Requirement of Ca2+ for the production and degradation of inositol 1,4,5-trisphosphate in macrophages

The requirement of Ca2+ for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) or the accumulation of inositol 1,4,5-trisphosphate (InsP3) in macrophages stimulated with fMet-Leu-Phe was examined using [32P]Pi or [3H]inositol-labeled cells. The dependence on Ca2+ of inositol-trisphosphate phosphatase was also examined. The application of 1 X 10(-8) M fMet-Leu-Phe caused a rapid decrease in the amount of PtdInsP2 to 70% of the control within 10 s, and the decrease was reverted to the control level by prolonged incubation. The decrease in the amount of PtdInsP2 accompanied the accumulation of phosphatidic acid and of InsP3, indicating that the loss of PtdInsP2 is due to phosphodiesteric breakdown. The dose-dependence of fMet-Leu-Phe or its analog on the hydrolysis of PtdInsP2 was much the same as that of the increase in intracellular free Ca2+ concentration in macrophages. The loss of PtdInsP2 as induced by fMet-Leu-Phe was similarly observed in macrophages treated with ionophore A23187 in the absence of external Ca2+ for 10 min. InsP3 was degraded by the particulate or cytosol fraction prepared from macrophages, and the activity of inositol-trisphosphate phosphatase in the particulate fraction was higher than that in the cytosol fraction. The enzyme in the cytosol fraction required Mg2+ for activity, and was activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6) M in the presence of 1 mM MgCl2.     

Ca2+-stimulated phospholipid phosphoesterase activities in rabbit erythrocyte membranes

The properties of the enzymes involved in Ca2+-stimulated breakdown of phosphatidylinositol 4'-phosphate (PIP), phosphatidylinositol 4',5'-bisphosphate (PIP2), and phosphatidic acid (PA) in rabbit erythrocyte ghosts were studied. At 25 degrees C, 1 to 180 microM Ca2+ rapidly stimulated the breakdown of PIP and PIP2, and maximal breakdown occurred within 10 minutes at all Ca2+ concentrations. The rate and the total amount of breakdown of PA, PIP, and PIP2 increased with Ca2+ concentration. MgCl2 inhibited the rate of Ca2+-stimulated breakdown of PIP and PIP2 at Ca2+ concentrations less than 10 microM, but did not have any appreciable effects at higher Ca2+ concentrations. MgCl2 also protected against Ca2+-stimulated breakdown of PA. In the presence and absence of 5 mM MgCl2, Ca2+ stimulated half-maximal breakdown of PIP and PIP2 at 2-3 microM under hypotonic and isotonic conditions. In the presence of 5 mM MgCl2, Ca2+-stimulated breakdown of PIP and PIP2 was associated with the release of Pi and inositol bisphosphate. In the absence of MgCl2, Ca2+ stimulated the release of 32P-labeled Pi, inositol bisphosphate, and inositol trisphosphate from labeled PIP, PIP2, and PA. Ca2+ increased phosphatidylinositol content and decreased PIP and PIP2 content in these membranes. The results of this investigation suggest that Ca2+ stimulates the breakdown of polyphosphoinositides by stimulating polyphosphoinositide phosphomonoesterase and phosphodiesterase activities in rabbit erythrocyte ghosts. These activities were activated by less than 3 microM Ca2+ in the presence of MgCl2 under hypotonic or isotonic conditions. These Ca2+-stimulated polyphosphoinositide phosphoesterase activities could therefore be active under physiological conditions in normal rabbit erythrocytes.     

Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane

Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.     

Subcellular localization and kinetic properties of phosphatidylinositol 4,5-bisphosphate phospholipase C and inositol phosphate enzymes from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells from normal donors exhibited phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC), inositol 1,4,5-trisphosphate (IP3) and inositol 1-phosphate (IP)-monophosphatase activities which were mostly recovered in the cytosol fraction. In both cytosol and particulate fractions PIP2-PLC displayed the highest activity at pH 6.2, whereas IP3 and IP-monophosphatases showed the same optimal pH at 7.0. While the PIP2-PLC displayed close apparent Km values in cytosol and particulate fractions, both inositol-monophosphatases were found to show substrate affinities for IP and IP3 characteristic of these two fractions, with an higher affinity in the soluble fraction.     

Phosphatidylinositol 4,5-bisphosphate-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum terminal cisternal membranes. Ca2+ flux and single channel studies.

We report here that the inositol 1,4,5-trisphosphate (IP3) precursor, L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent molecule (1 microM) which activates the ryanodine-sensitive Ca2+ release channel from rabbit skeletal muscle terminal cisternae incorporated into a phospholipid bilayer. It also stimulates Ca2+ release from these membrane vesicles. Therefore, it may play a modulating role in excitation-contraction coupling. In the bilayer, PIP2 added on the cytoplasmic side increased the mean channel opening probability 2-12-fold in the presence and absence of physiological Mg2+ and ATP. From flux studies, PIP2-induced Ca2+ release, occurring through the ryanodine-sensitive Ca2+ release channel, displayed saturation kinetics. The rate of Ca2+ release induced by PIP2 was approximately greater than 50% slower than the rates induced by other agents (e.g. caffeine, Ca2+, ATP). PIP2, and not IP3, effectively elicited Ca2+ release from terminal cisternae. On the contrary, IP3, and not PIP2, specifically mediated Ca2+ release from dog brain cerebellum microsomes, where IP3 receptors are known to be found. The PIP2-induced Ca2+ release from muscle membranes was not dependent on medium [Ca2+] (from less than 10(-9) to approximately 10(-4) M). However, IP3 could activate the terminal cisternae Ca2+ channel in the bilayer when there was low Ca2+ (less than 10(-7) M). The data suggest that the ionic microenvironment around the Ca2+ channel may be different for observing the two phosphoinositide actions.     

Soluble and membrane-bound polyphosphoinositide phosphohydrolases in mammalian erythrocytes

Phosphatases and phosphodiesterases that hydrolyse polyphosphoinositides are described in both membrane and cytosol fractions of human, pig, rat, rabbit, and sheep erythrocytes using exogenous substrates. With suitably optimized assay conditions, Ca2+-dependent phosphatidylinositol bisphosphate (PIP2) phosphodiesterase activity was found in the hemoglobin-free cytosol fraction, as well as the membrane. Membrane activity is completely dependent upon Triton X-100 and salt and inhibited by cetyltrimethylammonium bromide (CTAB), while the soluble activity requires CTAB and is inhibited by Triton. A low Ca2+-dependent PIP2 phosphatase activity, not present in other tissues, was also detected. The cation-independent phosphatidylinositol phosphate (PIP) phosphatase is localized in the membrane in most species, while the diesterase and the PIP2 phosphatases (both Mg2+ and Ca2+ dependent) are localized in the cytosol. Rat and rabbit erythrocytes are atypical in having a substantial proportion of their Mg2+-dependent PIP2 phosphatase activities in the membrane. All activities are lowest in sheep erythrocytes, except the PIP phosphatase, most of which is soluble in this species. Ca2+-dependent PIP2 phosphatase activity is not correlated with the activity or subcellular distribution of any of the other hydrolases and seems to be a separate enzyme. All the phosphoinositide hydrolase activities, particularly the diesterase, are orders of magnitude lower in erythrocytes than in other tissues. Both soluble and membrane diesterase activities are lost as erythrocytes age. Soluble polyphosphoinositide diesterase does not seem to be active with membrane-bound substrate, since pig and sheep erythrocytes that have negligible membrane activity do not respond to Ca2+ loading, yet have substantial diesterase activity in the cytosol. This supports the view that the diesterase is not physiologically functional in normal erythrocytes.     

Ca2+ dependence of inositol 1,4,5-trisphosphate 3-kinase activity in the cytosol fraction of pig coronary artery

1. The activity of inositol 1,4,5-trisphosphate 3-kinase in subcellular fractions of smooth muscles of the pig coronary artery was examined. 2. Incubation of [3H]inositol 1,4,5-trisphosphate (IP3) with muscle homogenates produced more polar 3H-radioactivity (probably as inositol 1,3,4,5-tetrakisphosphate, IP4) than IP3, in the Mg2+- and ATP-dependent manner, thereby indicating the presence of IP3 3-kinase activity in homogenates of the muscle. 3. Most of the kinase activity was present in the cytosol fraction. The enzyme activity was reversibly activated by Ca2+ with a half-maximal effective concentration of 2.5 x 10(-7) M. 4. The calmodulin antagonists, W-7 and chlorpromazine inhibited the Ca2+-activated enzyme activity.     

ADH-dependent phosphoinositide signalling system and prostaglandin E production in the frog urinary bladder.

Antidiuretic hormone (ADH) stimulated formation of inositol 1,4,5-trisphosphate (IP3), 1,2-diaclyglycerol (DAG) and an increase of phosphatidylinositol 4,5-biphosphate (PIP2) breakdown in the frog urinary bladder 20 s after addition. ADH also increased the prostaglandin E (PGE) secretion into serosal medium 3.5-fold and the release of arachidonic acid (AA) from 1,2-DAG, which was intensified in the presence of DAG kinase inhibitor R59022. Neomycin sulphate (10(-5) M) from the serosal side blocked ADH-stimulated PIP2 hydrolysis, IP3 production and increased the hydro-osmotic response to ADH. It also inhibited the ADH-stimulated PGE production (55%) and release of AA from 1,2-DAG. This data suggest that PIP2 breakdown is involved in the mechanism of feedback regulation of ADH action and is associated with PGE production via (i) the increase of AA release from PIP2-generated 1,2-DAG and (ii) possible activation of phospholipase A2 by IP3-induced elevation of cytosol Ca2+.     

Properties of enzyme activities involved in protein phosphorylation-dephosphorylation of thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca2+-dependent protein kinase activities as well as a basal (cAMP- and Ca2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kinase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent Km values of cAMP and Ca2+ for the membrane-bound protein kinase were 5 . 10(-8) M and 8.3 . 10(-4) M in the presence of 1 Mm EGTA), respectively. The apparent Km values of Mg2+ were 7.10-4M (without (in the cAMP and Ca2+), 5 . 10(-4) M (with cAMP) and 1.3 . 10(-3) M (with Ca2+), and those of ATP were 3.5 . 10(-5)M (with or without cAMP) and 8.5 . 10(-5) M (with Ca2+). The Ca2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca2+, but was stimulated by a rather broad range (5-25 mM) of Mg2+ and Mn2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Effect of insulin and insulin-like growth factors I and II on phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate breakdown in liver from humans with and without type II diabetes

We have characterized a plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and a cytosolic phosphatidylinositol (PI)-specific PLC in human liver. Epinephrine, 1 x 10(-5) M, and vasopressin, 1 x 10(-8) M, stimulated PIP2-PLC which was enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). PI-PLC stimulation was not observed by these agents. Insulin and insulin-like growth factors (IGF-I and IGF-II) in the presence and absence of GTP gamma S did not stimulate PIP2-PLC or PI-PLC in plasma membranes and cytosol preparations nor phosphoinositide breakdown in isolated human hepatocytes. Furthermore, serendipitly we found that PIP2-PLC activity was increased in liver membranes from obese patients with type II diabetes when compared to obese and lean controls. We conclude that in human liver, insulin and IGFs are not members of the family of hormones generating inositol trisphosphate (IP3) as a second messenger. Furthermore, the increased PIP2-PLC in diabetic liver may result in: (a) increased intracellular concentrations of IP3 and thus increased Ca2+, which has been postulated to induce insulin resistance; and (b) increased diacylglycerol and thus increased protein kinase C which phosphorylates the insulin receptor at serine residues inactivating the insulin receptor kinase. While the mechanism of increased PIP2-PLC activity in diabetes is unknown, it may initiate a cascade of events that result in insulin resistance.     

Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors.

Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2(+)-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (less than 5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+i of approximately 200 nM. This change in Ca2+i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10(-7) M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in less than 5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.     

Particulate guanylate cyclase of skeletal muscle: effects of Ca2+ and other divalent cations on enzyme activity.

The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.     

Stimulation of polyphosphoinositide hydrolysis by thrombin in membranes from human fibroblasts.

One of the earliest actions of thrombin in fibroblasts is stimulation of a phospholipase C (PLC) that hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. In membranes prepared from WI-38 human lung fibroblasts, thrombin activated an inositol-lipid-specific PLC that hydrolysed [32P]PIP2 and [32P]phosphatidylinositol 4-monophosphate (PIP) to [32P]IP3 and [32P]inositol 1,4-bisphosphate (IP2) respectively. Degradation of [32P]phosphatidylinositol was not detected. PLC activation by thrombin was dependent on GTP, and was completely inhibited by a 15-fold excess of the non-hydrolysable GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]). Neither ATP nor cytosol was required. Guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) also stimulated polyphosphoinositide hydrolysis, and this activation was inhibited by GDP[S]. Stimulation of PLC by either thrombin or p[NH]ppG was dependent on Ca2+. Activation by thrombin required Ca2+ concentrations between 1 and 100 nM, whereas stimulation of PLC activity by GTP required concentrations of Ca2+ above 100 nM. Thus the mitogen thrombin increased the sensitivity of PLC to concentrations of free Ca2+ similar to those found in quiescent fibroblasts. Under identical conditions, another mitogen, platelet-derived growth factor, did not stimulate polyphosphoinositide hydrolysis. It is concluded that an early post-receptor effect of thrombin is the activation of a Ca2+- and GTP-dependent membrane-associated PLC that specifically cleaves PIP2 and PIP. This result suggests that the cell-surface receptor for thrombin is coupled to a polyphosphoinositide-specific PLC by a GTP-binding protein that regulates PLC activity by increasing its sensitivity to Ca2+.     

Reappraisal of the effect of Ca2+ on the activity of liver microsomal glucose-6-phosphatase

It has recently been reported that free Ca2+, a second hormonal messenger in the liver, can modulate the activity of liver glucose-6-phosphatase by inhibition (van de Werve, G. (1989) J. Biol. Chem. 264, 6033-6036) or activation (Yamagushi, M., Mori, S., and Suketa, Y. (1989) Chem. Pharm. Bull. (Tokyo) 37, 388-390). Such a controversial role for Ca2+ is reinvestigated by comparing the effect of the addition of free Ca2+ (10(-10) to 20.10(-3) M) under the form of CaCl2 or of Ca-EGTA buffers. We show that the glucose-6-phosphatase activity is: 1) increased in the presence of CaCl2 at concentrations higher than 10(-4) M and unaffected in the presence of CaCl2 at lower concentrations; 2) decreased in the presence of Ca-EGTA buffers yielding free Ca2+ concentrations higher than 10(-8) M; 3) the latter effect is not depending on free Ca2+ or free EGTA concentrations, but on Ca.EGTA complex concentration. In addition, these effects can be reproduced in the same concentration ranges by MgCl2 and Mg-EDTA buffers, respectively. It is concluded that a physiological role for free Ca2+ on the activity of liver glucose-6-phosphatase remains to be established.     

A cytosolic protein activator of cardiac sarcolemmal phosphoinositide phospholipase C

Ca2+ dependent polyphosphoinositide phospholipase C (PLC) activity in cardiac sarcolemma hydrolyzed both endogenous and exogenous phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with an associated increase in inositol bisphosphate (IP2). Dialyzed cytosol and certain fractions of cytosol isolated by anion exchange or gel filtration chromatography activated sarcolemmal PLC activity by approx. 100%. The PLC activator eluted with an apparent molecular weight of 160 Kdal on a Sephacryl 300 column and was destroyed by heat or trypsin treatment. Exogenous 3H-PIP2 was not hydrolyzed by cytosolic fractions containing sarcolemmal PLC activator. These studies demonstrate that the polyphosphoinositide PLC in cardiac sarcolemma is regulated by a cytosolic protein.     

Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5''-[gamma-thio]triphosphate and by thrombin in the presence of GTP.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.     

Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store ([3H]IP3 binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum P2 and P3 fractions displayed levels of [3H]IP3 binding 10- to 30-fold higher, and rates of IP3-induced Ca2+ release greater than 15-fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (less than 30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified.     

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.     

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes.

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.     

Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.