Mechanisms involved in carbachol-induced Ca(2+) sensitization of contractile elements in rat proximal and distal colon.

1. Mechanisms involved in Ca(2+) sensitization of contractile elements induced by the activation of muscarinic receptors in membrane-permeabilized preparations of the rat proximal and distal colon were studied. 2. In alpha-toxin-permeabilized preparations from the rat proximal and distal colon, Ca(2+) induced a rapid phasic and subsequent tonic component. After Ca(2+)-induced contraction reached a plateau, guanosine 5'-triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca(2+) sensitization). Y-27632, a rho-kinase inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization more significantly in the proximal colon than in the distal colon. 3. Y-27632 at 10 microm had no effect on Ca(2+)-induced contraction or slightly inhibited phorbol-12,13-dibutyrate-induced Ca(2+) sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization in the distal colon, but not in the proximal colon. The component of Ca(2+) sensitization that persisted after the chelerythrine treatment was completely inhibited by Y-27632. 4. In beta-escin-permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh-induced Ca(2+) sensitization, but PKC(19-31) did not. In the distal colon, C3 exoenzyme abolished GTP-induced Ca(2+) sensitization. It inhibited CCh-induced sensitization by 50 % and the remaining component was inhibited by PKC(19-31). 5. These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca(2+) sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.

Origin of ATP for Ca2+-induced contraction in the guinea-pig femoral artery.

Previously, we have described differences between the rat proximal colon and femoral artery with respect to the role of ATP newly synthesized by creatine kinase. In the present study the role of newly synthesized ATP was studied in the guinea-pig femoral artery to examine species differences. In the alpha-toxin-permeabilized preparation of the guinea-pig femoral artery, the rapid Ca(2+)-induced contraction was suppressed when creatine kinase activity was inhibited. The contraction was restored completely by treatment with NaN(3), an inhibitor of ecto-ATPase, the enzyme that breaks down exogenous ATP. Thus, ATP newly synthesized by creatine kinase may have no role in contraction of the guinea-pig femoral artery. This is in marked contrast to the rat femoral artery, in which Ca(2+)-induced contractions are almost completely inhibited by inhibition of creatine kinase activity but only partly restored by NaN(3). To characterize the difference between the guinea-pig and rat tissue, the origin of ATP required for contraction was determined in intact preparations. Monoiodoacetic acid, an inhibitor of glycolysis, inhibited the high K(+)-induced contraction in the guinea-pig femoral artery more potently than in the rat tissue. In contrast, an inhibitor of mitochondrial respiration, carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), inhibited contraction in femoral arteries from rats, but not from guinea-pigs. These results suggest that contraction in the rat femoral artery is dependent largely on oxidative phosphorylation, while contraction in the guinea-pig tissue is dependent only on glycolysis. Because oxidative phosphorylation generates ATP and phosphocreatine, while glycolysis generates only ATP, the strong dependence of the contraction of the rat femoral artery on the oxidative phosphorylation is consistent with its dependence on ATP newly synthesized by creatine kinase from ADP and phosphocreatine, as previously shown. Thus, it is proposed that ATP, newly synthesized by creatine kinase, in addition to ATP generated by oxidative phosphorylation, is utilized for contraction in the rat femoral artery, while glycolysis produces sufficient ATP for contraction in the guinea-pig femoral artery.

Essential role of ATP synthesized by creatine kinase in contraction of alpha-toxin permeabilized preparations of tonic type smooth muscle.

The role of ATP newly synthesized from ADP and phosphocreatine (PC) by creatine kinase (CK) in the contraction of tonic type smooth muscle, rat femoral artery was studied, since its necessity for phasic type smooth muscle was previously shown. In alpha-toxin-permeabilized preparations obtained from rat femoral artery, Ca(2+) induced a tonic type contraction in the presence of ATP and PC. Omission of PC inhibited significantly the contraction. Treatment of the preparations with 2,4-dinitrofluorobenzene, an inhibitor of CK, also inhibited the contraction. In the presence of ADP and PC, Ca(2+) also induced the contraction to a level comparable to that in the presence of ATP and PC. The extent of phosphorylated myosin light chain was fairly consistent with that of Ca(2+)-induced contraction under all experimental conditions planned above. These results suggest that ATP newly synthesized by CK essentially participates in the whole of the contraction in tonic type smooth muscle, although it participates only in a rapid phasic contraction in phasic type muscle as previously shown.