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MATERIALS AND METHODS

A total of 71 male New Zealand White rabbits weighing 2.5 to 3.0 kg were used. They were maintained on standard chow and had free access to tap water. In vivo experimental interventions were followed by in vitro studies of cells or tissues isolated after rabbits were sacrificed. To administer aldosterone or spironolactone we implanted osmotic minipumps (Alza, Palo Alto, CA) subcutaneously in the interscapular region under a general anesthetic of 2% halothane with two parts nitrous oxide and one part oxygen. Aldosterone and spironolactone were dissolved in a stock solution of ethanol and diluted in 0.9% sterile saline. Control rabbits were infused with the ethanol vehicle only. Blood was collected for determination of serum concentrations of K+ and plasma concentrations of aldosterone at the time of implantation of minipumps and immediately before the rabbits were sacrificed. K+ concentrations were measured by flame photometry and aldosterone concentrations by radioimmunoassay. When treatment protocols had been completed we anesthetized the rabbits with intramuscular ketamine (50 mg kg-1) and xylazine hydrochloride (20 mg kg-1) and excised the heart. Experimental protocols were approved by the institutional ethics committee at Royal North Shore Hospital, Sydney and at Rigshospitalet, Copenhagen and were conducted in accord with the legislation given by the Danish Ministry of Justice.

Measurement of Na+-K+ pump current

Single myocytes from either ventricle were isolated and voltage clamped with wide-tipped (4-5 mm) patch pipettes as described previously (10). The pipettes were filled with a solution containing (in mmol/L): 70 K glutamate, 1 KH2PO4, 5 HEPES, 5 ethylene glycol-bis (ß-aminoethyl ether)-N,N, N',N'-tetraacetic acid (EGTA), 2 MgATP, 10 or 80 Na glutamate and 80 or 10 tetramethyl-ammonium chloride (TMA-Cl). The solution was titrated with 1 mol/L KOH to a pH 7.20  0.01 at 22 oC. The filled patch pipettes had resistances of 0.9 - 1.1 MW. For measurement of Ip myocytes were superfused with modified Tyrode’s solution which contained (in mmol/L): 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 1.0 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The solution was titrated with 1 mol/L NaOH to pH of 7.40  0.01 at 35 oC. This solution was used while the whole-cell configuration was established and the membrane capacitance was measured. The superfusate was then changed to one that was identical except that it was nominally Ca2+-free and contained 0.2 mmol/L CdCl2 and 2 mmol/L BaCl2. All solutions were warmed to 35 oC. Ip was identified in myocytes voltage clamped at -40 mV as the shift in holding current induced by 100 mmol/L ouabain. Reported currents are normalized for membrane capacitance. Details of the experimental set-up and of the experimental protocols used to measure membrane capacitance and Ip have been described previously (11).

Measurement of 3H-ouabain binding and K+-dependent pNPPase activity

Vanadate-facilitated 3H-ouabain binding to intact left ventricular samples of 2-4 mg (wet weight) was performed as previously described in detail for rat skeletal muscle (12). K+-dependent pNPPase activity was determined in crude homogenates (10 mg tissue/ml) as described previously (13). We incubated 100 ml homogenate at 37 oC in 800 ml buffer and calculated the difference using buffer containing (in mmol/L) 25 histidine, 15 MgCl2 and 100 NaCl (pH 7.4) versus buffer containing 25 histidine, 15 MgCl2 and 50 KCl (pH 7.4). The phosphatase reaction was started by addition of 100 ml 100 mmol/L pNNP and stopped after 30 min by addition of 2 ml ice cold buffer containing 500 mmol/L tris and 55 mmol/L EDTA. The liberated p-nitrophenyl was determined by spectrophotometry at a wavelength of 410 nm using a photometer 4020 (Hitachi, Tokyo, Japan).

Measurement of tissue K+ content and intracellular Na+ activity

Tissue K+ content was measured in samples of ~25 mg wet weight by flame photometry using lithium as an internal standard. All measurements were made in duplicate. Details have been previously described (14). Intracellular Na+ activity (aiNa) was measured in intact isolated right ventricular papillary muscles with Na+-sensitive microelectrodes as described previously (10, 11).

Reagents & Chemicals

Aldosterone, spironolactone, ouabain, dihydroouabain (DHO) and pNPP were purchased from Sigma Chemical Company, St. Louis, MO, USA. Tetramethyl-ammonium chloride (TMA-Cl) was "purum" grade and purchased from Fluka (Switzerland). 3H-ouabain was from Amersham International, Buckinghamshire, UK. Vanadate was purchased from Merck, Darmstadt, Germany. Chemicals used for the K+-dependent pNPPase activity, 3H-ouabain binding and tissue K+ content experiments were purchased from Bie and Berntsen (Denmark). All other chemicals were purchased from BDH (Australia). All chemicals were "analytical" grade.

Statistical analysis

Results are expressed as means  SE. Statistical comparisons were made by both paired and unpaired Student's t-test and one-way analysis of variance followed by Tukey's test. Differences were regarded as statistically significant when P<0.05.