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Nucleotide binding to purinergic P2Y receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Here, we investigate the regulatory mechanism of the P2Y1 receptor agonist 2-methylthioadenosine diphosphate (2-MeSADP) on Cl- transport in A6 cells, a commonly used model of the distal section of theXenopus laevisnephron. Protein and mRNA expression analysis together with functional measurements demonstrated the basolateral location of theXenopusP2Y1 receptor. 2-MeSADP increased intracellular [Ca2+] and cAMP and Cl- efflux, responses that were all inhibited by the specific P2Y1 receptor antagonist MRS 2179. Cl- efflux was also inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide. Inhibition of either protein kinase A (PKA) or the binding between A-kinase-anchoring proteins (AKAPs) and the regulatory PKA RII subunit blocked the 2-MeSADP-induced activation of CFTR, suggesting that PKA mediates P2Y1 receptor regulation of CFTR through one or more AKAPs. Further, the truncation of the PDZ1 domain of the scaffolding protein Na+/H+ exchanger regulatory factor-2 (NHERF-2) inhibited 2-MeSADP-dependent stimulation of Cl- efflux, suggesting the involvement of this scaffolding protein. Activation or inhibition of PKC had no effect per se on basal Cl- efflux but potentiated or reduced the 2-MeSADP-dependent stimulation of Cl- efflux, respectively. These data suggest that theX. laevisP2Y1 receptor in A6 cells can increase both cAMP/PKA and Ca2+/PKC intracellular levels and that the PKC pathway is involved in CFTR activation via potentiation of the PKA pathway. [ABSTRACT FROM AUTHOR]

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y1 agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y1 antagonists, MRS 2179 (N6-methyl-2?-deoxyadenosine-3?,5?-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-g-S but not by UTP. 2-MeSADP induced a P2Y1 antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that in A(6) renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A(1) and A(2A) receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A(3) receptor-selective agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca(2+) influx. The agonist-induced rise in [Ca(2+)](i) was significantly inhibited by the selective adenosine A(3) receptor antagonists, 2,3-diethyl-4, 5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A(1) or A(2) receptors supporting the hypothesis that Cl-IB-MECA increases [Ca(2+)](i) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elicited Ca(2+) entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca(2+)](i) response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data suggest that in A(6) cells a G(s)/protein kinase A pathway is involved in the A(3) receptor-dependent increase in calcium entry. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that in A6 renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A1 and A2A receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A3 receptor-selective agonist, 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca2+ influx. The agonist-induced rise in [Ca2+]i was significantly inhibited by the selective adenosine A3 receptor antagonists, 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A1 or A2 receptors supporting the hypothesis that Cl-IB-MECA increases [Ca2+]i by interacting exclusively with A3 receptors. Cl-IB-MECA-elicited Ca2+ entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited [Ca2+]i response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A3 receptor antagonist. Altogether, these data suggest that in A6 cells a Gs/protein kinase A pathway is involved in the A3 receptor-dependent increase in calcium entry. [ABSTRACT FROM AUTHOR]

NHE3 is most likely the isoform involved in renal reabsorption of HCO3 –and Na+. The functional properties of the ”cloned” NHE3 isoform, including its transport regulation by extra- and intracellular stimuli, have so far been studied using non-epithelial expression systems. In the present report we stably transfected NHE3 cDNA (rabbit isoform) into Madin-Darby canine kidney cells (MDCK) cells and compared the sensitivity to inhibitors and the regulation of the Na+/H+-exchanger by vasotocin in NHE3 transfectants to that of the intrinsic basolateral Na+/H+-exchanger in untransfected and control transfected MDCK cells. By Southern blot analysis we documented that the NHE3 transcript is expressed in NHE3 transfectants. Na+/H+-exchange activity, measured as sodium-dependent recovery of intracellular pH from an acid load using 2′, 7′-bis(carboxymethyl)-5(6)-carboxy-fluorescein (BCECF), was equally present at the basolateral cell surface of all cell lines; however, NHE3 transfectants demonstrated transport activity in the apical membrane that was significantly higher than that in untransfected or control transfected MDCK cells. Studies with ethylisopropylamiloride (EIPA) have shown that there is a similar sensitivity to inhibitors of the apical and/or basolateral Na+/H+-exchanger in transfected and untransfected MDCK cell lines. In contrast, the apical Na+/H+-exchanger (as compared to the basolateral Na+/H+-exchanger) of NHE3 transfectants was found to be relatively insensitive to the inhibitor HOE 694. Vasotocin decreased the activity of the apical Na+/H+-exchanger in NHE3 transfectants and stimulated the activity of the basolateral Na+/H+-exchanger in transfected (with NHE3 or pMAMneo) and untransfected MDCK cells. Phorbol ester, as expected, increased the activity of the Na+/H+-exchanger in the basolateral membrane of all cell lines; also, it stimulated transport activity at the apical cell surface of NHE3 transfectants. No change of Na+/H+-exchange activities was seen in studies with 8-bromo-cAMP. The PKC inhibitor calphostin C completely suppressed regulation of the apical and/or basolateral Na+/H+-exchanger by vasotocin, it partially blocked activation of the apical Na+/H+-exchanger in NHE3 transfectants by phorbol 12-myristate 13-acetate (PMA), and completely blocked stimulation of basolateral Na+-exchanger by PMA. Consistent with a V+receptor action, the effects of vasotocin in NHE3 transfectants and in MDCK cells were blocked by the V1 receptor action, the effects of vasotocin in NHE3 transfectants and in MDCK cells were blocked by the V1 receptor antagonist, d(CH2Tyr(Me)-AVP, but were not reproduced by the V5Tyr(Me)-AVP, but were not reproduced by the V2 receptor agonist desmopressin. It is concluded that NHE3 in the apical membrane of NHE3-transfected MDCK cells contributes to the differential regulation of the apical and basolateral Na+/H+-exchanger by vasotocin; NHE3 is inhibited and endogenous Na+-exchange activity is stimulated by vasotocin via V+-exchange activity is stimulated by vasotocin via V1 receptor activation of the protein kinase C pathway. [ABSTRACT FROM AUTHOR]

In the present study several aspects of the osmotic water-flow-regulation mechanism in frog urinary bladder were examined, utilizing forskolin either as a direct hydrosmotic agent or in association with vasopressin. It was found that forskolin induces a hydrosmotic effect similar to the one induced by vasopressin. This effect is rapid, reversible and dose-dependent. The half-maximally effective concentration (Ec) is 1.37 μM forskolin. No additional effect on the osmotic water flow was observed when maximal concentrations of forskolin and vasopressin were given simultaneously. Moreover, forskolin can also markedly potentiate vasopressin-induced hydrosmotic response. This potentiation was maximal with submaximal doses of vasopressin, whereas it disappeared when the hormonal concentration was increased to very high levels. Therefore, forskolin increases vasopressin potency without affecting vasopressin efficacy. The Ec for the forskolin-induced increase in vasopressin potency was 11 nmol, about two orders of magnitude lower than the Ec for the direct effect of forskolin on the osmotic water transport. On the whole, our results are compatible with a two-site model of forskolin action in frog urinary bladder: a low affinity site (Ec=1.37 μM) that mediates the direct action of forskolin on the osmotic water flow and a high affinity site (Ec=11 nmol), which mediates the synergic effect of forskolin with the antidiuretic hormone. [ABSTRACT FROM AUTHOR]

Treatment with the calcium ionophore A23187 on either the serosal or mucosal sides of frog skin, strongly inhibits the hydrosmotic response to vasopressin. On the contrary, the hydrosmotic response to 8-br-cAMP is not affected by treatment with the A23187. Trifluoperazine, a drug which inhibits the Ca-calmodulin complex, selectively inhibits vasopressin-induced water transport. Collectively, our results suggest that an increase in the intracellular concentration of Ca, obtained by treatment with the ionophore A23187, interferes with a pre-cAMP step of the hydrosmotic response to the antidiuretic hormone. Calcium ions could regulate adenyl-cyclase activity and consequently intracellular levels of cAMP. This effect may probably involve calmodulin. [ABSTRACT FROM AUTHOR]

This report presents evidence for urea active absorption by isolated skin of Rana esculenta. One of the supporting factors of such evidence is that at a low concentration the urea influx is five times greater than the outflux, in the absence of a chemical gradient. The transport shows a saturation kinetics with an apparent K=1.33mM and is inhibited by an uncoupling agent (ECCP). 5·10M Phloretin, added to the external side, markedly inhibits inward urea transport, whereas it is ineffective when added to the serosal fluid. This provides evidence for a phloretin-sensitive mechanism located at the external side of the epithelium. Phloretin stimulates the sodium active transport; the possible coupling of urea and sodium movement is analysed [ABSTRACT FROM AUTHOR]

The effect of microtubular-poisons, such as colchicine and vincristine, on frog skin permeability has been investigated. Three-hour treatment with the drugs has no effect on nonelectrolyte basal transepithelial permeability, but completely supresses the effect of ADH. Colchicine and vincristine, in addition, affect both basal sodium transport and the rise in short circuit current induced by vasopressin. The inhibition produced by microtubular-poisons disappears, however, when hydrocortisone, a glucocorticoid known to preserve junctional communications is used. Together with the results previously obtained with isolated epithelial cells (Svelto et al. 1979), these findings provide further support for our hypothesis that the microtubularmicrofilament-system, is involved in cell-to-cell exchange. [ABSTRACT FROM AUTHOR]

Transepithelial urea outfluxes across toad gallbladder were determined before and after the addition of cycloheximide. The drug inhibits the movement of urea but has not effect on thiourea and antipyrine outfluxes. The inhibition of amide transport is time dependent as also shown in counterflow experiments. These results are consistent with the hypothesis that cycloheximide inhibits the synthesis of membrane proteic sites involved in urea mediated transport. [ABSTRACT FROM AUTHOR]

The toad gallbladder epithelium is much more selective than that of the rabbit expecially as to the permeability of two molecules like urea and thiourea. These observations can probably be attributed to different permeation mechanisms of the 2 molecules. Neither active transport nor solvent drag can explain these phenomena. 10 M phloretin strongly inhibits urea movement, but does not alter either thiourea fluxes or isotonic net water transport: these results suggest that a specific mechanism is involved in urea movement. The urea transport shows saturation kinetic which is consistent with the presence of a facilitated mechanism. [ABSTRACT FROM AUTHOR]

Amphotericin B treatment increases the thiourea, d-xylose and mannitol fluxes and lowers those of urea, N-methyl-urea, acetamide, formamide, and N-N′-dimethyl-thiourea. The degree of flux inhibition is related to the cellular permeability of these compounds. Most probably Amphotericin B increases the permeability of all those molecules across the luminal plasma membrane, but simultaneously elicits a cellular swelling, which reduces the diffusion across the lateral plasma membranes. This effect masks the polyene effect especially for molecules showing a mainly cellular permeation pathway such as amides and lipid, soluble molecules. [ABSTRACT FROM AUTHOR]

The effect of adenosine regulation on sodium and chloride transport was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on short-circuit current (Isc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the Isc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A2 selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic Isc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A1 adenosine antagonist, CPX, completely prevented this response. This Isc response to apical CPA was also strongly reduced in Cl--free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca2+]i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A2 adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl- secretion via A1 receptor-mediated changes in [Ca2+]i. [ABSTRACT FROM AUTHOR]

We have used a well-differentiated A6-cell preparation (A6-C1) to study cellular location and vasopressin control of Na/H-exchange activity. After cell acidification, cell pHi (measured by BCECF-fluorescence) only recovered by the addition of Na medium to the basolateral cell surface; this pHi recovery was inhibited by dimethylamiloride (2 microM) consistent with basolateral location of Na/H-exchange activity. Addition of vasopressin produced stimulation of Na/H-exchange activity and increased the affinity of the exchanger for Na+. Stimulation of Na/H exchange was mimicked by pharmacological activation of protein kinase A (forskolin, 8-Br-cAMP) and not by pharmacological activation of protein kinase C (TPA). It is concluded that basolaterally located Na/H-exchange in A6-C1 cells is activated by vasopressin. [ABSTRACT FROM AUTHOR]

We have analyzed the mechanism of Na(+)-dependent pHi recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 2'7'-bis(2-carboxyethyl)5,6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na+/H(+)-exchange activity only on the basolateral membrane: Na+/H(+)-exchange activity follows simple saturation kinetics with an apparent Km for Na+ of approximately 11 mM; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na+/H(+)-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na+/H(+)-exchange activity; AVP stimulates IP3 production at low concentrations, whereas much higher concentrations are required to stimulate cAMP formation. These findings suggest that in A6 cells (i) Na+/H(+)-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP3 turnover) is likely to be the mediator of AVP action at low AVP concentrations. [ABSTRACT FROM AUTHOR]

Serosal preincubation of frog skin with tetradecanoyl phorbol acetate, TPA, an activator of protein kinase C, inhibits the hydrosmotic response elicited by vasopressin (AVP) but not that induced by 8br-cAMP. This proves that serosal TPA primarily influences a pre-cAMP step. The TPA-induced inhibition of AVP response appears to be related to TPA-induced prostaglandin synthesis. The pretreatment with naproxen, in fact, prevents the inhibition induced by serosal TPA on the AVP response. On the contrary, mucosal TPA produces a more marked inhibition of the response to AVP and significantly diminishes the water flow induced by 8br-cAMP; this suggests that mucosal TPA interferes mainly with a post-cAMP step. Furthermore, naproxen is unable to completely prevent the inhibition induced by mucosal TPA on AVP response thus indicating that mucosal TPA may also activate a prostaglandin-independent mechanism able to inhibit one of the last steps of the hydrosmotic response to AVP. [ABSTRACT FROM AUTHOR]