Stimulatory Effect Of Ca2 Research Articles

The suitability of the lead precipitation method for the localization of active ATPases in the meristematic cells of Allium cepa L. roots

Demonstrated in this study is the suitability of the lead precipitation method for the localization of active ATPases bound with the mitochondrial membranes, ER and plasma membranes of meristematic cells of <em>Allium cepa</em> L. root apices. It was found that the presence of glutaraldehyde in the fixative has a significant influence on the fixation of ATPases and that a somewhat smaller influence is exhibited by formaldehyde. Also shown in this study is the relationship between the course of the reaction indicating the presence of active ATPases and the precipitating activity of Pb ions as well as the stimulatory effect of Ca<sup>2</sup>, Mg<sup>2</sup> and K on the transport ATPase bound with the plasma and ER membranes.

Electrochemical potential of Microgramma vaccinifolia rhizome lectin

This work reports the isolation of Microgramma vaccinifolia rhizome lectin (MvRL) and the determination of electrochemical potentials of MvRL in the presence of Ca2+, Mg2+ and human type O erythrocytes. MvRL showed the highest specific hemagglutinating activity with human type O erythrocytes and showed a single polypeptide band of 17kDa on SDS-PAGE. MvRL hemagglutinating activity was neutralized after dialysis with EDTA, and addition of Ca2+ and Mg2+ restored the activity. Electrochemical potentials of MvRL in the presence of 100mM Ca2+ (882mV) and 60mMMg2+ (1051mV) were higher (p<0.05) than in the presence of only 0.15M NaCl (247mV), indicating that the electrochemical system was sensitive to structural and physico-chemical changes promoted by these ions. MvRL potential did not change in the presence of type O erythrocytes. The electrochemical system was able to detect changes in electrochemical potentials of MvRL promoted by Ca2+ and Mg2+, even in a complex environment (human serum supplemented with 40 and 60mM of these ions). The study reveals that the stimulatory effect of Ca2+ and Mg2+ on hemagglutinating activity may be linked to conformational change and/or alterations in surface charge distribution of MvRL.

Stimulation of H2O2 generation by calcium in brain mitochondria respiring on α‐glycerophosphate

It has been reported recently (Tretter et al., 2007b) that in isolated guinea pig brain mitochondria supported by alpha-glycerophosphate (alpha-GP) reactive oxygen species (ROS) are produced through the reverse electron transport (RET) in the respiratory chain and by alpha-glycerophosphate dehydrogenase (alpha-GPDH). We studied the effect of calcium on the generation of H(2)O(2) as measured by the Amplex Red fluorescent assay in this model. H(2)O(2) production in alpha-GP-supported mitochondria was increased significantly in the presence of 100, 250, and 500 nM Ca(2+), respectively. In addition, Ca(2+) enhanced the membrane potential, the rate of oxygen consumption, and the NAD(P)H autofluorescence in these mitochondria. Direct measurement of alpha-GPDH activity showed that Ca(2+) stimulated the enzyme by decreasing the Km for alpha-GP. In those mitochondria where RET was eliminated by the Complex I inhibitor rotenone (2 microM) or due to depolarization by ADP (1 mM), the rate of H(2)O(2) formation was smaller and the stimulation of H(2)O(2) generation by Ca(2+) was prevented partly, but the stimulatory effect of Ca(2+) was still significant. These data indicate that in alpha-GP-supported mitochondria activation of alpha-GPDH by Ca(2+) leads to an accelerated RET-mediated ROS generation as well as to a stimulated ROS production by alpha-GPDH.

The stimulatory effect of phosphatidylethanolamine on N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD)

N-Acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a membrane-bound enzyme which releases the endocannabinoid anandamide and other bioactive N-acylethanolamines from their corresponding NAPEs in animal tissues. Our previous studies showed that NAPE-PLD solubilized from the membrane is remarkably stimulated by millimolar concentrations of Ca2+ while the membrane-bound form is much less sensitive to Ca2+. This finding suggested that certain membrane constituents diminished the stimulatory effect of Ca2+. In the present studies, we examined the effects of membrane fractions from COS-7 cells and brain tissue on the purified recombinant rat NAPE-PLD, and found that heat-stable membrane component(s) dose-dependently activated NAPE-PLD up to 4.8–5.0 fold. In the presence of the membrane fractions, however, the stimulatory effect of Ca2+ on the purified NAPE-PLD was considerably reduced. When it was examined if the membrane fractions can be replaced with various pure phospholipids, phosphatidylethanolamine activated NAPE-PLD up to 3.3 fold, which was followed by decrease in the stimulatory effects of Ca2+ and several other divalent cations. These results suggest that membrane components including phosphatidylethanolamine keep the membrane-associated form of NAPE-PLD constitutively active.

Contribution of ATP synthase to stimulation of respiration by Ca2+ in heart mitochondria

A variety of experimental conditions were applied with the aim to estimate the correlation between the contribution of ATP synthase to the respiratory flux control and the calcium-induced activation of succinate oxidation in heart mitochondria isolated from rat, rabbit and guinea pig. The sensitivity of respiration in heart mitochondria to the decrease in temperature from 37 degrees C to 28 degrees C decreases in the order rabbit > guinea pig > rat. Ca2+ effect on succinate oxidation rate in state 3 respiration was species- and temperature-dependent and ranged from 0 (rat, 37 degrees C) to +44% (rabbit, 28 degrees C). For mitochondria from all experimental animals, the increase of Ca2+ in physiological range of concentration did not change state 2 respiration rate, and the stimulatory effect of Ca2+ on state 3 respiration was more pronounced at 28 degrees C than at 37 degrees C. The respiratory subsystem was sensitive to Ca2+ ions only in rabbit heart mitochondria. A high positive correlation between Ca2+ ability to stimulate succinate oxidation in state 3 and the control exerted by ATP synthase over the respiratory flux provides argument confirming stimulation of ATP synthase by Ca2+ ions.

Effect of calcium on growth of submerged Terfezia boudieri mycelium

The effect of Ca2+ on mycelial growth in Terfezia boudieri was studied. Terfezia boudieri Chatin (Ascomycotinae) occurs in mycorrhizal association with Helianthemum shrubs in deserts with calcareous soils. External Ca2+ stimulated mycelial growth in both liquid media and solidified substrates. The response to Ca2+ was very faint in well-aerated culture but pronounced in mycelia immersed in the medium, indicating dependence on mycelial aeration. 2,4-Dinitrophenol (DNP), an inhibitor of oxidative phosphorylation, and succinate, a potential cytoplasm acidifier, inhibited mycelial growth but enhanced the stimulatory effect of Ca2+. This effect was reduced by the Ca2+ channel blocker verapamil.

Requirement of calcium and phosphate ions in expression of sodium-dependent vitamin C transporter 2 and osteopontin in MC3T3-E1 osteoblastic cells

Osteoclasts dissolve mineralized bone matrix at bone resorption sites and release large amounts of calcium (Ca 2+) and phosphate (PO 4 3−) ions into the extracellular fluid. However, the exact nature of Ca 2+ and PO 4 3− on osteoblasts remains unclear. We proposed that Ca 2+ and PO 4 3− ions are required for the expression of sodium-dependent vitamin C transporter (SVCT) 2 and a differentiation marker, osteopontin (OPN), in osteoblasts as a response to the osteoclastic degradation. Results from Northern blotting indicated that a deficiency of Ca 2+ or PO 4 3− inhibited both SVCT2 and OPN expression in a time-dependent manner, whereas elevated Ca 2+ (1 to 4 mM) or PO 4 3− (1 to 4 mM) dose-dependently induced SVCT2, OPN expression and OPN promoter activity. In addition, the L-type calcium channel blocker, nifedipine (5 to 20 μM) and the phosphate transporter inhibitor, foscarnet (0.15 to 0.6 mM), dose-dependently abolished Ca 2+- and PO 4 3−-induced SVCT2, OPN expression and OPN promoter activity. Furthermore, the results from l-ascorbic acid uptake assay and Western blotting indicated that the stimulatory effect of Ca 2+ and PO 4 3− on functional SVCT2 protein expression. These findings suggested that Ca 2+ and PO 4 3− regulate osteoblastic phenotype by entering into cells to stimulate SVCT2 and OPN expression.

Phosphorylase kinase: the complexity of its regulation is reflected in the complexity of its structure.

Intracellular glycogen stores are used to maintain blood-glucose homeostasis during fasting, are a source of energy for muscle contraction, and are used to support a broad range of cellular activities in most tissues. A diversity of signals accelerate glycogen degradation that are mediated by phosphorylase b kinase (Phk), which phosphorylates and thereby activates glycogen phosphorylase. Phk is among the most complex of the protein kinases so far elucidated. It has one catalytic (gamma) subunit and three different regulatory (alpha, beta, and delta) subunits, a molecular mass of 1.3 X 106 daltons, and each holoenzyme molecule is presumed to contain four molecules of each subunit. The three regulatory subunits inhibit the phosphotransferase activity of the gamma subunit. Ca2+ relieves inhibition via the delta subunit, which is identical to calmodulin but remains an integral component of the holoenzyme even when the [Ca2+] is lowered to nanomolar levels. Phosphorylation of the alpha and beta subunits by the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) also relieves inhibition of the gamma subunit and thereby activates the enzyme. The stimulatory effects of Ca2+ and phosphorylation appear to be structurally coupled and are cooperative. In addition, Phk is activated in vitro by autophosphorylation, limited proteolysis of the regulatory subunits, and various allosteric effectors and these may also be mechanisms of physiological importance. The molecular mechanisms of regulation are currently poorly understood, but new insights are beginning to emerge. This review discusses current knowledge and concepts of the structure, function and regulation of Phk.

Interaction between Ca2+, K+, carbamazepine and zonisamide on hippocampal extracellular glutamate monitored with a microdialysis electrode.

1. Multiple components of hippocampal glutamate release were examined by study of Ca2+- and K+-evoked hippocampal extracellular glutamate release using an in vivo microdialysis glutamate biosensor in urethane-anaesthetized rats. In addition, the effects of the antiepileptic drugs, carbamazepine (CBZ) and zonisamide (ZNS) perfused through the probe on glutamate release were assessed. 2. Basal glutamate levels were below detection limits (approximately 0.1 microM). An increase in extracellular KCl (from 2.7 to 50 and 100 mM) increased extracellular hippocampal glutamate levels to 9.2+/-1.4 and 20.0+/-2.6 microM, respectively, calculated from the area under curve (AUC) for 60 min. 3. This KCl-evoked glutamate release consisted of three components: an initial transient rise, a late gentle rise, and late multiple phasic transient rises. 4. An increase in or removal of extracellular CaCl2 levels respectively enhanced and reduced the 50 mM KCl-evoked hippocampal glutamate release (AUC for 60 min) from 9.2+/-1.4 to 12.4+/-2.1 and 5.8+/-0.9 microM. 5. Perfusion with 100 microM CBZ or 1 mM ZNS inhibited both the 50 mM KCl-evoked hippocampal glutamate release (AUC for 60 min) from 9.2+/-1.4 to 5.5+/-1.1 and to 5.8+/-1.3 microM, respectively, as well as the stimulatory effects of Ca2+ on KCl-evoked hippocampal glutamate release. 6. These results suggest that both CBZ and ZNS may reduce epileptiform events by inhibiting excitatory glutamatergic transmission.

Calcium-Stimulated Protein Kinase Activity of the Hypodermal-Mesocarp Plasma Membrane from Preharvest-Mature and Postharvest Muskmelon

Vanadate-sensitive H+-ATPase and calcium-dependent protein kinase (kinase) activities of hypodermal-mesocarp plasma membrane (PM) vesicles isolated from preharvest mature, harvested, and stored muskmelons (Cucumis melo L. var. reticulatus Naud.) decreased following harvest. Kinase activity is markedly stimulated by Ca2+ and is responsible for phosphorylation of many melon PM proteins. Specifically, a PM protein band at 97 kDa, immunodetected as H+-ATPase, appeared phosphorylated in mature fruits, but phosphorylation and the sodium dodecyl sulfate (SDS) gel protein band were not detected in PM from harvested or stored fruit. Kinase peptide, immunodetected at 63 kDa, was present in all PM tissues, but kinase phosphorylation activity decreased with fruit harvest and storage. However, PM kinase activity in the presence of exogenous Ca2+ and histone III−S was greatly increased in harvested, and stored fruit, indicating both a stimulatory effect of Ca2+ and a loss of a suitable endogenous kinase substrate in po...

Inhibitory effect of calcium-binding protein regucalcin on ribonucleic acid synthesis in isolated rat liver nuclei.

The effect of regucalcin, a Ca2+-binding protein isolated from rat liver cytosol, on ribonucleic acid (RNA) synthesis in the nuclei of normal rat liver and of regenerating rat liver was investigated. The liver weight at 1 day after partial hepatectomy was increased about 50% of that of sham-operated (control) rats. Calcium chloride (1.0-20 microM Ca2+ as final concentration) was added into the reaction mixture of nuclear RNA synthesis. RNA synthesis was established by incorporation of [3H]-uridine 5'-triphosphate (UTP) into the nuclear RNA. Addition of Ca2+ (5 and 10 microM) caused a significant increase of RNA synthesis in the nuclei from control rat liver. Such effect of Ca2+ was potentiated in the nuclei of regenerating liver; nuclear RNA synthesis was increased about 2 fold by the 1.0 and 2.5 microM Ca2+ addition. The stimulatory effect of Ca2+ was significantly inhibited by the presence of alpha-amanitin (10(-8) M), an inhibitor of RNA polymerase II. The presence of regucalcin (0.25 and 0.5 microM) significantly inhibited RNA synthesis in the nuclei from control rat liver and from regenerating rat liver. The inhibitory effect of regucalcin was remarkable in the presence of EGTA (0.5 mM), and it was weakened by the addition of Ca2+ (5 microM). Such regucalcin effect was not seen in the presence of alpha-amanitin. The presence of anti-regucalcin IgG in the reaction mixture significantly increased RNA synthesis in the nuclei from control rat liver, indicating that the endogenous regucalcin may be involved in nuclear RNA synthesis. The present results demonstrate that regucalcin can inhibit nuclear RNA synthesis in rat liver. Regucalcin may have an inhibitory role in the regulation of liver nuclear RNA synthesis.

Studies of renal injury. II. Activation of the glucose transporter 1 (GLUT1) gene and glycolysis in LLC-PK1 cells under Ca2+ stress.

Injury to the renal proximal tubule is common and may be followed by either recovery or cell death. The survival of injured cells is supported by a transient change in cellular metabolism that maintains life even when oxygen tension is reduced. This adaptive process involves the activation of the gene encoding the glucose transporter GLUT1, which is essential to maintain the high rates of glucose influx demanded by glycolysis. We hypothesized that after cell injury increases of cell Ca2+ (Ca2+i) initiate the flow of information that culminates with the upregulation of the stress response gene GLUT1. We found that elevations of Ca2+i caused by the calcium ionophore A23187 activated the expression of the GLUT1 gene in LLC-PK1 cells. The stimulatory effect of Ca2+i on GLUT1 gene expression was, at least in part, transcriptional and resulted in higher levels of GLUT1 mRNA, cognate protein, cellular hexose transport activity, glucose consumption, and lactate production. This response was vital to the renal cells, as its interruption severely increased Ca2+-induced cytotoxicity and cell mortality. We propose that increases of Ca2+i initiate stress responses, represented in part by activation of the GLUT1 gene, and that disruption to the flow of information originating from Ca2+-induced stress, or to the coordinated expression of the stress response, prevents cell recovery after injury and may be an important cause of permanent renal cell injury and cell death.

Regulation of Lysophospholipase Activity of the 85-kDa Phospholipase A2 and Activation in Mouse Peritoneal Macrophages

The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A2 (PLA2) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA2 when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca2+ was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA2 can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA2. The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition of Ca2+ to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PLA2 in cytosols. Phosphorylation of recombinant PLA2 with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA2 activity. In macrophages stimulated with zymosan release of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA2 in regulating lysophospholipid levels in macrophages during zymosan stimulation.

Post-transcriptional Regulation of the Stanniocalcin Gene by Calcium

Stanniocalcin (STC) is a Ca(2+)-regulating hormone produced by the corpuscles of Stannius in bony fish. Calcium has been shown to stimulate STC synthesis at multiple levels including the level of gene expression. The purpose of this study was to determine the effects of Ca2+ on STC mRNA stability. The half-life of STC mRNA was measured in primary cultured trout corpuscles of Stannius cells maintained in either normal (1.2 mM) or high (1.9 mM) levels of extracellular calcium and treated with the transcriptional inhibitor alpha-amanitin. In cells maintained in 1.2 mM Ca2+, STC mRNA levels decreased progressively over time with an estimated half-life of approximately 71 h. However, message levels remained unchanged for up to 4 days in cells maintained in 1.9 mM Ca2+, indicating that the transcript had been stabilized in response to Ca2+ stimulation. Blocking transcription prior to exposing cells to high Ca2+ did not alter the stabilizing effects of the cation, indicating that synthesis and processing of the mRNA transcript were not involved in message stabilization. Inhibiting protein synthesis with cycloheximide also had no influence on the stabilizing effects of high calcium. The experiments involving cycloheximide further suggested that the mechanism of mRNA stabilization involved protein-nucleic acid interactions in the cytoplasm, whereby the polysomal complex protected the mRNA from degradation. These data demonstrate that the stimulatory effect of Ca2+ on STC gene expression is due, in part, to mRNA stabilization.

Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages

The effects of CdCl2 on the liberation of arachidonic acid (20:4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A2 (PLA2) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [3H]arachidonic acid (20:4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [3H]20:4 from their phospholipids. Exposure of cells to Cd2+ inhibited the liberation of [3H]20:4 in a dose-dependent manner. Liberation of [3H]20:4 from cell lipids was calcium dependent and the inhibitory effect of Cd2+ competed with the stimulatory effect of Ca2+. When Ca2+ was removed from the incubation medium, Cd2+ did not influence the liberation of [3H]20:4. Entry of 45Ca2+ into cells was enhanced by treatment of A23187. However, Cd2+ did not influence the cellular uptake of 45Ca2+. Treatment with A23187 markedly enhanced entry of 109Cd2+ into cells. The effect of Cd2+ on the activity of phospholipase A2 was determined with 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA2 was observed and Cd2+ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd2+ causes a reduction in the rate of liberation of 20:4 from cell lipids, as a possible result of the inhibition of PLA2 activity by Cd2+.

Role of calcium and cAMP messenger systems in intracellular pH regulation of osteoblastic cells.

We have recently shown that two mechanisms are involved in the regulation of pHi in the osteoblastic phenotype cell line UMR-106 (Na(+)-H+ antiporter and a Na(+)-independent Cl(-)-HCO 3(-)-OH- exchanger). In the present work, we used the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein as well as isotope fluxes to investigate the influence of second messengers on the activity of these transporters. Elevation in intracellular calcium concentration [( Ca2+]in) in UMR-106 cells (measured by fura-2 fluorescence) is followed by stimulation of the Cl(-)-HCO3- exchanger, leading to cytosolic acidification. Subsequently, cell alkalinization, mediated by the Na(+)-H+ exchanger, restores pHi to its resting value. An acute reduction in [Ca2+]in abruptly stops the activity of the anion exchanger while having no influence on the activity of the Na(+)-H+ exchanger. The stimulatory effect of Ca2+in on the anion exchanger is dose dependent and is abrogated by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide and calmidazolium. An increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) brought about by forskolin, 8-bromo-cAMP, or prostaglandin E2 leads to inhibition of activity of both the Na(+)-H+ antiporter and the anion exchanger. The suppressive effect of cAMP on Cl(-)-HCO3- exchange could be overcome by elevating [Ca2+]in. We conclude that 1) Ca2+in and cAMP can influence pHi in osteoblasts by altering the activities of pHi regulatory mechanisms and 2) the effect of Ca2+in is probably mediated by calmodulin.

Stimulatory effects of extracellular calcium on chorionic gonadotrophin and placental lactogen release by human placental explants

The effects of sudden modifications of extracellular calcium ion concentration on human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) release were investigated using term placental explants incubated in Krebs Ringer solution. The hCG and hPL releases were both stimulated when the extracellular Ca2+ concentration was increased. The hCG and hPL secretions elicited by the addition of extracellular Ca2+ were larger when placental explants were preincubated in alpha-calcium (no added calcium + EGTA) than in normo-calcium (1.5 mM). Removal of extracellular Ca2+ from the medium also elicited an increase in hCG and hPL release. However, this stimulatory effect of Ca2+ omission was partly suppressed by washing the explants prior to incubation in the alpha-calcium medium, and was completely abolished when alpha-calcium medium was supplemented with 1 mM cobalt. Our results indicate that changes in Ca2+ modify hCG and hPL release from term placental explants in a manner concordant with the 'stimulus-secretion coupling' concept.

Effect of sodium taurodeoxycholate, CaCl 2 and albumin on the action of pancreatic lipase on droplets of trioleoylglycerol and the release of lipolytic products into aqueous media

1. 1. Effects of various substances on the activity of pancreatic lipase and on the release of lipolytic products into aqueous media were studied with droplets of trioleoylglycerol suspended from a membrane filter at the top of a flow-through chamber. The droplets were perifused for 7 min with a commercial preparation of pancreatic lipase in 0.15 M NaCl solution at pH 6.5 and then perifused for 60 min with lipase-free media, either 0.15 M NaCl at pH 6.5 or basal medium at pH 7.4 (70 mM sodium barbital) containing different additives. 2. 2. About 6% of the trioleoylglycerol in droplets was hydrolyzed during the perifusion with lipase. Another 15% was hydrolyzed in 30 min, but none thereafter, when the droplets were perifused with 0.15 M NaCl alone. The rate of hydrolysis was doubled and prolonged when droplets were perifused with basal medium at pH 7.4. Lipolytic products formed at pH 7.4 were 62% oleic acid, 20% monooleoylglycerol and 18% dioleoylglycerol, yet only 4% of the lipolytic products were released into the perifusate. 3. 3. Sodium taurodeoxycholate (TDC) (17 mM) added to basal medium increased 18 × the amount of lipolytic products released into the perifusate but increased lipolysis only 13%. The molar ratio of oleic acid to monooleoylglycerol in the perifusate was 5.7 during the first 30 min and 4.0 during the last 30 min. 4. 4. Ca 2+ (3.3 mM) added to basal medium increased lipolysis 87% but did not affect the amount (4%) of lipolytic products released into the perifusing medium. 5. 5. TDC and Ca 2+ added to basal medium produced the largest increase in lipolysis, with 59% of trioleoylglycerol hydrolyzed in 15 min and 91% in 60 min. The amount of lipolytic products released into the perifusing medium, however, was not increased above that released into medium containing TDC alone. 6. 6. Serum albumin (0.6 mM) and Ca 2+ added to basal medium increased 14 × the amount of lipolytic products released into the perifusate without affecting the basal lipolytic rate. Albumin, however, suppressed by 40% the stimulatory effect of Ca 2+ on pancreatic lipase activity. 7. 7. The findings show that Ca 2+ enhanced the activity of pancreatic lipase on triacylglycerol without affecting release of lipolytic products into the medium, whereas TDC increased release without affecting formation of lipolytic products. TDC did increase, however, the stimulatory effect of Ca 2+ on lipolysis. Thus, bile salts could facilitate fat digestion in vivo, in the presence of Ca 2+, by enhancing the action of pancreatic lipase and dispersing lipolytic products into the surrounding aqueous phase.

Calcium-requirement and its pH dependency of lysophosphoinositide-specific phospholipase C in porcine platelet membranes.

The lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10,000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A.

Stimulation of hypothalamic histidine decarboxylase by calcium-calmodulin and protein kinase (cAMP-dependent) inhibitor.

Stimulatory effects of Ca2+-CaM and PKI on partially purified hypothalamic HD (10 fold purification) have been shown under conditions involving inhibition of the enzyme by cAMP-induced phosphorylation and under control conditions. A 1:1 (v/v) mixture of 0.1 mM CaCl2 and 10 units of CaM from human red blood cells reversed the inhibition of HD induced by cAMP-dependent protein phosphorylation activity to the control level. Verapamil (0.01 mM) could partially block the former effect without affecting the control level of enzyme activity. 0.01 mM TPA did not further increase the effect of Ca2+-CaM on HD, in the presence of 0.01 mM ATP, indicating that this stimulation does not require the action of Ca2+-dependent protein kinase. The control level of HD is not influenced by 0.1 mM CaCl2 or 0.02 mM EGTA but is raised by CaM in the presence of CaCl2 (0.1 mM). A highly purified protein kinase (cAMP-dependent) inhibitor (PKI) from bovine heart and a crude inhibitor from rat cerebellum could also reverse the inhibitory effect of cAMP-dependent protein kinase under phosphorylating conditions and enhanced HD activity above control levels. PKI and Ca2+-CaM, added together, produced single, not additive effects. We conclude that cAMP-induced phosphorylation is probable the main regulatory mechanism of histamine formation and this could be influenced by both Ca2+-CaM and PKI. Inhibition of cAMP-dependent protein kinase as well as stimulation of phosphoprotein phosphatase and Ca2+-CaM-dependent phosphodiesterase might be involved in the above actions.