Methods

Rat and rabbit vas deferens. Prostatic segments of rat (Chbb: Thom, 300 g) and rabbit(Chbb: NZW, 3-3.5 kg, live-stock breeding of Dr. Karl Thomas GmbH, Biberach, Germany) vasa deferentia were dissected and suspended in 25-mi organ baths at 37#{176} Ccontaining a modified Krebs’ buffer solution of the following composition(mill-molar); NaCI, 118; KC1, 4.8; MgSO4, 1.2; CaCl2, 2.5; NaH2PO4, 1.2; NaHCO3, 25; and glucose, 11.1; gassed continuously with 5% C02-95% 02. Yohimbine(1 tM) was added to the buffer solution to block prejunctional alpha receptors. Before the start of the experiments the preparations were allowed to equilibrate for about 60 mm with an initial tension of 10 mN. Electrical field stimulation(square-wave pulses at 0.15 Hz, 1 msec duration and submaximal voltage) was applied to stimulate smooth muscle contraction. After 15 mm of electrical field stimulation, cumulative concentrations of NPY (0.1 nM-3 ELM) were added to the organ bath. Twitch responses were recorded isometrically and inhibition was expressed in percentages. EC50 values were determined by nonlinear regression analysis. Antagonist affinity was determined by obtaining concentration-response curves to NPY in the presence of BIBP 3226 1 (R)-N2-(diphenylacetyi)-N-[(4-hydroxyphenyl) methyl]-D-arginine-amide)(0.3-10 tM) allowing a 15-mm equilibration ofthe antagonist. In order to determine the pA value for BIBP 3226, linear regression plots according to Arunlakshana and Schild(1959) were constructed. The slopes oftheA-S plots were not significantly different from unity, so pK,, values were obtained from plots constrained to a slope of 1 (Tallarida and Jacob, 1979). Isolated perfused rat kidney. Male rats (Chbb Thom, 300-350 g, live-stock breeding of Dr. Karl Thomas GmbH, Biberach, Germany) were anesthetized with pentobarbital sodium (60 mg/kg ip), and the right kidneys were taken for isolated perfusion. After the abdomen was incised at the midline, the abdomen was opened and the left renal artery, renal vein and ureter were prepared free and cannu-iated. Perfusion was started immediately upon interruption of aortic blood flow. The kidney was then removed and placed on a thermo-stated plastic holder. Perfusion pressure was kept constant at 100 mm Hg by adjustment of the pump rate (approximately 6 mI/mm). The kidneys were perfused at 37#{176} Cwith a modified Krebs’ buffer(for composition see above). After an equilibrium period of 60 mm, NPY was administered by a bolus injection(0.1 ml) at a concentration of

0.1 nM at 15-mm intervals. Two control applications were performed before the antagonist was investigated. Antagonists were infused during 5 mmn (0.025 mI/mm) in the kidney followed by NPY admin-istration. In each kidney, three to five different concentrations of the antagonist were examined. ICso values for the antagonists were calculated by plotting the percentage of inhibition of the NPY-in-duced increase in perfusion pressure against the concentration of the antagonist. Isolated perfused rat mesentery. Male rats (Chbb: Thom 300-350 g, live-stock breeding of Dr. Karl Thomas GmbH, Biberach, Germany) were anesthetized with pentobarbital sodium (60 mg/kg ip). The abdomen was opened and the mesenteric artery was cannulated at its origin with a PE-50 tubing. The mesenteric artery bed was excised, placed on a thermostated plastic holder and perfused at a rate of 5.0 mI/mm. The modified Krebs’ perfusion solution (for