title: Nitric oxide inhibits capacitative Ca2+ entry by suppression of mitochondrial Ca2+ handling.
abstract: 1. Nitric oxide (NO) is a key modulator of cellular Ca(2+) signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca(2+) entry (CCE) into HEK293 cells by impairment of mitochondrial Ca(2+) handling. 2. Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 micro M, respectively. 3. NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 micro M), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 micro M) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 micro M) failed to antagonize the inhibitory action of NO on CCE. 4. DEANO (1-10 micro M) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca(2+) levels, respectively, indicated that DEANO (10 micro M) depolarized mitochondria and suppressed mitochondrial Ca(2+) sequestration. The inhibitory effect of DEANO on Ca(2+) uptake into mitochondria was confirmed by recording mitochondrial Ca(2+) during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. 5. DEANO (10 micro M) failed to inhibit Ba(2+) entry into TG-stimulated cells when extracellular Ca(2+) was buffered below 1 micro M, while clear inhibition of Ba(2+) entry into store depleted cells was observed when extracellular Ca(2+) levels were above 10 micro M. Moreover, buffering of intracellular Ca(2+) by use of N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA/AM) eliminated inhibition of CCE by NO, indicating that the observed inhibitory effects are based on an intracellular, Ca(2+) dependent-regulatory process. 6. Our data demonstrate that NO effectively inhibits CCE cells by cGMP-independent suppression of mitochondrial function. We suggest disruption of local Ca(2+) handling by mitochondria as a key mechanism of NO induced suppression of CCE.
authors.affiliation: Department of Pharmacology and Toxicology, Karl-Franzens-University Graz, A-8010 Graz, Austria.
authors.forename: Baskaran
authors.initials: B
authors.lastname: Thyagarajan
authors.forename: Roland
authors.initials: R
authors.lastname: Malli
authors.forename: Kurt
authors.initials: K
authors.lastname: Schmidt
authors.forename: Wolfgang F
authors.initials: WF
authors.lastname: Graier
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
mesh_terms: D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004048:Diethylamines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009589:Nitrogen Oxides; D010069:Oxadiazoles / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004048:Diethylamines; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D010069:Oxadiazoles; D011810:Quinoxalines; D030421:Peroxynitrous Acid; D001464:Barium; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C002385:linsidomine; D019284:Thapsigargin; C087229:diethylamine dinitric oxide adduct; D008981:Molsidomine; D006152:Cyclic GMP; D002118:Calcium