Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions.

BACKGROUND: The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes. METHODS: We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-(32)P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation. RESULTS: This activity was linear with time up to 20min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5mM MgCl(2) was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN(3)) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN(3). The dose-response of ATP revealed a hyperbolic profile with maximal velocity of 25.2±1.2nmol Pixmg(-1)xmin(-1) and K(0.5) of 0.07±0.01mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60min of ischemia. CONCLUSION: Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia. GENERAL SIGNIFICANCE: This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.

Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling.

Guanine-induced inhibition of renal Na(+)-ATPase activity: evidence for the involvement of the Gi protein-coupled receptor.

There is some evidence to show a possible role of guanosine in the modulation of cellular function, in particular, in the neuronal system. However, nothing is known about the role of guanine in renal function. The aim of the present work was to investigate the role of guanine on modulation of Na+-ATPase activity in isolated basolateral membrane (BLM) of the renal cortex. Guanine inhibited the enzyme activity in a dose-dependent manner with maximal effect (56%) obtained at 10⁻6 M. This effect was reversed by DPCPX (8-cyclopentyl-1,3-dipropylxanthine), an antagonist of A1 receptors, but it was not changed by 10⁻8 M DMPX (3,7-dimethyl-1-propargylxanthine) or 10⁻8 M MRS (2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate), antagonists of A2 and A3 receptors, respectively. Furthermore, it was observed that guanine increased [γ-³5S]GTP-specific binding with the maximal effect observed at 10⁻6 M and this effect was abolished by 10⁻6 M GDPßS. The inhibitory effect of 10⁻6 M guanine on Na+-ATPase activity was reversed by 10⁻6 M GDPßS, 10⁻6 M forskolin, 10⁻6 M pertussis toxin and 10⁻8 M cholera toxin. These results indicate that guanine binds to a DPCPX-sensitive receptor promoting the activation of Gi protein and leading to a decrease in cAMP level and, consequently, inhibition of BLM Na+-ATPase activity.

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.

Adenine-induced inhibition of Na(+)-ATPase activity: Evidence for involvement of the Gi protein-coupled receptor in the cAMP signaling pathway.

In the present work, we demonstrate that adenine reduced Na(+)-ATPase activity in isolated basolateral membrane (BLM) of proximal tubule in a dose-dependent manner. Adenine metabolism was ruled out by TLC analysis of the potential [(3)H]adenine derived-metabolites. Specific binding of [(3)H]adenine to isolated BLM was observed in a dose-dependent manner with K(d) and B(max) of 242.6+/-27.6 nM and 2749.9+/-104.9 fmolmg(-1), respectively. Adenine increased the [(35)S]GTPgammaS specific binding and it was completely abolished by 10(-6)M GDPbetaS (G protein inhibitor) but it was not modified by DPCPX, DMPX and MRS1523, selective antagonists for A(1), A(2) and A(3) receptors, respectively. Furthermore, the inhibitory effect of adenine on the Na(+)-ATPase activity was blocked by 10(-6)M GDPbetaS, 1 microg/ml pertussis toxin (Gi protein inhibitor), 10(-6)M foskolin (adenylyl cyclase activator) and 10(-8)M cAMP. These data demonstrate that adenine inhibits the proximal tubule Na(+)-ATPase activity through the Gi protein-coupled receptor.

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.