Rhythmic oscillations of cytosolic free calcium in rat C-cells

The relationship between stimulation of single C-cells (rMTC-6-23 cell line) with extracellular calcium, glucagon or 8-bromo-cAMP and fluctuations of intracellular free calcium concentration was studied. After pretreatment of rMTC cells with either 1 microM glucagon (30-60 min) or 1 mM 8-bromo-cAMP (5 min) [Ca2+]i started to oscillate when extracellular calcium was raised to 3 mM. These fluctuations in [Ca2+]i could be stopped by chelating the external calcium with EGTA or by adding calcium channel blockers. The voltage-dependent calcium channels in the plasma membrane seem to play a major role in maintaining the oscillations of [Ca2+]i.     

Insulin does not activate a phosphoinositide-specific phospholipase C in adipocytes

Phosphoinositide-specific phospholipase C (PI-PLC) activity was determined in homogenates of adipocytes treated with maximal concentrations of insulin. PI-PLC activity measured using exogenous [3H]phosphatidylinositol [( 3H]PI) and exogenous [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PIP2) was not altered by prior exposure of adipocytes to insulin. It was possible to see oxytocin-induced breakdown of phosphoinositides but no effect of insulin was seen in intact adipocytes.     

Changes in phosphoinositide-specific phospholipase C and phospholipase A2 activity in ischemic and reperfused rat heart

Summary Phospholipid metabolism is altered during ischemia and post-ischemic reperfusion. Past studies demonstrating elevated myocardial free fatty acid and lysophospholipid content infer accelerated phospholipid degradation involving phospholipase A activity. Recently, ischemic and postischemic reperfusion (reperfusion) have been shown to affect levels of phosphoinositide (PPI) degradation products. Considering the role of PPI turnover in regulation of cellular calcium homeostasis, our laboratory and others have suggested that alteration in the metabolism of the inositol phospholipids could play a role in the development of ischemia-induced calcium overload injury. Using an isolated rat heart model (Langendorff perfusion), this study examines the effect of global ischemia and reperfusion on ventricular phosphoinositide-specific phospholipase C (PLC) activity and PLA₂ activity. The primary purpose was to determine if ischemia and reperfusion-induced changes in PLC activity could explain previously observed changes in PPI degradation products, and whether PLC and PLA₂ activities were similarly or differentially altered by ischemia and reperfusion. PLC and PLA₂ activities were measured in cytosolic and total membrane fractions from control (perfused), ischemic (5, 10, 30 and 60 min), and post-ischemic reperfused ventricular tissue. Phospholipase activity was determined under optimal in vitro conditions using exogenous radiolabeled substrates. Alterations in membrane-associated PPI-PLC activity correlated with reported ischemia and reperfusion-induced changes in ventricular content of PPI metabolites. Membrane PLC activity increased slightly at 5 min of ischemia, decreased significantly at 10 min of ischemia, and continued to decrease with longer duration of ischemia (73% of control after 60 min). Cytosolic PPI-PLC activity was decreased at 5 min, and then significantly increased by longer durations of ischemia, while cytosolic PLA₂ activity was reduced at all time points. Pretreatment with muscarinic, alpha₁-adrenergic, beta-adrenergic, and adenosine receptor blockers did not alter ischemiaelicited changes in PLC activity. Reperfusion caused a 140% to 200% rise in the activities of all phospholipases in all fractions after 40 min of ischemia, but not after 10 min of ischemia. Results suggest 1) ischemia and reperfusion-elicited alterations in membrane-associated PPI-PLC activity can explain previously observed changes in phosphoinositide turnover metabolites, 2) cytosolic and membrane-associated PPI-PLC and PLA₂ activities are not uniformly affected by ischemia, 3) reperfusion following ischemia of sufficient duration initiates uniform activation of PIP₂-PLC and PLA₂, and 4) because ischemia and reperfusion-induced changes in phospholipase activity can be detected under optimal in vitro assay conditions (removed from the in vivo ischemic microenvironment), it is likely that the enzymes themselves have been altered.Supported by NIH grant NR 02203     

Adenosine triphosphate-evoked cytosolic calcium oscillations in human granulosa-luteal cells: role of protein kinase C

ATP has been shown to modulate progesterone production in human granulosa-luteal cells (hGLCs) in vitro. After binding to a G protein-coupled P2 purinergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate activates protein kinase C (PKC) and intracellular calcium [Ca(2+)](i) mobilization, respectively. In the present study, we examined the potential cross-talk between the PKC and Ca(2+) pathway in ATP signal transduction. Specifically, the effect of PKC on regulating ATP-evoked [Ca(2+)](i) oscillations were examined in hGLCs. Using microspectrofluorimetry, [Ca(2+)](i) oscillations were detected in Fura-2 loaded hGLCs in primary culture. The amplitudes of the ATP-triggered [Ca(2+)](i) oscillations were reduced in a dose-dependent manner by pretreating the cells with various concentrations (1 nM to 10 microM) of the PKC activator, phorbol-12-myristate-13-acetate (PMA). A 10 microM concentration of PMA completely suppressed 10 microM ATP-induced oscillations. The inhibitory effect occurred even when PMA was given during the plateau phase of ATP evoked [Ca(2+)](i) oscillations, suggesting that extracellular calcium influx was inhibited. The role of PKC was further substantiated by the observation that, in the presence of a PKC inhibitor, bisindolylmaleimide I, ATP-induced [Ca(2+)](i) oscillations were not completely suppressed by PMA. Furthermore, homologous desensitization of ATP-induced calcium oscillations was partially reversed by bisindolylmaleimide I, suggesting that activated PKC may be involved in the mechanism of desensitization. These results demonstrate that PKC negatively regulates the ATP-evoked [Ca(2+)](i) mobilization from both intracellular stores and extracellular influx in hGLCs and further support a modulatory role of ATP and P2 purinoceptor in ovarian steroidogenesis.     

Abscisic acid regulation of guard-cell K+ and anion channels in Gbeta- and RGS-deficient Arabidopsis lines

In mammals, basal currents through G protein-coupled inwardly rectifying K(+) (GIRK) channels are repressed by Galpha(i/o)GDP, and the channels are activated by direct binding of free Gbetagamma subunits released upon stimulation of Galpha(i/o)-coupled receptors. However, essentially all information on G protein regulation of GIRK electrophysiology has been gained on the basis of coexpression studies in heterologous systems. A major advantage of the model organism, Arabidopsis thaliana, is the ease with which knockout mutants can be obtained. We evaluated plants harboring mutations in the sole Arabidopsis Galpha (AtGPA1), Gbeta (AGB1), and Regulator of G protein Signaling (AtRGS1) genes for impacts on ion channel regulation. In guard cells, where K(+) fluxes are integral to cellular regulation of stomatal apertures, inhibition of inward K(+) (K(in)) currents and stomatal opening by the phytohormone abscisic acid (ABA) was equally impaired in Atgpa1 and agb1 single mutants and the Atgpa1 agb1 double mutant. AGB1 overexpressing lines maintained a wild-type phenotype. The Atrgs1 mutation did not affect K(in) current magnitude or ABA sensitivity, but K(in) voltage-activation kinetics were altered. Thus, Arabidopsis cells differ from mammalian cells in that they uniquely use the Galpha subunit or regulation of the heterotrimer to mediate K(in) channel modulation after ligand perception. In contrast, outwardly rectifying (K(out)) currents were unaltered in the mutants, and ABA activation of slow anion currents was conditionally disrupted in conjunction with cytosolic pH clamp. Our studies highlight unique aspects of ion channel regulation by heterotrimeric G proteins and relate these aspects to stomatal aperture control, a key determinant of plant biomass acquisition and drought tolerance.     

The path of calcium in cytosolic calcium oscillations: a unifying hypothesis.

Data from 42 systems have been assembled in which the overall spatial course of relatively natural, intracellular calcium pulses has been or can be determined. These include 21 cases of solitary pulses in activating eggs and 21 cases of periodic (as well as solitary) pulses in various fully active cells. In all cases, these pulses prove to be waves of elevated calcium that travel from one pole of a cell to the other or from the periphery inward. The velocities of these waves are remarkably conserved--at approximately 10 microns/sec in activating eggs and approximately 25 microns/sec in other cells at room temperature. Moreover, in three cases, the data suffice to show that these velocities fit the Luther equation for a reaction/diffusion wave of calcium through the cytosol. It is proposed that (i) natural intracellular calcium pulses quite generally take the form of cytosolic calcium waves and (ii) cytoplasmically controlled calcium waves are triggered and then propagated by the successive action of two distinct modes of calcium-induced calcium release. First, in the lumenal mode, a slow increase of calcium within the lumen of the endoplasmic reticulum reaches a level that triggers fast lumenal release as well as fast localized release into the cytosol. Then, the well-known cytosolic mode drives a reaction/diffusion wave across or into the cell.     

Alterations of phosphoinositide-specific phospholipase C and protein kinase C in the myocardium of spontaneously hypertensive rats

Summary In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wister-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP₂) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP₂-hydrolyzing PLC activity (PIP₂-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP₂-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca²⁺-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP₂-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP₂-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.Until April 31, 1990 N. Makita, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-Ku Sapporo 060, Japan After May 1, 1990 N. Makita, Division of Nephrology, S-3223, Medical Center North, Vanderbilt University, Nashville. Tennessee 37232, USA     

Increased platelet cytosolic free calcium concentration in essential hypertension

Cytosolic free calcium concentration [Ca2+] was studied in platelets of hypertensive patients with the use of the fluorescent indicator Quin 2/AM. Cytosolic free Ca2+ was significantly higher in platelets of hypertensive patients than in those of normotensive subjects (241 +/- 9 versus 192 +/- 7 nmol/l, n = 58 and 57, respectively P less than 0.001). When all 115 subjects were included, there was a significant correlation between cytosolic free Ca2+ and systolic or diastolic blood pressure (r = 0.262, P less than 0.0025 and r = 0.251, P less than 0.0025, respectively). Intracellular Quin 2 concentration was measured to evaluate the formaldehyde production (a product of Quin 2/AM hydrolysis which has been described as reducing the adenosine triphosphate (ATP) production). The Quin 2 concentrations in platelets of the two groups of subjects were observed to be similar (0.41 +/- 0.03 versus 0.38 +/- 0.03 mmol/l, n = 8 and 7 for hypertensives and normotensives, respectively). The effects of prostaglandin E1 (PGE1), an adenylate cyclase stimulator, on cytosolic free Ca2+ were studied. The presence of 10(-7) mol/l PGE1 lowered the Ca2+ in platelets of hypertensive patients only, suppressing the difference between the two groups.     

Amiloride delays the ischemia-induced rise in cytosolic free calcium

An increase in cytosolic free calcium (Cai) has been shown to occur early during ischemia in perfused rat, ferret, and rabbit hearts. It has been proposed that this increase in Cai may occur as a result of exchange of Nai for Cao, which occurs as a result of an increase in Nai arising from exchange of Nao for H+i. The latter exchange is stimulated by the intracellular acidification that occurs during ischemia. To test this hypothesis, we examined Cai, Nai, ATP, and pHi during ischemia in rats in the presence and absence of 1 mM amiloride, a Na-H exchange inhibitor. Cai was measured using 19F nuclear magnetic resonance (NMR) of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetra-acetic acid (5F-BAPTA)-loaded rat hearts. Nai was measured using 23Na NMR, and the shift reagent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetramethylenephosph onate (Tm[DOTP]-5) was used to separate Nai and Nao. ATP and pH were determined from 31P NMR measurements. During 20 minutes of ischemia, amiloride did not significantly alter the ATP decline but did significantly attenuate the rise in Nai and Cai. After 20 minutes of ischemia, time-averaged Cai was 1.0 +/- 0.2 microM (mean +/- SEM) in amiloride-treated hearts compared with 2.3 +/- 0.9 microM in nontreated hearts. After 20 minutes of ischemia, Nai in the untreated heart was threefold greater than control, whereas in the amiloride-treated heart, Nai was not significantly different from control. These data are consistent with the involvement of Na-Ca exchange in the rise in Cai during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cytosolic calcium by parathyroid hormone and oscillations of cytosolic calcium in fibroblasts from normal and pseudohypoparathyroid patients.

The second messengers for PTH action on fibroblasts have not been determined. The hypothesis that Ca2+ is a second messenger was examined by spectrofluorometric measurement of cytosolic calcium ([Ca2+]i), of fura-2-loaded human dermal skin fibroblasts. PTH induced: 1) transient elevations of [Ca2+]i due to both Ca2+ influx and intracellular release which were independent of CAMP generation, and 2) membrane depolarization. PTH treatment of fibroblasts depolarized by KCl induced oscillations of [Ca2+]i, but spontaneous oscillatory activity was not observed. Transient elevations of [Ca2+]i similar to that induced by PTH were observed with PTH-related peptide (PTHrp). PTH regulation of [Ca2+]i was normal in fibroblasts from patients with pseudohypoparathyroidism (PHP) type Ia (deficiency of the stimulatory GTP-binding protein, Gs), but PTH induced transient decreases in [Ca2+]i in fibroblasts from patients with PHP type Ib (defective PTH receptor). Both PHP Ia and Ib fibroblasts exhibited: 1) spontaneous [Ca2+]i oscillations, possibly due to voltage gated Ca2+ entry and Ca2+ induced Ca2+ release; and 2) greater sensitivity than normal fibroblasts for release of Ca2+ from intracellular stores when [Ca2+]i was decreased by lowering external Ca2+. Conclusions: 1) PTH-induced transient elevations in [Ca2+]i in normal fibroblasts result from Ca2+ entry and intracellular release which are independent of CAMP generation; 2) [Ca2+]i homeostasis is altered in PHP fibroblasts, resulting in Ca2+ oscillations; 3) PTH regulation of [Ca2+]i is altered in PHP Ib, but not in PHP Ia fibroblasts.     

Platelet cytosolic proton and free calcium concentrations in essential hypertension

Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic H+ concentration [( H+]i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular H+ concentration in platelets, unstimulated ex vivo, from normotensive (n = 20) and hypertensive patients (n = 20). Cytosolic [H+] was 20% lower in hypertensive than in normotensive subjects (49.5 +/- 3.4 and 61.8 +/- 2.2 nmol/l cells, respectively, P less than 0.005; mean pHi values were 7.21 and 7.33, respectively). Platelet cytosolic H+ and free Ca2+ concentrations ([Ca2+]i) were determined in parallel in 15 normotensive and 15 hypertensive patients. [Ca2+]i was found to be 19% higher (P less than 0.01), and [H+]i 22% lower (P less than 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHi and [Ca2+]i were increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.     

Alpha-adrenoceptor-mediated increase in cytosolic free calcium in isolated cardiac myocytes.

The effect of alpha-adrenoceptor stimulation on the concentration of cytosolic free calcium (Cai2+) was determined by measuring indo-l fluorescence in isolated ventricular cardiomyocytes from normal and streptozotocin-diabetic rat; 1.3 x 10(5) alpha 1-adrenoceptors per normal myocyte and an unaltered number of these receptors in cells from diabetic rats were detected using the alpha 1-selective ligand WB-4101. Under basal conditions, Cai2+ was found to be 154 +/- 4 nM (n = 34) reaching a value of 192 +/- 10 nM (n = 15) after stimulation of myocytes with a maximal dose of methoxamine for 5 min. Under the same conditions the leakage of dye produced a significantly smaller increase of basal values of 169 +/- 5 nM (n = 17). Indo-l loaded cells did not respond to beta-stimulation unless in the presence of KCl (50 mM), demonstrating the specificity of methoxamine action. Treatment of cells with nifedipine or chelation of extracellular calcium by EGTA did not modify the alpha-adrenergic response. Experiments with cardiomyocytes from streptozotocin-diabetic rats showed an unaltered modulation of Cai2+ by both alpha- and beta-receptor stimulation. It is concluded that signalling by alpha 1-adrenoceptors in ventricular cardiomyocytes results in mobilization of intracellular calcium stores.     

Acetylcholine induces voltage-independent increase of cytosolic calcium in mouse myotubes.

Electrophysiological, biochemical, and Ca2+ imaging studies of cultured mouse myotubes were used to investigate whether the neurotransmitter acetylcholine causes an increase in intracellular Ca2+ concentration ([Ca2+]i) through activation of a second messenger system. Bath applications of acetylcholine to myotubes (i) elicited a significant membrane current even in a Na(+)-free Ca2+ medium, when the current was carried mainly by calcium ions; (ii) caused a rapid and transient cytosolic accumulation of inositol 1,4,5-trisphosphate; (iii) evoked a conspicuous alpha-bungarotoxin-sensitive long-lasting [Ca2+]i enhancement even in the presence of Cd2+; and (iv) transiently increased [Ca2+]i when cells were equilibrated in a Ca(2+)-free atropine-containing medium. We propose that, in addition to opening ion channels, the nicotinic action of acetylcholine on the muscle cell membrane increases [Ca2+]i through activation of the inositol 1,4,5-trisphosphate second messenger system and mobilization of Ca2+ from intracellular stores.     

Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K+ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.     

Prolactin increases cytosolic free calcium concentration in hepatocytes of lactating rats

PRL at a physiological concentration (10(-8) M) produced a very rapid and transient increase in 45Ca efflux in freshly isolated hepatocytes, which reached the highest value within 5 min and returned to baseline level after 20 min. PRL-induced 45Ca2+ efflux resulted in a loss of 15% of total cell calcium, which was similar to that found in vasopressin-treated cells. However, in contrast with the PRL effect, 45Ca2+ efflux induced by vasopressin was sustained. We demonstrate by using two different approaches, glycogen phosphorylase-a activation and direct cytosolic calcium concentration [( Ca2+]i) measurements, that PRL elicits a [Ca2+]i increase. The treatment of hepatic cells with PRL caused a 4-fold stimulation in glycogen phosphorylase-alpha activity after 2 min of PRL addition. Direct [Ca2+]i determination in fluo-3-loaded hepatocytes showed a 11% increase after 5 min of PRL addition. Similar data were observed in hepatocytes stimulated either with vasopressin (10(-7) M) or calcium ionophore A23187 (200 nM). The increase in [Ca2+]i promoted by PRL was independent of extracellular calcium or voltage-operated calcium channels. The data demonstrate that calcium is involved in the intracellular signaling of PRL in liver cells and that PRL initiates its action by a Ca2+ mobilization from the intracellular stores.     

Role of increased cytosolic free calcium concentration in myocardial ischemic injury

Summary Increases in cytosolic free calcium concentration ([Ca²⁺]_I) may play an important role in myocardial ischemic injury. An early effect of the rise in [Ca²⁺]_I may be impaired postischemic contractile function if the ischemic myocardium is reperfused during the reversible phase of ischemic injury; furthermore, if the rise in [Ca²⁺]_I is prolonged, a cascade of events may be initiated which ultimately results in lethal injury. With the development of methods for measuring [Ca²⁺]_I, it has become possible to evaluate directly the role of increased [Ca²⁺]_I in myocardial ischemic injury. Although it has been possible to show that inhibition of the transport processes which contribute to the early rise in [Ca²⁺]_I attenuates stunning and the rise in [Ca²⁺]_I concurrently, if increased [Ca²⁺]_I plays an important role in ischemic injury, then it should be possible to show that interventions which alter the timecourse of ischemic injury also alter the timecourse of the rise in [Ca²⁺]_I in a parallel manner. Recently, considerable effort has been expended to investigate the mechanisms underlying the preconditioning phenomenon, whereby repetitive brief periods of ischemia prior to a sustained period of ischemia protects the myocardium from injury during the sustained period of ischemia, and this has stimulated additional work to understand the possible involvement of adenosine as a mediator of preconditioning as well as to understand the protective effects of adenosine. Measurements of [Ca²⁺]_I using 19F NMR of 5FBAPTA-loaded hearts have shown that preconditioning attenuates the rise in [Ca²⁺]_I during 30 min of ischemia and reduces stunning during reflow. Adenosine pretreatment mimics the effects of preconditioning on the rise in [Ca²⁺]_I and on stunning, but adenosine receptor antagonists do not eliminate the protective effects of preconditioning, although some adenosine antagonists also block hexose transport and under these conditions, the ability of preconditioning to attenuate the rise in [Ca²⁺]_I is abolished and there is a corresponding loss of the protective effect of preconditioning on stunning. Although it has been suggested that the beneficial effect of preconditioning on infarct size can be eliminated by pretreatment with glibenclamide, in the isolated rat heart glibenclamide does not affect the attenuation of the rise in [Ca²⁺]_I induced by preconditioning and does not affect stunning. All of these studies show a consistent relationship between the magnitude of the rise in [Ca²⁺]_I during ischemia and the degree of stunning during reperfusion. The data suggest that increased [Ca²⁺]_I plays a very important role in myocardial ischemic injury.     

Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets

Lysophosphatidic acid is a biologically active phospholipid able to induce cell proliferation and platelet aggregation. In this study we investigated the biochemical mechanisms of platelet activation by lysophosphatidic acid. We found that lysophosphatidic acid stimulated 4-azidoanilido-{ alpha 32P}GTP to a 40-kDa protein on platelet membranes. Moreover, lysophosphatidic acid induced the rapid decrease of the intracellular concentration of cAMP in intact platelets, indicating that this lipid activates platelets by binding to a membrane receptor coupled to the inhibitory GTP-binding protein Gi. In agreement with a receptor-mediated action, we found that platelet activation by lysophosphatidic acid underwent homologous desensitization. In the absence of extracellular CaCl , lysophosphatidic acid did not induce platelet aggregation, and did not stimulate phospholipase C. However, under the same conditions, lysophosphatidic acid produced the rapid tyrosine phosphorylation of several platelet proteins. This effect was not mediated by the formation of thromboxane A . Our results demonstrate that, in lysophosphatidic acidstimulated platelets, activation of protein-tyrosine kinases occurs in the absence of phospholipase C activation and platelet aggregation, and may be directly related to the activation of the G-protein-coupled lysophosphatidic acid-receptor. the binding of the photoreactive GTP-analog 2 2.     

Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets

Lysophosphatidic acid is a biologically active phospholipid able to induce cell proliferation and platelet aggregation. In this study we investigated the biochemical mechanisms of platelet activation by lysophosphatidic acid. We found that lysophosphatidic acid stimulated the binding of the photoreactive GTP-analog 4-azidoanilido-[α³²P]GTP to a 40-kDa protein on platelet membranes. Moreover, lysophosphatidic acid induced the rapid decrease of the intracellular concentration of cAMP in intact platelets, indicating that this lipid activates platelets by binding to a membrane receptor coupled to the inhibitory GTP-binding protein Gi. In agreement with a receptor-mediated action, we found that platelet activation by lysophosphatidic acid underwent homologous desensitization. In the absence of extracellular CaCl₂, lysophosphatidic acid did not induce platelet aggregation, and did not stimulate phospholipase C. However, under the same conditions, lysophosphatidic acid produced the rapid tyrosine phosphorylation of several platelet proteins. This effect was not mediated by the formation of thromboxane A₂. Our results demonstrate that, in lysophosphatidic acid-stimulated platelets, activation of protein-tyrosine kinases occurs in the absence of phospholipase C activation and platelet aggregation, and may be directly related to the activation of the G-protein-coupled lysophosphatidic acid-receptor.     

1,25-Dihydroxyvitamin D3 enhances cytosolic free calcium in HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) caused a rise in the concentration of intracellular free calcium ions ([Ca2+]i) in HL-60 cells. This effect of 1,25(OH)2D3 parallels its suppression of cell proliferation and its induction of cell differentiation into monocyte-like cells. The changes in [Ca2+]i are dose and time dependent. The concentration of 1,25(OH)2D3 (10(-7) M) that induced maximal differentiation also caused the maximal increase in intracellular Ca2+. The rise in cytoplasmic free Ca2+ concentration was not immediate and reached statistical significance only after 24 h. The [Ca2+]i reached its peak at 48 h (134 +/- 4 nM vs 101 +/- 3 nM in controls) and remained stable at this level. The increase in intracellular Ca2+ was found to be related to new protein synthesis, because it was inhibited in the presence of specific RNA and protein synthesis inhibitors. The rise in [Ca2+]i was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), a vitamin D metabolite that does not induce the differentiation of HL-60 cells. In contrast, 25-hydroxyvitamin D3 (25-OH-D3) and phorbol 12-myristate 13-acetate (TPA), both of which induce differentiation in this cell line, also increase [Ca2+]i. In conclusion, the present study emphasizes that a significant increase in intracellular free Ca2+ occurs in the effect of 1,25(OH)2D3 on HL-60 cells.