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How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal [Ca.sup.2+] entry were studied in cultured rat brown adipocytes. Cytoplasmic [Ca.sup.2+] or [Mg.sup.2+] and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) produced by thapsigargin was abolished by removing extracellular [Ca.sup.2+], depressed by depleting extracellular [Na.sup.+], and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [[[Ca.sub.2+]].sub.i], in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a [Ca.sup.2+]-free EGTA solution, and an Nat-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular [Ca.sup.2+] and fully blocked by oligomycin but not by thapsigargin or an [Na.sup.+]-deficient solution, was accompanied by a rise in cytoplasmic [Mg.sup.2+] concentration, and was summated with a high pH-induced rise in [[[Ca.sup.2+]].sub.i], whereas the extracellular [Ca.sup.2+]-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing [Ca.sup.2+] but not by thapsigargin, depressed by decreasing [Na.sup.+], and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [[[Ca.sup.2+]].sub.i] in a [Ca.sup.2+]-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes [Ca.sup.2+] release, activating [Ca.sup.2+] release from the ER and store-operated [Ca.sup.2+] entry, and directly elicits a novel plasmalemmal [Ca.sup.2+] entry, whereas [Ca.sup.2+] release from the ER activates [Ca.sup.2+] accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP

Abstract: We studied how mitochondrial uncoupling by β3-adrenergic stimulation elicits Ca2+ signals in rodent brown adipocytes by fluorometry of Ca2+ concentrations ([Ca2+]i, [Ca2+]m and [Ca2+]ER) in the cytoplasm, mitochondria and the endoplasmic reticulum (ER), respectively, and mitochondrial membrane potential, using fura-2, rhod-5N, cameleon and rhodamine 123. Immunoblotting demonstrated α1A- and β3-adrenergic receptor and UCP1 in adipocytes, while RT-PCR revealed the mRNA of type 3, 7 and 9 adenylate cyclase, UCP1, UCP2, UCP3 and type 1 and 2 inositoltrisphosphate receptors. Isoproterenol and BRL37344, β-agonist, caused triphasic rises in [Ca2+]i (β-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca2+]m. β-Responses were blocked by propranolol, β-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of β-responses was depressed by a Ca2+ free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca2+ pump blocker, and by transfecting siRNA for type 2 IP3R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. β-Agonists, phenylephrine, α-agonist, and cyclopiazonic acid, ER-Ca2+ pump blocker, decreased [Ca2+]ER. Thus, the mitochondrial uncoupling by β3-adrenergic activation causes Ca2+ release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca2+ entries, including the store-operated Ca2+ entry. [Copyright &y& Elsevier]

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca2+ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca2+ or Mg2+ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca2+ concentration ([Ca2+]i1) produced by thapsigargin was abolished by removing extracellular Ca2+, depressed by depleting extracellular Na+, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rote- none caused bi- or triphasic rises in [Ca2+]i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca2+-free EGTA solution, and an Na+-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca2+ and fully blocked by oligomycin but not by thapsigargin or an Na+-deficient solution, was accompanied by a rise in cytoplasmic Mg2+ concentration, and was summated with a high pH-induced rise in [Ca2+]i whereas the extracellular Ca2+-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca2+ but not by thapsigargin, depressed by decreasing Na+, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca2+]i in a Ca2+-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca2+ release, activating Ca2+ release from the ER and store-operated Ca2+ entry, and directly elicits a novel plasmalemmal Ca2+ entry, whereas Ca2+ release from the ER activates Ca2+ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. [ABSTRACT FROM AUTHOR]

Nω-Phosphoarginine hydrolase from rat liver hydrolyzed Nω-phosphoarginine into arginine and inorganic phosphate, whereas it did not release inorganic phosphate from 19 other phosphorylated compounds containing a N-P bond, an O-P bond or a C-P bond. In addition, it was not able to transfer the phosphoryl moiety from Nω-phosphoarginine to ADP. These results indicated that this enzyme was distinct from both phosphoamidase and arginine kinase. Its properties were as follows: thiol compounds were essential for its activity; it was stimulated by 1.5-2-fold in the presence of 0.001% Lubrol, Tween 20, poly(oxyethylene) 9-1auryl ether and Nonidet P-40, while 0.004% sodium lauryl sulfate inhibited the activity completely; concentrations of sodium molybdate and sodium vanadate necessary for 50% inhibition were 7 μM and 12 μM, respectively; some proteins stimulated the activity, while lysophosphatidic acid, lysophosphatidylinositol, and phosphatidic acid suppressed the activity even in the presence of poly(oxyethylene) 9-1auryl ether. [ABSTRACT FROM AUTHOR]

Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0-0.30 M) at various pH (7.4-8.6) were distinguished from each other by their permeability through a Sepharose 4B column. 1. The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and. upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates. 2. Light meromyosin, which corresponded to the C-terminal two-thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected. 3. Truncated light meromyosin which had lost a small segment from either the C-terminal or N-terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH. 4. It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH-dependent manner by the subfragment 2 segment, the N-terminal third of the rod. [ABSTRACT FROM AUTHOR]