Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.     

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.     

The effects of extracellular calcium and epinephrine on cytosolic-free calcium in single rat adipocytes.

Changes in cytosolic calcium concentration ([Ca2+]i) in response to extracellular calcium and epinephrine were monitored in individual rat adipocytes by both photon counting and digital imaging techniques utilizing the intracellular fluorescent calcium probes Fura-2 and Indo-1. Adipocytes containing Fura-2 were attached to coverslips and shown to be as hormonally responsive to insulin as adipocytes in suspension [3.5 +/- 0.8 (n = 5) vs. 4.2 +/- 0.6 (n = 8)-fold increase in glucose oxidation over basal in response to 0.7 nM insulin]. Basal [Ca2+]i in single rat adipocytes was found to be 128 +/- 6 nM (n = 100). The addition of either extracellular calcium or epinephrine elicited transient, concentration-dependent increases in [Ca2+]i. Although the characteristics of calcium- and epinephrine-induced calcium transients are generally similar, the peak [Ca2+]i increase over basal is higher in response to calcium vs. epinephrine [37 and 64% (1 and 27 microM epinephrine), vs. 132 and 236% (2 and 4 mM calcium)]. All the cells tested responded to calcium but only 67% responded to epinephrine. Both alpha- and beta-adrenergic agonists were able to increase [Ca2+]i. The epinephrine-induced [Ca2+]i transients appear to be dependent upon extra-cellular calcium. Neither cholera nor pertussis toxin treatments altered basal [Ca2+]i. However, after treatment of adipocytes with either pertussis or cholera toxin, epinephrine stimulated oscillations in [Ca2+]i. Digital imaging revealed that adipocytes demonstrate a high degree of intracellular spatial heterogeneity and intercellular variability in the magnitude of response to both calcium and epinephrine. These studies demonstrate the feasibility of using single rat adipocytes to monitor intracellular free calcium, using both photon counting and digital imaging.     

Fluoride stimulates the accumulation of inositol phosphates, increases intracellular free calcium, and inhibits parathyroid hormone release in dispersed bovine parathyroid cells

The stimulation of polyphosphoinositide (PPI) turnover is associated with cellular activation and hormone secretion in numerous systems. GTP-binding proteins appear to couple receptors to phospholipase-C-mediated PPI breakdown. We assessed the effects of fluoride, an activator of GTP-binding proteins, on inositol phosphate accumulation, intracellular free Ca2+ [(Ca2+)i], cAMP content, and PTH release in dispersed bovine parathyroid cells. Sodium fluoride (5-30 mM) produced marked dose-dependent increases in inositol phosphates. With anion exchange HPLC, we confirmed that 30 mM fluoride stimulated a rapid increase in 1,4,5-inositol trisphosphate, a potent Ca2+-mobilizing compound. Using the Ca2+-sensitive probe fura-2, we determined that 30 mM fluoride increased [Ca2+]i from 339 +/- 9 to 650 +/- 39 nM (n = 8) within 30-60 sec at 1 mM extracellular Ca2+. After the depletion of extracellular Ca2+ by the addition of 1 mM EGTA, 30 mM fluoride increased [Ca2+]i 45 +/- 9% (n = 4), indicating that fluoride can mobilize intracellular Ca2+ stores. Fluoride (1-30 mM) also inhibited PTH release in dose-dependent fashion. Fluoride (30 mM) produced 72.8 +/- 4.2% suppression of maximal low Ca2+-stimulated PTH release comparable to the 83.7 +/- 3.7% inhibition by 2.0 mM extracellular Ca2+. Since changes in both [Ca2+]i and cAMP regulate PTH release, we measured the effect of fluoride on intracellular cAMP. Fluoride did not detectably change basal cAMP content, but it reduced forskolin-stimulated increases in cAMP. We conclude that fluoride may activate at least two GTP-dependent processes in parathyroid cells, resulting in PPI breakdown and cAMP accumulation. While both may contribute to the fluoride-induced suppression of PTH release, our findings suggest that the stimulation of PPI turnover leads to inhibition of PTH secretion.     

Increased basal and thrombin-induced free calcium in platelets of essential hypertensive patients

Intracellular free calcium, [Ca2+]i, was studied in platelets of essential hypertensive subjects and normotensive controls under basal conditions and after stimulation with epinephrine, norepinephrine, angiotensin II, ouabain, and thrombin, using the fluorescent calcium indicator quin 2. Basal [Ca2+]i was significantly higher in hypertensive subjects (n = 32) than in normotensive controls (n = 30; 167.4 +/- 5.0 vs 143.2 +/- 3.1 nmol/L; p less than 0.001). Epinephrine, norepinephrine, angiotensin II, and ouabain had no effect on platelet calcium, whereas thrombin induced a dose-dependent increase in [Ca2+]i in both the presence and absence of extracellular calcium. This [Ca2+]i increase in the presence of extracellular calcium, which depends mainly on calcium influx, was significantly higher (p less than 0.05) in platelets of hypertensive subjects at all thrombin concentrations (ranging from 0.025-0.1 U/ml), while the [Ca2+]i increase in the absence of extracellular calcium, which depends only on release from intracellular stores, was similar in hypertensive subjects and controls. These results suggest that, in essential hypertension, there is not only increased platelet resting [Ca2+]i but also an increase in agonist-mediated calcium influx, which appears to indicate a cell membrane abnormality in the platelets of subjects with essential hypertension.     

Abnormal calcium handling by platelets in thrombotic disorders

This study presents a quantitative comparison of the free cytoplasmic calcium concentration ([Ca2+]cyt) and the free concentration in the lumen of the dense tubules of the human platelet. The former was measured by the fluorescence of the high affinity indicator quin2 and latter by the fluorescence of chlorotetracycline (CTC). The CTC technique monitors calcium-CTC complex accumulation in the lumen of dense tubules and mitochondria when washed platelets were incubated in 2 mM Ca2+. Resting cytoplasmic and dense tubular Ca2+ concentrations were studied in platelets from patients suffering from venous and arterial thrombosis. Compared with normal controls (0.40 +/- 0.10, n = 54), the values of the calcium-CTC ratios were 0.68 +/- 0.19 (n = 16, p less than 0.005) in venous thrombosis; 0.75 +/- 0.18 (n = 14, p less than 0.005) in cardiovascular accident; 0.84 +/- 0.18 (n = 6, p less than 0.005) in occlusive peripheral vascular diseases; and 0.42 +/- 0.10 (n = 21, p greater than 0.1) in patient controls. The dense tubular Ca2+ levels for both patients and controls were perfectly correlated with the cytoplasmic levels using an equation that assumes that the dense tubular free calcium concentration ([Ca2+]dt) has a second power dependence on [Ca2+]cyt. The abnormal Ca2+ handling of platelets obtained from thrombotic patients could be completely reversed by preincubation with the calcium channel blocker verapamil. These observations suggest that the primary Ca2+ handling defect is the leakage through activated channels in the plasma membrane. The defect and the elevated resting [Ca2+]cyt and [Ca2+]dt are adequate to explain the observation of increased rates of collagen-activated aggregation in the above-mentioned group of patients. The results can be explained by platelets from thrombosis patients being exposed to activating factors in the circulation, resulting in Ca2+ channel activation. Channel activation persists through the process of platelet isolation and washing and is manifested in higher measured values of [Ca2+]cyt and [Ca2+]dt in the "resting state." This would bring the platelet closer to its aggregation when aggregation-inducing agents are added. The CTC test is shown to be a useful and convenient means of detecting this abnormality.     

Abnormal secretagogue-induced intracellular free Ca2+ regulation in cystic fibrosis nasal epithelial cells.

These studies identify a further abnormality in cystic fibrosis (CF). The increase in intracellular free calcium concentration ([Ca2+]i) after exposure to histamine and PGE1 is demonstrated to be abnormally low in nasal cells, studied in short-term culture, from patients with CF compared with control subjects. [Ca2+]i is measured by using the Ca(2+)-sensitive fluorescent dye fura-2 and a fluorescence microscope imaging system. The percentage of CF cells that increase [Ca2+]i in response to histamine is decreased compared with controls, and, even in those CF cells that increase [Ca2+]i, the magnitude of the increase in [Ca2+]i in response to histamine is smaller than in controls. When exposed to PGE1, a similar number of control and CF cells responded with an increase in [Ca2+]i, but again the magnitude of the response was smaller in the CF cells. The mechanism of the PGE1-induced increase in [Ca2+]i is not mediated by cAMP, since 8-bromo-cAMP failed to increase [Ca2+]i in these cells. This abnormality in [Ca2+]i response did not apply to all secretagogues, with the response to carbachol being similar in CF and normal cells. How the abnormal CF gene product accounts for the abnormality in intracellular Ca2+ response to some but not all secretagogues is unknown.     

Actions of endothelin-1 on swine ovarian (granulosa) cells.

We have investigated the regulatory actions of endothelin-1 (ET-1) on inositol phosphate accumulation, cytosolic free Ca2+ ion concentrations ([Ca2+]i), and basal and FSH-stimulated progesterone and cAMP accumulation by swine granulosa cells in serum-free cultures. ET-1 induced a rapid stimulation of phosphoinositide hydrolysis in populations of granulosa cells, as inferred by the rapid appearance of soluble inositol polyphosphates in response to ET-1 exposure. At the single cell level, fura-2 videomicroscopy was used to measure [Ca2+]i in individual granulosa cells. We observed cell-cell variability in the threshold concentration of ET-1 required to induce a rise in [Ca2+]i. More than 75% of granulosa cells responded to maximal doses of ET-1. The following parameters of [Ca2+]i were influenced by ET-1 concentration: percentage of responding cells, lag time for the onset of response, amplitude, and kinetics of the response. Two types of ET-1-mediated [Ca2+]i rises were observed. One type exhibited rapid Ca2+ kinetics, reaching at least a 2-fold increase above basal (spike phase) within 1-10 sec and returning to a new steady state (plateau phase) 2 min after onset. The other mode of response had slower [Ca2+]i kinetics, in which 50 sec or more were required to double [Ca2+]i, which remained at this level throughout the observation period (2.5 min). These responses to ET-1 were specific and were not initiated by vasopressin or tumor necrosis factor-alpha. In cell population studies using monolayer cultures of swine granulosa cells, ET-1 inhibited FSH-stimulated accumulation of progesterone and cAMP. The ET-1-mediated inhibition of FSH-stimulated accumulation of progesterone required at least 4 h of ET-1 exposure. The ET-1-mediated inhibition of both the FSH-stimulated accumulation of progesterone and cAMP after 24-h incubation was mimicked by an activator of protein kinase-C, phorbol 12-myristate 13-acetate, but not by an inactive phorbol. These observations in either single cells or populations of swine ovarian (granulosa) cells are consistent with a possible regulatory role of an ET-1-activated intracellular signaling pathway involving inositol phosphates, [Ca2+]i, and protein kinase-C in the mammalian granulosa cell.     

Effects of thrombin on intracellular calcium and pH in human and murine platelets

The aim of this study was to compare the effect of thrombin (Thr) on cytosolic calcium [Ca2]+i and intracellular pH [pH]i in human and murine platelets. Rich-platelet suspensions from both species were loaded with Fura-2 (2 microM) or BCECF (0.75 microM) by incubation with their respective acetoxymethyl esters to measure cytosolic calcium [Ca2+]i or intracellular pH [pH]i, respectively. Suspensions were challenged with increasing concentrations of Thr, from 0.1 to 10 IU/ml. Basal [Ca2+]i in human platelets was 98 +/- 6 and 99.1 +/- 9 nM in rat platelets (n = 20). Thr increased [Ca2+]i, EC50 was 1.1 +/- 0.04 in human and 0.97 +/- 0.06 IU/ml in rat platelets (n = 7). Extracellular Mg2+ (4 or 8 mM) abolished Thr response on [Ca2+]i. [pH]i in human was 7.09 +/- 0.08 and 7.11 +/- 0.04 in rat platelets. Thr induced alkalinization of platelets in both species. Our results indicate that the potency of Thr to change [Ca2+]i and [pH]i was similar in both species, allowing for comparisons between human and murine platelets and to extrapolate results from an animal model to human pathology.     

Alpha 1-adrenergic potentiation of vasoactive intestinal peptide stimulation of rat pinealocyte adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate: evidence for a role of calcium and protein kinase-C

alpha 1-Adrenergic agonists have recently been found to potentiate vasoactive intestinal peptide (VIP) stimulation of rat pinealocyte cAMP and cGMP. alpha 1-Adrenergic agonists also elevate pineal intracellular Ca2+ [( Ca2+]i) and activate protein kinase-C. In the present study, the possible involvement of Ca2+ and protein kinase-C in the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP accumulation was examined with agents that alter [Ca2+]i or activate protein kinase-C. It was found that treatment with a Ca2+ chelator or with inorganic Ca2+ channel blockers inhibited alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP responses. Increasing [Ca2+]i by treatment with A23187, ouabain, or K+ potentiated VIP stimulation of cAMP and cGMP response. These observations indicate that Ca2+ mediates the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP accumulation, as is true for the alpha 1-adrenergic potentiation of beta-adrenergic stimulated cAMP and cGMP accumulation. Activators of protein kinase-C mimicked the large effect alpha 1-adrenergic agonists have on cAMP accumulation in VIP-treated pinealocytes and had a small effect on cGMP accumulation in VIP-treated cells. These effects were not blocked by the Ca2+ chelator EGTA. However, the effects of a protein kinase-C activator on the cGMP response in VIP-stimulated cells were amplified by K+ (15 mM) or ouabain (1 microM), presumably through an action causing an increase in [Ca2+]i. These results suggest protein kinase-C is involved in the alpha 1-adrenergic potentiation of VIP-stimulated cAMP accumulation, as is the case for the alpha 1-adrenergic potentiation of beta-adrenergic stimulated cAMP. Protein kinase-C is also involved in cGMP accumulation, provided that there is a modest increase in [Ca2+]i.     

Platelet activation and subsequent inhibition by plasmin and recombinant tissue-type plasminogen activator.

The success of plasminogen activators in recanalizing occluded coronary arteries may be influenced by their effect on blood platelets; however, some previous studies have shown platelet activation by plasmin and thrombolytic agents while others have shown an inhibitory effect. Moreover, it has not been determined whether these effects reflect an alteration of intracellular signal transduction, fibrinogenolysis, degradation of adhesive protein receptors, or a combination of these events. To distinguish among these possibilities, the increase of cytoplasmic [Ca2+] [( Ca2+]i), which is an intracellular marker of platelet activation that precedes fibrinogen binding to the surface of activated platelets, was measured along with aggregation and release of 5-hydroxytryptamine (5-HT) in washed human platelets incubated with plasmin or recombinant tissue-type plasminogen activator (rt-PA). Plasmin (0.1 to 1.0 CU/mL) induced a prompt, concentration-dependent [Ca2+]i increase when added to platelets, but subsequently inhibited the [Ca2+]i increase in response to thrombin or the endoperoxide analog U44069. Platelet aggregation accompanied the [Ca2+]i increase if the platelets were stirred, while the aggregation of platelets unstirred during plasmin incubation was inhibited upon agonist addition and resumption of stirring. The release of 5-HT paralleled the [Ca2+]i increase induced by plasmin and was also inhibited after the subsequent addition of a second agonist. The effects of rt-PA, added with plasminogen (100 micrograms/mL), were similar to those of plasmin, and could be accounted for by the concentration of plasmin generated. The ADP scavengers apyrase and CP/CK each prevented the [Ca2+]i increase, and aggregation caused by plasmin or rt-PA, and also prevented their inhibitory effects on thrombin-induced activation. Thus, plasmin and rt-PA initially activate platelets, inducing a [Ca2+]i increase, and, if the platelets are stirred, aggregation. Such activation is followed by subsequent inhibition of cellular activation by a second agonist; the inhibitory effect is in proportion to the degree of initial activation, and ADP is an important cofactor in both processes. These platelet effects occur at rt-PA concentrations achievable clinically, and may affect the success of therapy with thrombolytic and adjunctive agents.     

Desensitization of rat osteoblast-like cells (ROS 17/2.8) to parathyroid hormone uncouples the adenosine 3',5'-monophosphate and cytosolic ionized calcium response limbs.

We have investigated the effects of PTH-induced desensitization on second messenger interactions in the rat osteosarcoma cell line ROS 17/2.8. Adenylate cyclase activation was assessed by accumulation of immunoassayable cAMP, and cytosolic calcium ion ([Ca2+]i) concentrations were measured in adherent perifused cells loaded with the Ca2(+)-sensitive bioluminescent protein aequorin. Preexposure to rat PTH-(1-34) [rPTH-(1-34); 10(-8) M for 48 h, then 10(-7) M for 24 h] dramatically reduced (by 85%) the cAMP response to fresh challenge [2 min; 10(-9)-10(-7) M rPTH-(1-34)], but the peak PTH-induced rise of [Ca2+]i was not diminished significantly (0-20%). Nevertheless, we did observe other changes in the PTH-induced [Ca2+]i response. Exposure of treated cells to (Bu)2cAMP nearly abolished the [Ca2+]i response to PTH (greater than 80% reduction), but had much less effect on the PTH-stimulated [Ca2+]i increment of the naive cells (less than 35% reduction). Treated cells also had a blunted [Ca2+]i response to PTH in the presence of low extracellular calcium (greater than 60% reduction), but in the naive cells, low extracellular Ca2+ did not significantly diminish the peak PTH-induced [Ca2+]i rise, although low extracellular Ca2+ dramatically reduced the area under this [Ca2+]i transient (greater than 50%). Low extracellular Ca2+ had no influence on the peak [Ca2+]i responses of treated cells to bradykinin or prostaglandin F2 alpha. Although the peak PTH-stimulated [Ca2+]i rise of treated cells in normal Ca2+ medium was not significantly attenuated, the time to half-maximum [Ca2+]i concentration was significantly increased (greater than 100%), and the area under the [Ca2+]i transient was diminished. These alterations in the [Ca2+]i response of treated cells were not observed upon challenge with bradykinin or prostaglandin F2 alpha. Thus, 1) the cAMP and [Ca2+]i responses of ROS 17/2.8 cells to rPTH-(1-34) are not obligatorily coupled; 2) the response of naive cells to PTH includes both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+; and 3) pretreatment of these cells with rPTH-(1-34) augments the dependence on Ca2+ entry during hormone rechallenge. We propose that the preserved PTH-stimulated [Ca2+]i rise in treated cells results partly from loss of cAMP-mediated inhibition of extracellular Ca2+ entry.     

Effect of amlodipine and cilazapril treatment on platelet Ca2+ handling in spontaneously hypertensive rats.

Abnormal Ca2+ handling and enhanced aggregation response have been reported in platelets from spontaneously hypertensive rats (SHR) and patients with essential hypertension, and thought to be involved in the progression of target organ damage of hypertension. It is important to examine whether antihypertensive therapy can improve the abnormal platelet response in hypertension. We investigated the effect of antihypertensive treatment such as amlodipine and cilazapril on Ca2+ handling and aggregation response in SHR platelets. Four-week-old male SHR were divided into three groups. Each group was treated with amiodipine (A: 10 mg/kg/day), cilazapril (C: 10 mg/kg/day) or vehicle (V) for 8 weeks by gavage. At 12-week-old, platelet [Ca2+]i was measured with fura-2 in each group of SHR and age-matched Wistar-Kyoto rats (WKY) as normal control. Systolic blood pressure in amlodipine and cilazapril treated groups were similar with WKY and significantly lower than vehicle treated group (A: 124 +/- 9, C: 126 +/- 9, WKY: 122 +/- 10 and V: 180 +/- 9 mmHg, respectively). The basal [Ca2+]i in the three groups of SHR were similar and higher than WKY (A: 47 +/- 1.7, C: 47 +/- 1.2, V: 48 +/- 3.9 and WKY: 40 +/- 4.0 nmol/l, respectively). There were no significant differences in thrombin (0.1 U/ml)-stimulated [Ca2+]i rise in the presence or absence of extracellular Ca2+ among the three groups of SHR and these were higher than WKY. Intracellular Ca2+ discharge capacity, assessed by the ionomycinstimulation was similar in the all groups. Thrombin-induced maximum platelet aggregation responses in the three groups of SHR were similar and higher than WKY. The antihypertensive treatment of Ca2+ antagonist or ACE inhibitor gave no change in intraplatelet Ca2+ metabolism in SHR. These results support the hypothesis that an abnormal Ca2+ handling in SHR platelet is genetically determined and not improved by hypotensive therapy.     

Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.     

Intracellular Ca2+ mobilization and its regulation in platelet activation]

It has been generally accepted that platelets play etiological roles for the development of atherosclerosis and arterial thrombosis. Platelet activation may be dependent upon the cytosolic free Ca2+ concentration ([Ca2+]i), and regulated by PGI2 and endothelium-derived relaxing factor (EDRF) released by vascular endothelium. We have studied here the effect of endothelial cells (EC) on platelet activation and intracellular Ca2+ mobilization. Effluent of non-stimulated EC column inhibited thrombin-induced platelet aggregation and intracellular Ca2+ mobilization. An addition of this effluent to platelet suspension leaded to increase in intraplatelet cyclic AMP (cAMP) which was inhibited by the treatment of indomethacin to EC, suggesting that this effect was involved in PGI2 released by EC. On the other hand, effluent of thimerosal-stimulated EC column inhibited platelet aggregation and increase in [Ca2+]i stimulated with thrombin, and leaded to increase in intraplatelet cyclic GMP (cGMP). But the treatment of indomethacin to EC had no effect of this inhibition. The effect of thimerosal-stimulated EC was inhibited by the addition of 1-NG-monomethylarginine (NMA), EDRF/NO inhibitor, suggesting that EDRF released by thimerosal-stimulated EC produced an increase in cGMP and inhibited platelet activation. Although forskolin-induced in cAMP caused a marked prevention of inositol 1, 4, 5-trisphosphate (IP3) production stimulated with thrombin, 8-bromo cGMP and EDRF-induced increase in cGMP had no effect of IP3 production. An increase in cAMP and cGMP was considered to inhibit intracellular Ca2+ mobilization by different mechanisms in platelets.     

Effects of phosphatidic acid on parathyroid hormone release, intracellular free Ca2+, and inositol phosphates in dispersed bovine parathyroid cells

The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+ mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 +/- 6%, 48 +/- 5%, 59 +/- 10%, and 79 +/- 6% at concentrations of 1, 10, 50, and 100 micrograms/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 micrograms/ml) inhibited PTH secretion by 39 +/- 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 +/- 8 to a peak of 356 +/- 26, 454 +/- 22, and 587 +/- 57 nM with the addition of 1, 10, and 100 micrograms/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 +/- 4% in [Ca2+]i, indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 micrograms/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 +/- 9%, 37 +/- 9%, and 59 +/- 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (greater than or equal to 50 micrograms/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 +/- 5% inhibition observed at lower doses (10 micrograms/ml) and a 27 +/- 8% stimulation of secretion at 100 micrograms/ml (n = 6).(ABSTRACT TRUNCATED AT 400 WORDS)     

Lowering of cytosolic free Ca2+ by carbachol, a muscarinic cholinergic agonist, in clonal pituitary cells (GH3 cells)

Muscarinic cholinergic agonists have been shown to inhibit PRL secretion in normal and tumor-derived pituitary cells. Evidence from experiments with the fluorescent Ca2+ probe quin 2 shows that carbachol, acting through muscarinic acetylcholine receptors, lowers the cytosolic free Ca2+ concentration ([Ca2+]i), in GH3 cells. A decrease in [Ca2+]i is observed rapidly after carbachol addition, the lowered steady state [Ca2+]i is maintained, and upon the addition of atropine [Ca2+]i returns to the initial basal value. The lowering from a basal [Ca2+]i, averaging 110 +/- 2 nM (+/- SEM, n = 9), to a steady state [Ca2+]i of 63 +/- 4 nM (+/- SEM, n = 5) at 10 micron carbachol is dose dependent, a significant decrease from basal [Ca2+]i being observed at 0.1 micron. Carbachol does not prevent TRH-induced mobilization of Ca2+ but attenuates the resulting rise in [Ca2+]i. The lowering of steady state [Ca2+]i and the attenuation of the rise in [Ca2+]i provoked by stimulators of PRL secretion could explain the inhibition of both basal and stimulated PRL secretion. Concomitantly with the action on [Ca2+]i, carbachol causes hyperpolarization of GH3 cells. Together with the established inhibition of adenylate cyclase by muscarinic cholinergic agonists, these findings suggest a relation between changes in trans-membrane Ca2+ fluxes and cAMP generation.     

Activation of human platelets by the rabbit anticardiolipin antibodies.

Affinity purified anticardiolipin antibodies (ACLA) raised in rabbits showed cross-reactivities with various negatively charged phospholipids as shown by both the solid phase enzyme-linked immunosorbent assay (ELISA) and inhibition studies. In ELISA, ACLA showed strong cross-reactivity to both sphingomyelin (SM) and phosphatidylethanolamine (PE), but the inhibition studies showed that ACLA failed to bind the aqueous suspensions of SM, PE, and PE/PC (1:1). ACLA bound to resting gel-filtered human platelets (GFP) as shown by both inhibition study and flow cytofluorometric analysis. Western blotting procedure showed that ACLA strongly cross-reacted to an 80-Kd plasma membrane protein. ACLA activated platelet response in a concentration-dependent manner. At less than 10 micrograms/mL, ACLA induced both platelet shape change to spiculate irregular forms as shown by scanning electron microscopy and the phosphorylation of 20-Kd protein. ACLA at more than 10 micrograms/mL caused platelet aggregation and secretion. The aggregation was inhibited by EDTA; aspirin; antimycin A plus 2-deoxyglucose; PGE1; and the F(ab')2 fragment of ACLA. It was not inhibited by monoclonal antibody to Fc receptor (MoAb FcR2). The biochemical events of ACLA-induced platelet response involved the elevation of (1) thromboxane A2 formation, (2) cytosolic free calcium ion concentration ([Ca2+]i), and (3) 47-Kd protein phosphorylation. In addition, the subaggregatory concentration of ACLA showed synergistic platelet activation with that concentration of thrombin, collagen, and epinephrine. The study showed the mechanism involved in ACLA-induced platelet responses.     

Adenosine triphosphate activates the phospholipase-C cascade system in human amnion cells without increasing prostaglandin production

Human amnion is hypothesized to be a target tissue for hormone messages from the fetus regarding labor. We have previously demonstrated prostaglandin E2 (PGE2) release in amnion after treatment with phorbol and oxytocin, but other potential agonists of the inositol phospholipid/protein kinase-C system have not been investigated. The effects of extracellular ATP on cytosolic calcium concentration [( Ca2+])i) inositol phosphate (IP) accumulation, and PGE2 production were studied in cultured human amnion cells. Intracellular free calcium [Ca2+]i was measured using the fluorescent dye fura-2. Addition of 0.01-30 microM ATP resulted in a [Ca2+]i transient which peaked within 15 sec and returned to baseline over 10 min. UTP (1 microM) was more effective than ATP (1 microM); [Ca2+]i levels rose from 233 to 2880 nM (UTP) and 2320 nM (ATP). A reduced effect was observed with other nucleotides in a rank order of agonist potency of ITP greater than CTP greater than ADP greater than GTP greater than TTP. No effect was seen with AMP, cAMP, or adenosine. This is consistent with P2 purinoceptors, as described in other tissues. ATP (100 microM) also dramatically increased IP accumulation. Inositol triphosphate, inositol bisphosphate, and inositol monophosphate were increased 7-, 9-, and 16-fold respectively. The agonist potency order of other nucleotides for IP accumulation was the same as that of [Ca2+]i. Pharmacological concentrations of ATP (1 mM) were required to increase PGE2 production. Many other nucleotides were equally effective at this concentration. ATP activates the phospholipase-C system in human amnion, as demonstrated by the increase in [Ca2+]i and inositol phosphates. The physiological significance of purinergic stimulation of this tissue remains unclear.     

Oxidized low density lipoprotein (LDL) and platelet intracellular calcium: interaction with nitric oxide

The present study tested the effects of ox-low density lipoprotein (LDL) on nitric oxide (NO)-dependent decrease in agonist-stimulated [Ca2+]i. The effects of ox-LDL on platelet aggregation were also evaluated. Platelets loaded with FURA 2 AM (2 micromol/litre) were incubated with NO-donors for 2-10 min to obtain a 40-50% reduction in \[Ca2+]i and with NO-donors plus ox-LDL (100 microg of protein/ml). Thrombin (0.03 U/ml) was used as an agonist. In some experiments 8-Br-cGMP (0.5-1 mmol/l) was used to investigate the NO-dependent intraplatelet signalling system. Slightly oxidized LDL was obtained by leaving native LDL in the light at room temperature for at least 7 days. Ox-LDL did not cause any increase in thrombin-induced [Ca2+] (control: 215.4 +/- 44.3 nmol/l, ox-LDL 223.4 +/- 35.3 nmol/l, M +/- SEM; n = 8) and platelet aggregation (control: 78.7 +/- 4.9% , ox-LDL: 78.9 +/- 4.2% , n = 12). Ox-LDL antagonized the effects of NO-donors on platelet [Ca2+]i (NO-donor: 137.4 +/- 22.1 nmol/l, NO + ox-LDL: 177.3 +/- 27.6 nmol/l, n = 11; P < 0.001) and platelet aggregation (NO-donor: 15.4 +/- 3.4% , NO + ox-LDL: 28.9 +/- 3.8%, n = 24; P < 0.001). Ox-LDL did not affect the inhibitory activities of 8-Br-cGMP on platelet aggregation (8-Br-cGMP: 22.0 +/- 8.5%, 8-Br-cGMP + ox-LDL: 19.3 +/- 7.8%, n = 5) and platelet [Ca2+]i . In conclusion, slightly oxidized LDL does not directly activate platelets and does not i affect the intracellular NO-dependent signalling system. The present results suggest that LDL reduces the antiplatelet activity of NO mainly by preventing its biological effects.