Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60 src tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60 src and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.

Cytoplasmic free calcium concentration ((Ca2+]i) was evaluated by dual-wavelength microspectrofluorometry of fura-2-loaded individual rat pancreatic acinar cells. Resting [Ca2+]i in unstimulated acini was 94.1 +/- 4.1 nM. Stimulation with high concentrations of cholecystokinin (CCK, 100 pM to 1 nM) led to an immediate rise in [Ca2+]i to 400-1,000 nM followed by a fall within 2-5 min to a plateau only slightly above the prestimulation level. Lower and more physiological concentrations of CCK (1-30 pM), after a latent period of 60-90 s, induced a smaller sustained increase in [Ca2+]i (30-40 nM) with superimposed repetitive transient [Ca2+]i spikes. These oscillations averaged 120-150 nM in amplitude, occurred at a frequency which averaged 1.5 times/min, and were maintained as long as the stimulus was applied. Similar [Ca2+]i oscillations were observed when acini were stimulated with submaximal concentrations of carbamylcholine (0.1-1 microM) and neuromedin C (0.1-1 nM). Intracellular Ca2+ stores were not depleted during [Ca2+] oscillations, since a subsequent increase to 1 nM CCK led to an immediate rise in [Ca2+]i indistinguishable from the response of cells initially stimulated at this concentration. Although extracellular Ca2+ was required for maintenance of frequency of the spikes, the major source of Ca2+ utilized for oscillations was intracellular, since elimination of medium Ca2+ or Ca2+ entry blockade with lanthanum failed to inhibit oscillations. Vasoactive intestinal polypeptide (10 nM) and high K+ (50 mM) did not affect [Ca2+]i oscillations. Antimycin (10 microM), which depletes cytoplasmic ATP, increased basal [Ca2+]i and inhibited the oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of gastrin, histamine, and carbachol to stimulate acid secretion by direct action on gastric parietal cells is well established but the role of intracellular Ca2+ concentration ([Ca2+]i) in mediating these effects is the subject of some controversy. To examine this issue further, secretagogue-mediated changes in [Ca2+]i in single isolated canine gastric parietal cells were examined by microspectrofluorometry of fura-2-loaded cells. Resting [Ca2+]i in single parietal cells was 63 +/- 6 (SE) nM. Carbachol, 10(-5) M, induced a maximum elevation in [Ca2+]i with an initial transient rise of 178 +/- 24 (SE) nM, which was maintained in the absence of extracellular Ca2+ and a sustained plateau of 112 +/- 20 (SE) nM, which was abolished by removal of extracellular Ca2+. Both effects were reversed by the muscarinic receptor antagonist atropine. Gastrin (10(-9)-10(-7) M) also induced a bimodal rise in [Ca2+]i with a maximal initial transient rise of 206 +/- 14 nM and a sustained plateau of 94 +/- 9 nM. Both components of the [Ca2+]i response to gastrin were reversed by the gastrin specific antagonist L 365260. Lower concentrations of gastrin (10(-10) M) induced repetitive transient increases (oscillations) in cytosolic Ca2+. The amplitude of the first spike was less than 50% of the transient rise in [Ca2+]i stimulated by 10(-8) M gastrin. The oscillations occurred at a rate of 0.9/min, gradually decreasing in amplitude within 15 min of secretagogue administration. Histamine (10(-4) M) led to a minimal rise in [Ca2+]i (less than 5% of control) in less than 10% of the canine parietal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The effects of bombesin on physiological responses (amylase secretion, protein synthesis) and intracellular mediators [inositol 1,4,5-trisphosphate (1,4,5-IP3), [Ca2+]i, and diacylglycerol] were studied in isolated rat pancreatic acini and compared with the actions of cholecystokinin (CCK). Bombesin stimulated amylase secretion to the same extent as CCK. However, it failed to reproduce the inhibition of amylase secretion by high concentrations of CCK and likewise did not inhibit incorporation of [3H]leucine into protein in contrast to high concentrations of CCK. Low concentrations of bombesin (1-100 pM) induced repetitive oscillations in [Ca2+]i, whereas higher concentrations of bombesin (1-10 nM) induced a large transient increase in [Ca2+]i followed by a small sustained plateau. Bombesin (1-100 nM) induced an early peak of 1,4,5-IP3 at 5-15 s but was without measurable effect at lower concentrations. These effects on [Ca2+]i and 1,4,5-IP3 were similar to those seen with CCK except that bombesin was approximately 10-fold less potent than CCK. Bombesin induced an increase in acinar 1,2-diacylglycerol with a biphasic time course similar to CCK. However, the magnitude of the response to bombesin was much smaller than the response to CCK. The results suggest that bombesin receptors initiate similar intracellular messengers as does CCK. However, CCK induces a larger increase of diacylglycerol and probably an as yet unidentified messenger responsible for its inhibitory effects.