Activation of cyclic amp dependent protein kinase (cA-PK) is associated with relaxation of canine lower esophageal sphincter (LES) by isoproterenol but not by electrical field stimulation (EFS)

Abstract

ACTIVATION OF CYCLIC AMP DEPENDENT PROTEIN KINASB (CA-PK) IS ASSOCIATED WITH RELAXATION OF CANINE LOWER ESOPHAGEAL SPHINCTER (LES) BY ISOPROTERENOL BUT NOT BY ELECTRICAL FIELD STIMULATION (EFS). M.S. Bsmette, M. Graus, T.J. Torphy and H.S. Ormsbee, III. Departmentofmaxgy, Smith Kline 6 French Laboratories, Philadelphia, PA 19101. Relaxation of LES smooth muscle is associated with elevations of either CAMP or cGMP content. Since the biological effects of the cyclic nucleotides are produced through the activation of their respective kinases, we have begun to examine the ability of several drugs and EFS to increase the activity of CA-PK. The gastroesophageal junction was removed from anesthetized dogs. The tissue was opened along the lesser curvature and the mucosa removed. Strips of circular smooth muscle (1.0-1.5 cm hy 0.3 cm) were cut and placed in a” oxygenated Krebs solution at 37“C with a” initial tension of 2 g. Muscle strips were electrically stimulated (4.0 Hz, 1.0 msec, 65V) and only those that gained tqne spontaneously and relaxed to EFS were used. Frequency-response ~“1‘“~s to EFS and cumulative cancentration-response curves to sodium nitroprusside (SNP), and to selective phosphodiesterase inhibitors (M&B 22,948 and SK&F 94120) were constructed in the presence of atropine (luH), phentolamine (IouM), propranolol (luM), and meclofenamic acid (1uM). In experiments examining the action of isoproterenol, propranolol was not added to the buffer. At the conclusion of the concentration-response studies, tissues were flashfrozen for determination of :yclic nucleotide content and CA-PK activity. Cyclic nucleotida’content was measured by RIA. Protein kinase activity was measured by the incorporation of 32Pi into histone (Type IIA) in the presence and absence of CAMP and the activity ratios (-CANP/+CAMP) were determined. I” tissues treated with isoproterenol and SK&F 94120, the activity ratio of CA-Pd was significantly increased. On the other hand EFS, SNP, or M&B 22,94R did not produce a significant change in the activity ratio of CA-PK. This is the first demonstration qf a” activation of PK in smooth muscle of canine LES and supports OUT hypothesis that there exist both CAMP and cGMP mediated pathways of relaxation in LES.