Distinct signalling cascades downstream to Gsalpha coupled dopamine D1-like NHE3 inhibition in rat and opossum renal epithelial cells.

Dopamine D1-like receptors are linked via G proteins to multiple cellular signaling pathways, namely adenylyl cyclase (AC) and phospholipase C (PLC). We have previously shown that the D1-mediated inhibition of Na+-K+-ATPase activity in OK cells involves the sequential activation of the AC-protein kinase A (AC-PKA) and the PLC-protein kinase C (PLC-PKC) pathways. The present study evaluated signaling cascades involved in dopamine-mediated inhibition of Na+/H+ exchanger isoform 3 (NHE3) in rat and opossum renal cells. Na+/H+ exchanger activity was assayed as the initial rate of intracellular pH (pHi) recovery after an acid load. Vmax values (in pH units/s) for Na+-dependent pHi recovery in rat cells (0.0097+/-0.0007) were greater (P<0.05) those in opossum cells (0.0063+/-0.0007), with similar Km values (in mM) for Na+ (rat, 35+/-9; opossum, 24+/-9). The IC50 values for EIPA and amiloride induced decrease in NHE activity in rat and opossum kidney cells are in agreement with the observation that rat renal proximal tubules and opossum kidney cells express mainly the NHE3 isoform. The D1-like receptor agonist SKF 38393 inhibited NHE3 activity in a concentration-dependent manner in both rat and opossum cells. The D1-mediated inhibition of NHE3 was prevented either by the D1-like receptor antagonist SKF 83566 (1 microM), overnight treatment with cholera toxin (500 ng/ml) and the PKA antagonist H-89 (10 microM) in rat and opossum kidney cells. The effect of SKF 38393 was abolished by the PKC antagonist chelerythrine (1 microM), or the PLC inhibitor U-73,122 (3 microM) in opossum cells, but not in rat cells. In addition, dibutyril cAMP (dB-cAMP; 500 microM) was found to increase PLC activity in OK cells but not in rat cells. The effect of D1-like dopamine agonist was accompanied by increases in cyclic AMP production in rat and opossum cells. The inhibitory effect of SKF 38393 (1 microM) on NHE3 activity was abolished in rat and opossum cells pre-treated with the anti-GSalpha antibody, but not in cells treated with the anti-Gq/11 alpha antibody. It is concluded that D1 agonists decrease NHE3 activity by classical stimulation of AC and PKA via GSalpha proteins in rat kidney cells. By contrast, the D1-mediated inhibition of NHE3 in renal opossum cells involves a peculiar mechanism with AC-PKA and PLC-PKC pathways. Copyright 2004 S. Karger AG, Basel