Regulation of nitric oxide-responsive recombinant soluble guanylyl cyclase by calcium.
Calcium (Ca2+) and cyclic GMP (cGMP) subserve antagonistic functions that are reflected in their coordinated reciprocal regulation in physiological systems. However, molecular mechanisms by which Ca2+ regulates cGMP-dependent signaling remain incompletely defined. In this study, the inhibition of recombinant nitric oxide (NO)-stimulated soluble guanylyl cyclase (SGC) by Ca2+ was demonstrated. The alpha- and beta-subunits of recombinant rat SGC were heterologously coexpressed in HEK 293 cells which do not express NO synthase, whose Ca2+-stimulated activity can confound the effects of that cation on SGC. Ca2+ inhibited basal and NO-stimulated SGC in a concentration- and guanine nucleotide-dependent fashion. This cation inhibited SGC in crude cell extracts and immunopurified preparations. Ca2+ lowered both the Vmax and Km of SGC via an uncompetitive mechanism through direct interaction with the enzyme. In intact HEK 293 cells, increases in the intracellular Ca2+ concentration induced by ionomycin, a Ca2+ ionophore, and thapsigargin, which releases intracellular stores of that cation, inhibited NO-stimulated intracellular cGMP accumulation. Similarly, carbachol-induced elevation of the intracellular Ca2+ concentration inhibited NO-stimulated intracellular cGMP accumulation in HEK 293 cells. These data demonstrate that SGC behaves as a sensitive Ca2+ detector that may play a central role in coordinating the reciprocal regulation of Ca2+- and cGMP-dependent signaling mechanisms.
Parkinson
S J
SJ
Division of Clinical Pharmacology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Jovanovic
A
A
Jovanovic
S
S
Wagner
F
F
Terzic
A
A
Waldman
S A
SA
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology*; D002457:Cell Extracts / Q000037:antagonists & inhibitors; D002460:Cell Line; D006152:Cyclic GMP / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D012995:Solubility
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D006150:Guanine Nucleotides; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium