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Nitric oxide inhibits capacitative Ca 2+ entry by suppression of mitochondrial Ca 2+ handling
Author(s) -
Thyagarajan Baskaran,
Malli Roland,
Schmidt Kurt,
Graier Wolfgang F,
Groschner Klaus
Publication year - 2002
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0704949
Subject(s) - peroxynitrite , mitochondrion , nitric oxide , thapsigargin , chemistry , membrane potential , soluble guanylyl cyclase , biophysics , inner mitochondrial membrane , stimulation , biochemistry , superoxide , biology , endocrinology , endoplasmic reticulum , guanylate cyclase , organic chemistry , enzyme
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. 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 μ M , respectively. NO‐induced inhibition of CCE was not mimicked by peroxynitrite (100 μ M ), the peroxynitrite donor 3‐morpholino‐sydnonimine (SIN‐1, 100 μ M ) or 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐BrcGMP, 1 m M ). In addition, the guanylyl cyclase inhibitor 1H‐[1,2,4] oxadiazole[4,3‐a] quinoxalin‐1‐one (ODQ, 30 μ M ) failed to antagonize the inhibitory action of NO on CCE. DEANO (1–10 μ 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 μ 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. DEANO (10 μ M ) failed to inhibit Ba 2+ entry into TG‐stimulated cells when extracellular Ca 2+ was buffered below 1 μ M , while clear inhibition of Ba 2+ entry into store depleted cells was observed when extracellular Ca 2+ levels were above 10 μ 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. 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.British Journal of Pharmacology (2002) 137 , 821–830. doi: 10.1038/sj.bjp.0704949