A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin.
1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase.
Broad
L M
LM
Department of Pharmacology, Tennis Court Road, Cambridge CB2 1QJ, UK.
Cannon
T R
TR
Taylor
C W
CW
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005682:Gadolinium / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D008345:Manganese / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D013324:Strontium / Q000378:metabolism
D001127:Arginine Vasopressin; D016718:Arachidonic Acid; D008345:Manganese; D005682:Gadolinium; D010727:Phosphoric Diester Hydrolases; C021491:glycerophosphoinositol glycerophosphodiesterase; D002118:Calcium; D013324:Strontium