PT) Na+-ATPase activity. The aim of this work was to investigate the molecular mechanisms involved in the effect of PGE2 on PT Na+-ATPase. We used isolated basolateral membrane (BLM) from pig PT, which expresses several components of different signaling pathways. The inhibitory effect of PGE2 on PT Na+-ATPase activity involves G-protein and the activation of protein kinase A (PKA) because: (1) PGE2 increased [³5S]GTPγS binding; (2) GDPßS abolished the inhibitory effect of PGE2; (3) PGE2 increased PKA activity; (4) the inhibitory effect of PGE2 was abolished by PKA inhibitor peptide. We observed that the PKA-mediated inhibitory effect of PGE2 on PT Na+-ATPase activity requires previous activation of protein kinase C. In addition, we observed that PGE2 stimulates Ca²+-independent phospholipase A2 activity representing an important positive feedback to maintain the inhibition of the enzyme. These results open new perspectives to understanding the mechanism involved in the effect of PGE2 on proximal tubule sodium reabsorption.

Lack of Na(+),K (+)-ATPase expression in intercalated cells may be compensated by Na(+)-ATPase: a study on MDCK - C11 cells.

The lack of Na(+),K(+)-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na(+),K(+)-ATPase and Na(+)-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na(+),K(+)-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its beta-subunit is similar in both cells. On the other hand, a subset of C11 without alpha-subunit expression has been found. In C11 cells the Na(+)-ATPase activity is higher than that of the Na(+),K(+)-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na(+)-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na(+)-ATPase may explain the adequate survival of cells that lack Na(+),K(+)-ATPase.

Crosstalk between the signaling pathways triggered by angiotensin II and adenosine in the renal proximal tubules: implications for modulation of Na(+)-ATPase activity.

We have previously demonstrated that adenosine (Ado) reverses the stimulatory effect of angiotensin II (Ang II) on Na(+)-ATPase activity via the A(2A) receptor. In this work, the molecular mechanism involved in Ado-induced shutdown in the signaling pathway triggered by 10(-8)M Ang II was investigated. It was observed that: (1) both 10(-12)M PMA (a PKC activator) and 5x10(-8)M U73122 (an inhibitor of PI-PLCbeta) prevent the reversion effect induced by 10(-6)M Ado (only observed in the presence of 10(-6)M DPCPX (an A(1) receptor antagonist)) on Ang II-stimulated Na(+)-ATPase and PKC activities; (2) Ang II-stimulated PKC activity was reversed by 10(-6)M forskolin (an adenylyl cyclase activator) or 10(-8)M PKA inhibitory peptide and 10(-8)M DMPX (an A(2) receptor-selective antagonist). Considering that PMA prevents the inhibitory effect of Ado on Ang II-stimulated Na(+)-ATPase and PKC activities, it is likely that the PMA-induced effect, i.e. PKC activation, is downstream of the target for Ado-induced reversion of Ang II stimulation of Na(+)-ATPase activity. We investigated the hypothesis that PI-PLCbeta could be the target for Ado-induced PKA activation. Our data demonstrate that Ang II-stimulated PI-PLCbeta activity was reversed by Ado or 10(-7)M cAMP; the reversibility of the Ado-induced effect was prevented by either DMPX or PKA inhibitory peptide. These data demonstrate that Ado-induced PKA activation reduces Ang II-induced stimulation of PI-PLCbeta.

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na+ -ATPase: sequential activation of the phosphoinositide-specific phospholipase Cbeta/protein kinase C and Ca2+ -independent phospholipase A2 pathways.

In a previous paper we showed that bradykinin (BK), interacting with its B2 receptor, inhibits proximal tubule Na+ -ATPase activity but does not change (Na+ +K+)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B2-mediated modulation of proximal tubule Na+ -ATPase by BK. To abolish B1 receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg9-[Leu8]-BK (DALBK), a specific antagonist of B1 receptor. A dual effect on the Na+ -ATPase activity through the B2 receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C beta (PI-PLCbeta)/protein kinase C (PKC); its inhibitory action is mediated by Ca2+ -independent phospholipase A2 (iPLA2). Prior activation of the PI-PLCbeta/PKC pathway is required to activate the iPLA2-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B2 receptor and activation of membrane-associated iPLA2.

Characterization and partial isolation of ouabain-insensitive Na(+) -ATPase in MDCK I cells.

We show that MDCK I cells express, besides the classical (Na(+)+K(+))ATPase, a Na(+)-stimulated ATPase activity with the following characteristics: (1) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 23.12+/-1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, respectively). This enzyme forms a Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na(+)+K(+))ATPase with monoclonal anti-alpha(1) antibody reduced its activity in the supernatant by 90%; the Na(+)-ATPase activity was completely maintained. In addition, the formation of the Na(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na(+)-ATPase and (Na(+)+K(+))ATPase activities are independent, with Na(+)-ATPase belonging to a different enzyme entity.

Modulation of multidrug resistance protein (MRP1/ABCC1) expression: a novel physiological role for ouabain.

Besides being a (Na(+),K(+))-ATPase inhibitor, high doses of the hormone ouabain have also been reported to modulate both the expression and activity of proteins belonging to the ATP binding cassette family of transporters, such as ABCC7 (CFTR), ABCB1 (P-glycoprotein), and ABCC1 (MRP1). Although these proteins are present in the kidney, only ABCB1 has a putative physiological role in this organ, secreting endobiotics and xenobiotics. In the present work, we studied the relationship between ouabain and ABCC1 expression and function, aiming to establish a physiological role for ouabain. It was observed that prolonged (24 h) but not short (30 min) incubation with 1 nmol/L or higher ouabain concentrations decreased the expression of ABCC1 protein and induced its mRNA expression. This decrease was rapidly reversible, reaching control levels after incubation of cells in ouabain-free medium for 3 h, denoting a hormonal action. Moreover, concentrations equal or higher than 100 nmol/L ouabain also induced impairment of ABCC1 activity, increasing the accumulation of carboxyfluorescein diacetate, an ABCC1 fluorescent substrate. Because ouabain is now accepted as an endogenous hormone, our results suggest that ABCC1 is regulated by hormones related to body volume control, which may have implications for the treatment of hypertensive cancer patients. Moreover, providing ABCC1 is expressed in several other tissues, such as brain, testis, and the immune system, and is related to the transport of glutathione, it is possible that ouabain release may control a number of functions within these organs and tissues by modulating both the expression and the activity of ABCC1.

Vanadate-induced cell death is dissociated from H2O2 generation.

Vanadium is an environmentally toxic metal with peculiar and sometimes contradictory cellular effects. It is insulin-mimetic, it can either stimulate cell growth or induce cell death, and it has both mutagenic and antineoplastic properties. However, the mechanisms involved in those effects are poorly understood. Several studies suggest that H(2)O(2) is involved in vanadate-induced cell death, but it is not known whether cellular sensitivity to vanadate is indeed related to H(2)O(2) generation. In the present study, the sensitivity of four cell lines from different origins (K562, K562-Lucena 1, MDCK, and Ma104) to vanadate and H(2)O(2) was evaluated and the production of H(2)O(2) by vanadate was analyzed by flow cytometry. We show that cell lines very resistant to H(2)O(2) (K562, K562-Lucena 1, and Ma104 cells) are much more sensitive to vanadate than MDCK, a cell line relatively susceptible to H(2)O(2), suggesting that vanadate-induced cytotoxicity is not directly related to H(2)O(2) responsiveness. In accordance, vanadate concentrations that reduced cellular viability to approximately 60-70% of the control (10 mumol/L) did not induce H(2)O(2) formation. A second hypothesis, that peroxovanadium (PV) compounds, produced once vanadate enters into the cells, are responsible for the cytotoxicity, was only partially confirmed because MDCK cells were resistant to both vanadate and PV compounds (10 micromol/L each). Therefore, our results suggest that vanadate toxicity occurs by two distinct pathways, one dependent on and one independent of H(2)O(2) production.

Adenosine reverses the stimulatory effect of angiotensin II on the renal Na+-ATPase activity through the A2 receptor.

In the present paper, we report the modulation of the Angiotensin II (Ang II)-stimulated Na+-ATPase activity of the proximal tubule basolateral membrane by adenosine (Ado). Preincubation of isolated basolateral membrane with 10(-8)M Ang II increases the Na+-ATPase activity from 7.5+/-0.3 (control) to 14.6+/-0.9 nmol Pi x mg(-1)x min(-1)nmol Pi x mg(-1) x min(-1) (p<0.05). Incubation of Ang II-stimulated enzyme with 10(-6)M Ado, in the presence of the A1 receptor antagonist DPCPX (10(-6)M), completely reverses the Ang II-induced effect bringing the Na+-ATPase activity to the basal level. The following evidences demonstrate involvement of the A2 receptor/Gs protein/adenylyl cyclase/PKA signaling pathway in the inhibitory effect induced by Ado on the Ang II-stimulated Na+-ATPase activity in the presence of the DPCPX: 1) the inhibitory effect of Ado is abolished by the A2 receptor selective antagonist DMPX (10(-8)M); 2) the effect induced by Ado is blocked by 10(-8)M GDPbetaS and mimicked by 10(-9)M cholera toxin and 10(-8)M GTPgammaS; 3) the stimulatory effect of Ang II is reduced by 10(-6)M forskolin, an activator of adenylyl cyclase, or 10(-6)M cAMP; 4) Ado stimulates PKA activity; 5) the inhibitory effect induced by this nucleoside is reversed by the PKA inhibitor peptide.

Modulation of the (Na(+)+K+)ATPase activity by Angiotensin-(1-7) in MDCK cells.

In the present paper the effect of Ang-(1-7) on the distal tubule (Na(+)+K+)ATPase activity was evaluated by using MDCK cells as a model. Confluent cell monolayers were incubated with increasing concentrations of Ang-(1-7) for 30 min. Thereafter, the (Na(+)+K+)ATPase activity was evaluated and a dose-dependent (from 10(-12) to 10(-7) M) inhibition was observed. The maximal inhibitory effect (54%) was reached at the concentration of 10(-8) M. The inhibitory effect of Ang-(1-7) was not affected by the AT2 receptor selective antagonist PD123319 (from 10(-10) to 10(-7) M) but was blocked in a dose-dependent manner by the AT1 receptor selective antagonists losartan (10(-10) M), candesartan (10(-17) M), irbesartan (2 x 10(-12) M) and telmisartan (2 x 10(-16) M). The signaling pathway triggered by stimulation of the AT(1) receptor was also investigated. The PI-phospholipase C (PI-PLC) inhibitor U73122 (5 x 10(-8) M) blocked the inhibitory effect elicited by Ang-(1-7). Involvement of the protein kinase C (PKC) was evidenced by the sensitivity of the inhibitory effect of Ang-(1-7) to calphostin C (6.32 x 10(-7) M) and the lack of additive effects when the cells were co-incubated with Ang-(1-7) and 3.2 x 10(-8) M PMA. Altogether, these results demonstrate that Ang-(1-7) inhibits the (Na(+)+K+)ATPase activity of the prototypic distal tubule cell MDCK through the AT1 receptor-mediated stimulation of PI-PLC/PKC signaling pathway.

PI-PLCbeta is involved in the modulation of the proximal tubule Na+-ATPase by angiotensin II.

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.

Stimulation of the proximal tubule Na+-ATPase activity by adenosine A(2A) receptor.

The aim of this work was to determine the molecular mechanism involved in the stimulation of the pig kidney proximal tubule Na+-ATPase by adenosine (Ado). To study the role of A2 Ado receptors, we added in all experiments 10(-6)M DPCPX, an A1 receptor-selective antagonist, since we have previously shown that Ado inhibits the enzyme activity through this receptor. Ado increased the Na+-ATPase activity with maximal effect observed at 10(-6)M. The presence of both A(2A) and A(2B) receptors were demonstrated by immunoblotting using specific polyclonal antibodies. The stimulatory effect of Ado was completely abolished by 5 x 10(-9)M DMPX, an antagonist of A2 receptor, and 10(-7)M SCH 58261, an A(2A) receptor-selective antagonist. DMPA (10(-7)M), a specific agonist of A(2A) receptor mimicked the stimulatory effect of Ado. Involvement of a Gs protein/adenylate cyclase/PKA pathway was evidenced by: (a) the reversion of Ado-induced effect by GDPbetaS; (b) stimulation of the Na+-ATPase activity in a similar and non-additive manner to Ado by 10(-8)M cholera toxin, 10(-7)M GTPgammaS, 10(-6)M forskolin, 10(-7)M cAMP or 1.25 U catalytic subunit of PKA; (c) the reversion of the stimulatory effect of Ado by 10(-8)M PKA inhibitor peptide; (d) Ado-produced two-fold increase of the PKA activity, which was completely reversed by 10(-6)M DMPX. These are the first evidences showing the modulation of a renal primary active sodium transporter by Ado through A(2A) receptor.

Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+ -ATPase activity through AT2 receptor.

In the present paper, the modulation of the basolateral membrane (BLM) Na+ -ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) was evaluated. Ang II and Ang-(1-7) inhibit the Na+ -ATPase activity in a dose-dependent manner (from 10(-11) to 10(-5) M), with maximal effect obtained at 10(-7) M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1-7) are mediated by AT2 receptor: The effect of both polypeptides is completely reversed by 10(-8) M PD 123319, a selective AT2 receptor antagonist, but is not affected by either (10(-12) - 10(-5) M) losartan or (10(-10)-10(-7) M) A779, selective antagonists for AT1 and AT(1-7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3',5'-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10(-9) M guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS; an inhibitor of the G protein activity), and mimicked by 10(-10) M guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the Gs protein in the isolated basolateral membrane fraction; (4) (10(-10)-10(-6) M) cAMP has a similar and non-additive effect to Ang II and Ang-(1-7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1-7); and (6) both angiotensins stimulate PKA activity.

Ouabain-insensitive Na+-ATPase of proximal tubules is an effector for urodilatin and atrial natriuretic peptide.

In the present paper we studied the effect of urodilatin and atrial natriuretic peptide (ANP) on the proximal tubule Na+-ATPase and (Na+K+)ATPase activities. Urodilatin and ANP inhibit the Na+-ATPase activity but not the (Na+K+)ATPase activity. Maximal effect was observed at a concentration of 10(-11) M for both peptides. In this condition, the enzyme activity decreases from 10.8 +/- 1.6 (control) to 5.7 +/- 0.9 or 6.1 +/- 0.7 nmol Pi mg(-1) min(-1) in the presence of urodilatin or ANP, respectively. This effect was completely reversed by 10(-6) M LY83583, a guanylyl cyclase inhibitor, and mimicked by 10 nM cGMP. Furthermore, both ANP and urodilatin increase cGMP production by 33% and 49%, respectively. This is the first demonstration that it was shown that urodilatin and ANP directly modulate primary active sodium transport in the proximal tubule. The data obtained indicate that this effect is mediated by the activation of the NPR-A/guanylate cyclase/cGMP pathway.

PLA2/PGE2 are involved in the inhibitory effect of bradykinin on the angiotensin-(1-7)-stimulated Na(+)-ATPase activity of the proximal tubule.

Recently, we demonstrated that bradykinin (BK) counteracts the stimulatory effect of Ang-(1-7) on the Na(+)-ATPase activity from basolateral membrane of the proximal tubule through B2 receptor. In the present paper, the signaling pathway involved in the inhibitory response of the Na(+)-ATPase activity to BK was investigated. The following results indicate that the phospholipase A2 (PLA2)/COX/prostaglandin E (PGE2) pathway is implicated in this process: (1) The inhibitory effect of BK on Ang-(1-7)-stimulated enzyme is abolished in a dose-dependent manner by quinacrine (10(-9)-10(-6)M), a nonspecific PLA2 inhibitor, and by PACOCF3 (10(-7)M), an inhibitor of a Ca(2+)-independent PLA2. However, AACOCF3 (2 x 10(-4) M), an inhibitor of the cytosolic PLA2, does not modify the inhibitory effect of BK. (2) The inhibitory effect of BK on the Ang-(1-7)-stimulated enzyme is reversed by cyclooxygenase (COX) inhibitors diclofenac (10(-12) M) and indomethacin (10(-12) M). (3) PGE2 (10(-12)-10(-5) M) inhibits the Na(+)-ATPase activity in a dose dependent manner. (4)The inhibitory effects of PGE2 and BK on the Na(+)-ATPase activity are not cumulative. (5) PGE2 (10(-12)-10(-8) M) counteracts the stimulatory effect of Ang-(1-7) on the enzyme activity in a dose-dependent manner.

Bradykinin B1 receptor stimulates the proximal tubule Na(+)-ATPase activity through protein kinase C pathway.

Recently, our group described a B1-mediated stimulatory effect of des-Arg(9)-bradykinin (DABK) on the Na(+)-ATPase activity of proximal tubule basolateral membranes (BLM) [Biochim. Biophys. Acta 1431 (1999) 483.]. Data in the present report suggest the participation of a phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway as the molecular mechanism of DABK-mediated stimulation of the Na(+)-ATPase activity since (i) 10(-8) M DABK activates PI-PLC activity; (ii) 10(-9) M U73122, a PI-PLC inhibitor, abolishes the effect of 10(-8) M DABK on the Na(+)-ATPase activity; (iii) 10(-8) M DABK increases phosphoprotein formation by 34%. This effect is completely reversed by 10(-7) M calphostin C, an inhibitor of PKC; (iv) 20 ng/ml TPA, an activator of PKC, and 10(-8) M DABK stimulate the Na(+)-ATPase activity in a similar and nonadditive manner. Furthermore, the effect of 10(-8) M DABK is completely reversed by calphostin C; (v) 10(-8) M DABK increases phosphoserine residue levels by 54%. This effect is completely reversed by 10(-7) M calphostin C.

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor.

Recently, we demonstrated that angiotensin-(1-7) (Ang-(1-7)) stimulates the Na(+)-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B(2) receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1-7)-stimulated Na(+)-ATPase activity was evaluated. Preincubation of Na(+)-ATPase with 10(-9) M Ang-(1-7) increases enzyme activity from 7.9+/-0.9 to 14.1+/-1.5 nmol Pi mg(-1) min(-1), corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10(-14)-10(-8) M), reaching maximal inhibitory effect at 10(-9) M. Des-Arg(9) bradykinin (DABK), an agonist of B(1) receptor, at the concentrations of 10(-9)-10(-7) M, does not mimic the BK inhibitory effect, and des-Arg(9)-[Leu(8)]-BK (DALBK), a B(1) receptor antagonist, at the concentrations of 10(-10)-10(-7) M, does not prevent the inhibitory effect of BK on Ang-(1-7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B(2) receptor, abolishes the inhibitory effect of BK on the Ang-(1-7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10(-7) M. Taken together, these data indicate that stimulation of B(2) receptors by BK can counteract the stimulatory effect of Ang-(1-7) on the proximal tubule Na(+)-ATPase activity.

Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide.

In addition to the (Na(+)+K(+))ATPase another P-ATPase, the ouabain-insensitive Na(+)-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg(2+), MgATP and furosemide on the Na(+)-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na(+)-ATPase at pH 7.0 are: (a) K(0.5) for Na(+)=8.9+/-2.2mM; (b) K(0.5) for MgATP=1.8+/-0.4mM; (c) two sites for free Mg(2+): one stimulatory (K(0.5)=0.20+/-0.06 mM) and other inhibitory (I(0.5)=1.1+/-0.4 mM); and (d) I(0.5) for furosemide=1.1+/-0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na(+) (K(0.5)=19.5+/-0.4) and MgATP (K(0.5)=3.4+/-0.3 mM) but increases the apparent affinity for furosemide (0.18+/-0.02 mM) and Mg(2+) (0.05+/-0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na(+) (K(0.5)=18.7+/-1.5 mM) and furosemide (I(0.5)=3.04+/-0.57 mM) but does not change the apparent affinity to MgATP and Mg(2+). The data presented in this paper indicate that the modulation of the Na(+)-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg(2+) and MgATP complex may play an important role in the Na(+)-ATPase physiological regulatory mechanisms.

Angiotensin II stimulates renal proximal tubule Na(+)-ATPase activity through the activation of protein kinase C.

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.