Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.
Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002217:Carbachol / Q000494:pharmacology; D002108:Ceruletide / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D001095:Arachidonic Acids; D004987:Ethers; D007295:Inositol Phosphates; D007476:Ionophores; D010716:Phosphatidylinositols; D013873:Thionucleotides; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015759:Ionomycin; D006160:Guanosine Triphosphate; D002108:Ceruletide; D002217:Carbachol; D010738:Type C Phospholipases; D002118:Calcium