SENSITIZATION OF THE CONTRACTILE SYSTEM OF CANINE COLONIC SMOOTH-MUSCLE BY AGONISTS AND PHORBOL ESTER

1. Sensitization of the contractile system in response to combinations of excitatory agonists acetylcholine (ACh), methacholine, histamine a nd neurokinin A (NKA) was investigated in colonic circular smooth musc le of the dog. NKA (1 nM) potentiated the contractile response to 1 mu M ACh, but did not increase the fura-2 fluorescence ratio (R(340/380) ). Contraction in response to low concentrations of either methacholin e or histamine was potentiated significantly by 0.1 mu M 4-phorbol 12, 13-dibutyrate (PDBu), suggesting that activation of protein kinase C c an potentiate contraction at threshold concentrations of agonists. 2. Variability in the sensitivity of the contractile system to Ca2+ was d emonstrated over a range of agonist concentrations. KCl, ACh, histamin e and NKA each produced a concentration-dependent increase in the ampl itude of phasic contractions and R(340/380). However, ACh, histamine a nd NKA each induced maximal increases in R(340/380) at concentrations less than that needed to induce maximum force. 3. In depolarized muscl es, NKA (50 nM) and PDBu (1 mu M) each increased the magnitude of toni c contraction with no change or a decrease in both R(340/380) and myos in light chain phosphorylation. In alpha-toxin-permeabilized fibres, 0 .1 mu M PDBu and 1 mu M NKA shifted the Ca2+-force response to the lef t. Ca2+-induced contractions were also potentiated by 100 mu M GTP-gam ma-S or 1 mu M NKA plus 10 mu M GTP. Potentiation of contraction by NK A and GTP was antagonized by 10 mu M GDP-beta-S. 4. The results sugges t that endogenous agonists acting via G-proteins sensitize the contrac tile element of colonic smooth muscle in part by activation of protein kinase C. In some cases, sensitization may be secondary to increased myosin phosphorylation (ACh), but in other cases it appears to be inde pendent of increased myosin light chain phosphorylation (NKA and PDBu) . Therefore regulatory mechanisms in addition to myosin phosphorylatio n contribute to the apparent sensitization of the contractile system t o Ca2