Purinergic receptor signaling at the basolateral membrane of macula densa cells

Purinergic receptors are important in the regulation of renal hemodynamics, therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca2+ sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca2+](i)) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2-MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca2+](i) with addition of adenosine (10(-7) to 10(-3) M). UTP and ATP (10(-4) M) caused [Ca2+](i) to increase by 268 +/- 40 nM (n = 21) and 295 +/- 53 nM (n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca2+](i) increased by only 67 +/- 13 nM (n = 8) and 93 +/- 36 nM (n = 14), respectively. Dose response curve for ATP (10(-7) to 10(-3) M) added in bath showed an EC50 of 15 muM. No effect on macula densa [Ca2+](i) was seen when ATP was added from the lumen. ATP caused similar increases in macula densa [Ca2+](i) in the presence or absence of bath Ca2+ and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca2+](i) dynamics. Also, ATP-Ca2+ responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that macula densa cells possess P2Y(2) purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca2+.