Autoradiography. For autoradiographic localization of [3H]BK receptor binding, guinea pigs were perfused through the heart with 500 ml of a mixture of equal parts of isotonic phosphate-buffered saline (pH 7.4) and 10%o (wt/vol) sucrose. After perfusion, the tissues were removed, embedded in homogenized calf cortex, and frozen on dry ice. Canine tissues were removed without perfusion and immersed in chilled perfusion medium for 20 min, then mounted and frozen as described. Ten-micrometer sections were cut in a cryostat at -12°C, thaw-mounted onto chrome alum/gelatin-coated microscope slides, and stored frozen at -20°C.When ready for use, the slides were warmed to room temperature, incubated for 120 min at 4°C in medium containing 20 mM 2-{[tris(hydroxymethyl)methyl]amino}ethane sulfonic acid (Tes) (pH 6.8), 1 mM 1,10-phenanthroline, 0.2% bovine serum albumin (protease free), 1 mM dithiothreitol, bacitracin (140 ,ug/ml), 0.1 mM SQ20,881, 300 mM sucrose, and 0.5 nM [3H]BK (52 Ci/mmol; 1 Ci = 37 GBq).Blanks were incubated in the same medium with 1 ,uM unlabeled BK or Lys-BK.After incubation, the tissue sections were washed in a solution of 25 mM Tes (pH 6.8) and 10% (vol/vol) sucrose for two 10-min periods at 4°C, dried rapidly under cold dry air, and apposed to either NTB-3 emulsion-coated coverslips or LKB Ultrafilm for 1 month. After exposure, the coverslip/slide or Ultrafilm was developed, and the tissue was stained with cresyl violet.Isolated Tissues. Smooth muscle effects of the BK analogs were determined on the isolated rat uterus and isolated guinea pig ileum by standard methods (10). BK analogs were incubated with the preparation for 30 sec before adding an ED50 dose of BK. BK analogs that decreased BK were then examined in detail to determine pA2 values for antagonism (pA2 is the negative log of the antagonist concentration that causes a 2-fold shift in the agonist dose-response curve) (11). Dose-response curves were determined for analogs that produced contractions at 10 uM.BK-Induced Vascular Pain. Male Sprague-Dawley rats (200-300 g) (Charles River Breeding Laboratories) were housed five per cage with constant access to food and water with a 12-hr light-dark cycle (7 a.m.-7 p.m.). Rats received an indwelling carotid artery cannula routed subcutaneously to exit behind the head. One to 3 days following surgery, the cannula was connected through tubing to a remote syringe allowing intraarterial injections while the rats were conscious and freely moving. In preliminary experiments, BK at 2 nmol/kg caused stereotypic flexion of the right forelimb and dextrorotation of the head in essentially 100% of the rats tested. Rats that displayed this BK response were injected 5 min later, through the same arterial cannula, with BK and a BK antagonist at 2, 20, or 200 nmol/kg.
