Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.     

The inhibition of progesterone secretion and the regulation of cyclic nucleotides by atrial natriuretic factor in gonadotropin responsive murine Leydig tumor cells

We have found that atrial natriuretic factor (ANF) has a profound effect on testicular cells in altering intracellular cyclic nucleotide levels as well as progesterone secretion. Using clonal cultured Leydig tumor cells we found that 1 X 10(-8)M ANF caused a two thousand-fold elevation in the accumulation of cellular cGMP and inhibited cAMP in treated cells by more than 90% as compared to the controls. ANF (1 X 10(-8)M) also significantly inhibited gonadotropin-stimulated accumulation of cAMP in response to bovine luteinizing hormone (bLH) or human chorionic gonadotropin (hCG). Gonadotropin-stimulated progesterone secretion was inhibited by ANF (1 X 10(-10) - 1 X 10(-9)M) in these cultured Leydig tumor cells. Approximately 50% inhibition of progesterone secretion was observed at the peptide concentration of 1 X 10(-9) M.     

Atrial natriuretic factor and cGMP inhibit amiloride-sensitive Na+ transport in the cultured renal epithelial cell line, LLC-PK1

The renal cell culture model, LLC-PK1, which contains an amiloride-sensitive conductive Na+ transport pathway and a Na+/H+ exchanger, was utilized to examine the direct effects of atriopeptin II and cGMP on Na+ transport in epithelial cells. Exposure of cells to atriopeptin II (10(-7) M) increased cGMP production within 2 min of addition to cells in monolayer. Atriopeptin II (10(-7) M) or exogenous 8-bromo-cGMP (10(-3) M) maximally inhibited the uptake of 22Na+ through the conductive pathway which accounted for up to 60% of total 22Na+ uptake. The apparent Ki for this inhibition by atriopeptin II was 2 X 10(-11) M. Amiloride inhibited 22Na+ uptake to a similar extent as atriopeptin II, and the effects of the presence of both agents was not additive. In contrast, neither atriopeptin II nor cGMP blunted the increment in 22Na+ uptake induced by a pH gradient. Thus atriopeptin II can directly inhibit Na+ transport in renal epithelial cells, probably through its stimulation of cGMP.     

Regulation of cGMP production by intracellular alkalinization in cultured rat inner medullary collecting duct cells

We evaluated the relationship between cell pH and cGMP production in cultured rat renal inner medullary collecting duct cells. The cGMP level, 21 +/- 6, was not different in control vs. alkalinized cells, 49 +/- 17 fmol/mg protein (p greater than 0.5). 10(-11) M atrial natriuretic peptide (ANF) enhanced cGMP production in alkalinized cells, 426 +/- 34 vs. 141 +/- 9*. Conversely, alkalinization inhibited 10(-4)M nitroprusside (SNP) induced cGMP formation, 29 +/- 9 vs. 332 +/- 67*. Phosphodiesterase inhibition abolished the difference in cGMP production by ANF but did not reverse the inhibitory effect of alkalinization on SNP induced cGMP production. In rat renal inner medullary collecting duct cells, cellular alkalinization plays a significant role in the regulation of guanylate cyclase mediated cGMP production. * = p less than 0.05).     

Signaling pathways involved in atrial natriuretic factor and dopamine regulation of renal Na+, K+ -ATPase activity

Dopamine (DA) and atrial natriuretic factor (ANF) share a number of physiological effects. We hypothesized that ANF and the renal dopaminergic system could interact and enhance the natriuretic and diuretic effects of the peptide. We have previously reported that the ANF-stimulated DA uptake in renal tubular cells is mediated by the natriuretic peptide type-A receptor (NPR-A). Our aim was to investigate the signaling pathways that mediate ANF effects on renal 3H-DA uptake. Methylene blue (10 microM), an unspecific inhibitor of guanylate cyclase (GC), blunted ANF elicited increase of DA uptake. ODQ (10 microM) a specific inhibitor of soluble GC, did not modify DA uptake and did not reverse ANF-induced increase of DA uptake; then the participation of nitric oxide-dependent pathways must be discarded. The second messenger was the cGMP since the analogous 125 microM 8-Br-cGMP mimicked ANF effects. The specific inhibitor of the protein kinase G (PKG), KT 5823 (1 microM) blocked ANF effects indicating that PKG is involved. We examined if ANF effects on DA uptake were able to modify Na+, K+ -adenosine triphosphatase (Na+, K+ -ATPase) activity. The experiments were designed by means of inhibition of renal DA synthesis by carbidopa and neuronal DA uptake blocked by nomifensine. In these conditions renal Na+, K+ -ATPase activity was increased, in agreement with the decrease of DA availability. When in similar conditions, exogenous DA was added to the incubation medium, the activity of the enzyme tended to decrease, following to the restored availability of DA. The addition of ANF alone had similar effects to the addition of DA on the sodium pump, but when both were added together, the activity of Na(+), K(+)-ATPase was decreased. Moreover, the extraneuronal uptake blocker, hydrocortisone, inhibited the latter effect. In conclusion, ANF stimulates extraneuronal DA uptake in external cortex tissues by activation of NPR-A receptors coupled to GC and it signals through cGMP as second messenger and PKG. Dopamine and ANF may achieve their effects through a common pathway that involves reversible deactivation of renal tubular Na+, K+ -ATPase activity. This mechanism demonstrates a DA-ANF relationship involved in the modulation of both decreased sodium reabsorption and increased natriuresis.     

Phorbol esters inhibit phosphate uptake in opossum kidney cells: a model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.     

Atrial natriuretic factor stimulates renal dopamine uptake mediated by natriuretic peptide-type A receptor

To determine the effects of atrial natriuretic factor (ANF) on renal dopamine (DA) metabolism, 3H-DA and 3H-L-DOPA uptake by renal tubular cells was measured in experiments carried out in vitro in Sprague-Dawley rats. The receptor type involved was also analyzed. The results indicate that ANF increased at 30 min, DA uptake in a concentration-response fashion having 10 pM ANF as the threshold concentration. Conversely, the uptake of the precursor L-DOPA was not modified by the peptide. ANF effects were observed in tissues from external and juxtamedullar cortex and inner medulla. On this basis, 100 nM ANF was used to continue the studies in external cortex tissues. DA uptake was characterized as extraneuronal uptake, since 100 microM hydrocortisone blocked ANF-induced increase of DA uptake. Renal DA uptake was decreased at 0 degrees C and in sodium-free medium. The effects of ANF in these conditions were not present, confirming that renal DA uptake is mediated by temperature- and sodium-dependent transporters and that the peptide requires the presence of the ion to exhibit its actions on DA uptake. The biological natriuretic peptide type A receptor (NPR-A) mediates ANF effects, since 100 nM anantin, a specific blocker, reversed ANF-dependent increase of DA uptake. The natriuretic peptide type C receptor (NPR-C) is not involved, since the specific analogous 100 nM 4-23 ANF amide has no effect on renal DA uptake and does not alter the effects of 100 nM ANF. In conclusion, ANF stimulates DA uptake by kidney tubular cells. ANF effects are mediated by NPR-A receptors coupled to guanylate cyclase and cGMP as second messenger. The process involved was characterized as a typical extraneuronal uptake, and characterized as temperature- and sodium-dependent. This mechanism could be related to DA effects on sodium reabsorption and linked to ANF enhanced natriuresis in the kidney. The increment of endogenous DA into tubular cells, as a consequence of increased DA uptake, would permit D1 receptor recruitment and Na+,K+-ATPase activity inhibition, which results in decreased sodium reabsorption and increased natriuresis.     

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Modulation of rabbit ventricular cell volume and Na+/K+/2Cl- cotransport by cGMP and atrial natriuretic factor.

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2Cl- cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volume(test)/volume(control)) were determined. Exposure to 10 microM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 +/- 0.007; the ED50 was 0.77 +/- 0.33 microM. Activating guanylate cyclase with 100 microM sodium nitroprusside also decreased cell volume to 0.889 +/- 0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 microM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2Cl- cotransport was tested by blocking the cotransporter with 10 microM bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 microM 8-Br-cGMP failed to decrease cell volume. Replacement of Na+ with N-methyl-D-glucamine or Cl- with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2Cl-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 microM 8-Br-cGMP prevented the effect of 1 microM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 microM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprinast; 100 microM) amplified the volume decrease caused by a low dose of ANF (0.01 microM) approximately fivefold. In contrast, neither 100 microM 8-Br-cAMP nor 50 microM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2Cl- cotransport. Basal cGMP levels also appear to modulate cell volume.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

Effect of native and synthetic atrial natriuretic factor on cyclic GMP

Mammalian atrial cardiocyte granules contain a potent natriuretic and diuretic peptide. Since cGMP appears to be involved in the modulation of cholinergic and toxin-induced sodium transport, we examined the effect of atrial natriuretic factor (ANF) on this nucleotide. Atrial but not ventricular extracts elicited approximately a 28-fold increase of urinary cGMP excretion parallel to the natriuresis and diuresis. The atrial extracts also elevated cGMP levels in kidney slices and primary cultures of renal tubular cells. The effect of ANF on cGMP appeared to be specific since antibodies which were capable of inhibiting the ANF-induced diuresis also suppressed cGMP excretion. Furthermore, during the course of ANF purification, the ANF-induced increase of cGMP production by kidney cells paralleled the heightened specific natriuretic activity of the atrial factor. A synthetic peptide (8-33)-ANF similarly increased urinary plasma and kidney tubular cGMP levels. The exact mechanism of action of ANF on cGMP remains to be elucidated, but indirect inhibition of cGMP phosphodiesterase appears to participate in its effect.     

Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells

Atrial natriuretic factor (ANF) rapidly enhanced phosphorylation of plasma membrane proteins of bovine adrenal cortical cells. Pretreatment of the membranes with ANF (1 x 10(-8)M to 1 x 10(-7)M) resulted two- to four-fold in an incorporation of 32p-radioactivity from [gamma -32p]ATP as compared to the controls. The guanosine 3', 5' monophosphate (cGMP) which has been considered a second messenger of ANF also enhanced the phosphorylation of several proteins which were stimulated by ANF. However, the phosphorylation of certain proteins was stimulated differentially only by either ANF or cGMP. These results suggest that ANF-induced protein phosphorylation may play a role in transmembrane signalling pathway involving other second messenger(s) in addition to cGMP during the biological action of ANF.     

Atrial natriuretic peptide reduces cyclosporin toxicity in renal cells: role of cGMP and heme oxygenase-1.

Using cultured proximal renal tubular epithelial cells (LLC-PK1), the present study investigates the effect of atrial natriuretic peptide (ANP) on cytotoxicity induced by cyclosporin A (CsA). Preincubation with ANP (1-100 nM) protected LLC-PK1 cells from CsA-induced toxicity in a concentration-dependent manner. A cytoprotective effect comparable to ANP was observed when preincubating the cells with 8-bromo cGMP (1-100 microM) or the antioxidant heme oxygenase (HO) metabolite bilirubin (0.1-10 microM). ANP or cGMP produced increases in HO-1 protein levels at concentrations that were also effective in cellular protection. Moreover, incubation with ANP or 8-bromo cGMP led to increased HO activity, i.e., formation of bilirubin in the cell lysate (up to 3-fold over basal). Tin protoporphyrin-IX (SnPP; 19 microM), an inhibitor of HO activity, completely abolished ANP-induced cytoprotection. Our results demonstrate that HO-1 is a cellular target of ANP and cGMP in renal cells. HO-1 induction and ensuing formation of antioxidant metabolites may be a novel pathway by which ANP protects from CsA-dependent nephrotoxicity and preserves renal function.     

Atrial natriuretic factor inhibits vasopressin secretion from rat posterior pituitary

The effects of synthetic atrial natriuretic factor (ANF) were studied in superfused rat posterior pituitary gland. ANF (10(-6)M, 10(-10)M) significantly inhibited basal as well as KC1 (50 mM) or angiotensin II-stimulated immunoreactive arginine vasopressin secretion. The magnitude of inhibition was greater at 10(-6)M than at 10(-10)M. ANF also decreased cAMP secretion and increased cGMP secretion from the posterior pituitary. These results suggest that ANF directly acts on the posterior pituitary to inhibit arginine vasopressin secretion and that this effect is, at least, partly mediated by the changes in cyclic nucleotide production.     

Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulateproduction of guanosine 3',5'-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate(J_va). Comparedwith control solutions, SNP(10⁴ M) decreasedJ_va by 23% whenadministered luminally and by 35% when added to the peritubularperfusate. Stimulation of fluid uptake by luminal angiotensin II (ANGII; 10⁹ M) was abolished bySNP (10⁴ and10⁶ M). In proximal tubulesuspensions, ANF (10⁶ M)increased cGMP concentration to 143%, whereas SNP(10⁶,10⁵,10⁴,10³ M) raised cGMP to 231, 594, 687, and 880%, respectively.S-nitroso-N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANFonly affected ANG II-stimulated transport may be because of productionof higher concentrations of cGMP by SNP.

     

Down-regulation of atrial natriuretic factor receptors and correlation with cGMP stimulation in rat cultured vascular smooth muscle cells

The relationship between the binding of 125I-labeled rat ANF and the responsiveness in cGMP production of ANF receptors were examined in cultured rat thoracic smooth muscle cells after preexposure with the peptide. Binding assay of 125I-labeled ANF showed a specific, reversible and saturable binding with a KD value of 3.1 +/- 0.3 10(-10) M and a maximum binding (Bmax) of 240 +/- 30 fmol/10(6) cells. Pretreatment of the cells with increasing concentrations of unlabeled ANF (10(-9) M to 10(-7) M) resulted in a dose-dependent decrease of the number of binding sites without a change in the affinity. This effect was clearly associated with a desensitization of ANF-induced cGMP production.     

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.     

Atrial natriuretic factor decreases renal dopamine turnover and catabolism without modifying its release

Atrial natriuretic factor (ANF) and dopamine (DA) are both important regulators of sodium and water transport across renal proximal tubules. Many evidences suggest that some of ANF inhibitory effects on sodium and water reabsorption are mediated by dopaminergic mechanisms. We have previously reported that ANF stimulates extraneuronal DA uptake in external renal cortex by activation of NPR-A receptors coupled to cGMP signal and PKG. Moreover, ANF enhanced DA-induced inhibition of Na(+)-K(+) ATPase activity. The aim of the present study was to evaluate if ANF could alter also renal DA release, catabolism and turn over. The results indicate that ANF did not affect basal secretion of the amine in external renal cortex or its KCl-induced release, but diminished DA turn over. Moreover, ANF diminished COMT and did not alter MAO activity. In conclusion, present results as well as previous findings show that ANF modifies DA metabolism in rat external renal cortex by enhancing DA uptake and decreasing COMT activity. All those effects, taken together, may favor DA accumulation into renal cells and increase its endogenous content and availability. This would permit D1 receptor recruitment and stimulation and in turn, Na(+), K(+)-ATPase activity over inhibition that results in decreased sodium reabsorption. Therefore, ANF and DA could act via a common pathway to enhance natriuresis and diuresis.