Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb

The effect of antidiuretic hormone ([Arg]vasopressin, ADH) on intracellular calcium activity [Ca²⁺]_i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500–530 nm (R) was monitored as a measure of [Ca²⁺]_i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PD_te) and transepithelial resistance (R_te) of the tubule were measured simultaneously. After addition of ADH (1–4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84±0.05 to 1.36±0.08 (n = 46). Subsequently, within 7–12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca²⁺]_i from a basal level of 155±23 nmol/l [Ca²⁺]_i up to 429±53 nmol/l [Ca²⁺]_i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PD_te and R_te) of the tubules were not changed by ADH. The ADH-induced Ca²⁺ transient was dependent on the presence of Ca²⁺ on the basolateral side, whereas luminal Ca²⁺ had no effect. d(CH₂)₅[Tyr(Me)²]²,Arg⁸vasopressin, a V₁ antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca²⁺]_i completely (n = 5). The V₂ agonist 1-desamino-[d-Arg⁸]vasopressin (10 nmol/l, n=4), and the cAMP analogues, dibutyryl-cAMP (400 mol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mol/l, n = 1) or 8-bromo-cAMP (200 mol/1, n = 4) had no influence on [Ca²⁺]_i. The ADH-induced [Ca²⁺]_i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mol/l, n = 4). We conclude that ADH acts via V₁ receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca²⁺ release from intracellular stores and by further Ca²⁺ influx through Ca²⁺ channels in the basolateral membrane. These channels are insensitive to L-type Ca²⁺ channel blockers, e.g. nifedipine and verapamil.Supported by DFG GR 480/10