Does nitric oxide regulate capacitative Ca influx in HEK 293 cells?
It has been proposed that capacitative Ca influx into both pancreatic acinar cells and HT-29 colonic cells is regulated by stimulation of nitric oxide synthase (NOS). NO, in turn, controls cGMP levels through effects on guanylate cyclase. We tested this possibility by measuring Ca (and Ba) entry into human embryonic kidney 293 cells and into 293 cells that had been transfected with the neuronal NOS gene (293/NOS). 293 cells had undetectable levels of NOS, while 293/NOS cells exhibited very high levels [Bredt D.S., Ferris C.D., Snyder S.H. Nitric oxide synthase regulatory sites. J Biol Chem 1992; 267: 10976-10981]. Ca (or Ba) entry into single cells was measured as the rate of increase of the Fura-2 fluorescence ratio (digital imaging microscopy) during rapid changes from Ca-free (or Ba-free) to Ca- (or Ba-) containing solutions (using high K to depolarize the membrane potential). cGMP levels (EIA method) were measured to correlate to rates of Ca entry. 100 microM ATP caused release of Ca from internal stores, but no sustained plateau due to Ca entry in either 293 or 293/NOS cells. Cyclopiazonic acid (CPA, which inhibits the Ca pump of the internal store, allowing Ca to leak from the store) caused apparent Ca entry to increase 5-10-fold from similar, low levels in both 293 and 293/NOS cells. CPA-stimulated Ca entry was unaffected by the NOS inhibitor N-nitro-L-arginine (L-NA) in either 293 or 293/NOS cells. In 293 cells [cGMP] was low; ATP and CPA both increased [cGMP] by 2-fold, and the guanylate cyclase inhibitor LY83583 and L-NA decreased [cGMP] by 50-75%. [cGMP] was 20-fold higher in 293/NOS cells than in 293 cells; these [cGMP] were not affected by ATP and CPA, but were effectively decreased by 80-90% by L-NA and by LY83583. Thus, [cGMP] and Ca or Ba entry showed no relationship to each other: Ca entry was small into cells in which [cGMP] was either low (resting 293, CPA + L-NA or CPA + LY83583), intermediate (ATP-treated 293) or high (resting 293/NOS). Similarly, Ca entry was high into cells in which [cGMP] was low (CPA + L-NA- or CPA + LY83583-treated 293), intermediate (CPA-treated 293 and CPA + L-NA-treated 293/NOS) or high (CPA- or ATP-treated 293/NOS). We conclude that, as in most other non-excitable cells, Ca entry into 293 cells is stimulated by loss of Ca from the store but, unlike pancreatic and colonic cells, this capacitative Ca entry does not appear to be regulated by NO and cGMP. Therefore, although capacitative entry across the plasma membrane may be regulated by NO and cGMP in Gl epithelial cells, this regulation does not occur in all cells.
Bischof
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Department of Molecular and Cell Biology, University of California, Berkeley 94720-3200, USA.
Serwold
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Machen
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000634:Aminoquinolines / Q000494:pharmacology; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D006152:Cyclic GMP / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D019335:Nitroarginine / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D014162:Transfection
D000634:Aminoquinolines; D004791:Enzyme Inhibitors; D007211:Indoles; D019335:Nitroarginine; D001464:Barium; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; C041715:6-anilino-5,8-quinolinedione; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D000252:Calcium-Transporting ATPases; D006152:Cyclic GMP; D011188:Potassium; D002118:Calcium; C000543:cyclopiazonic acid