Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Hypoxic vasorelaxation: Ca2+-dependent and Ca2+-independent mechanisms

The mechanisms of oxygen sensing in vascular smooth muscle have been studied extensively in a variety of tissue types and the results of these studies indicate that the mechanism of hypoxia-induced vasodilation probably involves several mechanisms that combined to assure the appropriate response. After a short discussion of the regulatory mechanisms for smooth muscle contractility, we present the evidence indicating that hypoxic vasorelaxation involves both Ca2+-dependent and Ca2+-independent mechanisms. More recent experiments using proteomic approaches in organ cultures of porcine coronary artery reveal important changes evoked by hypoxia in both Ca2+-dependent and Ca2+-independent pathways.     

Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa

We reported previously that a Ca²⁺-ATPase in rat testes and goat spermatozoa could be activated by Ca²⁺ alone without Mg²⁺, though it has a lot of similarities with the well known Ca²⁺, Mg²⁺-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca²⁺ and at low temperature. Increase of the concentration of Ca²⁺ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca²⁺, Mg²⁺-ATPase. The findings strengthened our previous report that Mg²⁺-independent Ca²⁺-ATPase is involved in Ca²⁺ transport and Ca²⁺ uptake like Ca²⁺, Mg²⁺-ATPase.     

Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca2+ to provide the simultaneous activation of the (Ca2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 degrees C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle

Excitatory agonists can induce significant smooth muscle contraction under constant free Ca(2+) through a mechanism called Ca(2+) sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca(2+)-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA(2) activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca(2+) sensitization of contraction from 49 +/- 3.6 to 12 +/- 1.0% (p < 0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca(2+), phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA(2) in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA(2) is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca(2+)-sensitization of contraction in vascular smooth muscle.     

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

A cyclic nucleotide- and Ca²⁺-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. $\text{254}$, 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca²⁺-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.     

Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.     

Effects of phalloidin on K+-dependent, Ca2+-independent neurotransmitter efflux and K+-facilitated, Ca2+-dependent neurotransmitter release.

Phalloidin tightly binds to actin and converts soluble actin into depolymerization-resistant actin filaments. Phalloidin promotes the potassium-dependent, calcium-independent efflux of γ-amino butyric acid and nore-pinephrine from synaptosomes but inhibits the potassium-facilitated, calcium-dependent release of these neurotransmitters. This suggests that an actomyosin system is involved in synaptic transmission.     

Isolation of purified brush-border membranes from rat jejunum containing a Ca2+-independent phospholipase A2 activity

A novel phospholipase activity was recognized in intact, rat jejunal brush-border membranes and its effect on membrane lipid composition was evaluated following various incubation protocols. Brush-border membranes were isolated from mucosal scrapings by a combination of existing techniques. A brush-border plus nuclei fraction was first prepared by homogenization and low-speed centrifugation in isotonic mannitol, in the presence of 5 mM EDTA. Brush-border membrane vesicles were isolated from this fraction by homogenization, followed by precipitation of the remaining undesired membranes with 10 mM CaCl2. Membranes were judged to be highly purified by marker enzyme content, protein profile, and electron microscopy. In total lipid extracts, prepared immediately following membrane isolation, the ethanolamine phosphatides were found to be the major phospholipid class, accounting for nearly 45% of the total lipid phosphorus. Storage of the intact membranes, at either room temperature or at -20 degrees C, but not at -70 degrees C, resulted in a gradual and progressive hydrolysis of phosphatidylethanolamine to lysophosphatidylethanolamine. Over 60% of the total ethanolamine phospholipid was converted to the lyso form during a 2 week storage period. Incubation of the intact membranes at 37 degrees C produced a similar effect in one hour. Only small amounts of other glycerophospholipids were degraded under these conditions. Hydrolysis was specific for the sn-2 position as more than 80% of the fatty acids in the lysophosphatidylethanolamine were found to be saturated. Substitution of MgCl2 for CaCl2 in the precipitation step did not block the hydrolysis. It was concluded that rat brush-border membranes contain a Ca2+-independent phospholipase A2 with a high substrate preference for phosphatidylethanolamine. The physiological significance of this enzyme is not known.     

TRPM8-independent menthol-induced Ca2+ release from endoplasmic reticulum and Golgi

Menthol, a secondary alcohol produced by the peppermint herb, Mentha piperita, is widely used in the food and pharmaceutical industries as a cooling/soothing compound and odorant. It induces Ca2+ influx in a subset of sensory neurons from dorsal root and trigeminal ganglia, due to activation of TRPM8, a Ca2+-permeable, cold-activated member of the TRP superfamily of cation channels. Menthol also induces Ca2+ release from intracellular stores in several TRPM8-expressing cell types, which has led to the suggestion that TRPM8 can function as an intracellular Ca2+-release channel. Here we show that menthol induces Ca2+ release from intracellular stores in four widely used cell lines (HEK293, lymph node carcinoma of the prostate (LNCaP), Chinese hamster ovary (CHO), and COS), and provide several lines of evidence indicating that this release pathway is TRPM8-independent: 1) menthol-induced Ca2+ release was potentiated at higher temperatures, which contrasts to the cold activation of TRPM8; 2) overexpression of TRPM8 did not enhance the menthol-induced Ca2+) release; 3) menthol-induced Ca2+ release was mimicked by geraniol and linalool, which are structurally related to menthol, but not by the more potent TRPM8 agonists icilin or eucalyptol; and 4) TRPM8 expression in HEK293 cells was undetectable at the protein and mRNA levels. Moreover, using a novel TRPM8-specific antibody we demonstrate that both heterologously expressed TRPM8 (in HEK293 cells) and endogenous TRPM8 (in LNCaP cells) are mainly localized in the plasma membrane, which contrast to previous localization studies using commercial anti-TRPM8 antibodies. Finally, aequorin-based measurements demonstrate that the TRPM8-independent menthol-induced Ca2+ release originates from both endoplasmic reticulum and Golgi compartments.     

Thiophosphorylation causes Ca2+-independent norepinephrine secretion from permeabilized PC12 cells

Adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) was used to examine the role of phosphorylation in the regulation of norepinephrine secretion by digitonin-permeabilized PC12 cells. While most kinases will use ATP gamma S to thiophosphorylate proteins, thiophosphorylated proteins are relatively resistant to dethiophosphorylation by protein phosphatases. Norepinephrine secretion by permeabilized PC12 cells was ATP- and Ca2+-dependent but resistant to calmodulin antagonists. Half-maximum secretion was obtained in 0.75 microM Ca2+. Permeabilized PC12 cells were incubated with ATP gamma S in the absence of Ca2+, the ATP gamma S was removed, and norepinephrine secretion was determined. Preincubation with ATP gamma S increased the amount of norepinephrine secreted in the absence of Ca2+, but it had no effect on the amount released in the presence of Ca2+. After a 15-min preincubation in 1 mM ATP gamma S, there was almost as much secretion in the absence of Ca2+ as in its presence. Inclusion of ATP in the preincubation inhibited the effect of ATP gamma S. Ca2+ stimulated the rate of modification by ATP gamma S as brief preincubations with ATP gamma S in the presence of Ca2+ resulted in higher levels of Ca2+-independent secretion than did preincubations with ATP gamma S in the absence of Ca2+. Similarly, brief preincubations of permeabilized cells with ATP in the presence of Ca2+ resulted in elevated levels of Ca2+-independent secretion. Secretion of norepinephrine from ATP gamma S-treated cells was ATP-dependent. These results suggest that norepinephrine secretion by PC12 cells is regulated by a Ca2+-dependent phosphorylation. Once this phosphorylation has occurred, secretion is still ATP-dependent, but it no longer requires Ca2+.     

Ca2+-independent,protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093–4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca²⁺, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.     

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta

During an agonist stimulation of endothelial cells, the sustained Ca2+ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca2+ stores depletion to the opening of such channels are still elusive. We have used Ca2+ and tension measurements in intact aortic strips to investigate the role of the Ca2+-independent isoform of phospholipase A2 (iPLA2) in endothelial store-operated Ca2+ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca2+ entry when Ca2+ stores are artificially depleted. We also show that the sustained store-operated Ca2+ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca2+ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA2 upon Ca2+ stores depletion may act as an intracellular messenger that stimulates store-operated Ca2+ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca2+ entry is a key physiological mechanism regulating arterial tone.     

The Ca2+-independent PKC (p105) mediates the PMA-activation of marine mussel hemocytes and the Ca2+-dependent PKC (p60) does not intervene

Molecular and Cellular Biochemistry - Previous works revealed the presence of a Ca2+-dependent protein kinase (p60) and a Ca2+-independent protein kinase (p105) in the mantle tissue from the sea...     

The molecular factors of the cell adhesion of neural tissues and the Ca2+-independent adhesion system

The review discusses various technical approaches to identification of plasma membrane and extracellular matrix proteins involved in cell adhesion. Physicochemical characteristics of macromolecular factors of the Ca2(+)-independent adhesion system in neural tissue cells are discussed alongside with the findings on their biosynthesis and expression on the cell surface at different stages of embryogenesis.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.