Stimulation of spreading and aggregation of platelets on immobilized type V collagen: mechanisms depending on Ca2+ and protein kinase C

The effects of phorbol ester (PMA) and stable prostaglandin endoperoxide analog (U46619) on platelet interaction with a surface coated with monomeric type V collagen (CV substrate) and free Ca2+ concentration in platelet cytoplasm ([Ca2+]in) have been studied. In the absence of PMA and U46619, the discoid and spherical platelets from suspension are attached to CV substrate but are incapable of spreading and aggregation on the substrate. An addition of PMA (0.15-1.5 nM) or U46619 (1.5 microM) to the reaction mixture stimulates platelet spreading and the formation of multilayer (thrombi-like) aggregates on CV substrate. Using the fluorescent probe Quin 2, it was found that U46619 (0.1 microM) increases [Ca2+]in from the basal level (100-120 mM) to 600 nM. PMA (0.75-15 nM) exerts only a slight effect increasing [Ca2+]in by 30-40 nM. The data obtained suggest that the PMA-induced spreading and aggregation of platelets on CV substrate can occur via activation of protein kinase C at relatively low [Ca2+]in values. These results also testify to the existence of a substrate-independent mechanism of spreading of platelets activated in suspensions by soluble inducers.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Characterization of the effect of the adenosine agonist cyclohexyladenosine on platelet activating factor-induced increases in [Ca]i in human platelets in vitro

Previous studies have shown that adenosine agonists acting at A-2 receptors inhibit platelet aggregation. Since an increase in cytosolic Ca2+ concentration (delta [Ca2+]i) is closely associated with the time frame of platelet aggregation, we have examined the effect of adenosine receptor function on induced increases of [Ca2+]i by a potent platelet activator, platelet activating factor (PAF). We loaded washed platelets with Fura-2, then induced increases in [Ca2+]i with various concentrations of PAF, and then determined EC50 values (PAF concentration at half-maximal response) and values for maximal response of delta[Ca2+]i (max-delta[Ca2+]i). The EC50 for PAF-delta[Ca2+]i was 112 +/- 37 (SD) pM and the max-delta[Ca2+]i was 284 +/- 138 (SD) nM. Our results show that PAF-delta[Ca2+]i was inhibited in a non-competitive manner by the adenosine receptor agonist cyclohexyladenosine (CHA) with an IC50 of 14.9 microM. This inhibition was partially reversed by theophylline, an adenosine receptor antagonist, with an IC50 of 19 microM. Based on the results of these studies together with evidence from other research groups that platelets do not possess A-1 receptors, our results suggest that CHA inhibited PAF-delta[Ca2+]i in platelets through an activation of A-2 receptors.     

Role of glycosaminoglycans in calcium metabolism of human platelets

Changes in intracellular Ca2+, [Ca2+]i, were measured in control and chondroitin ABC lyase-pretreated platelets. [Ca2+]i was measured with the fluorescent calcium probe Quin2. Chondroitin ABC lyase removed chondroitin 4-sulfate from the platelet surface without inducing shape change or release of serotonin. Compared to similarly prepared controls, enzyme treated platelets showed an increase of [Ca2+]i in response to stimulation by various agonists at high (1 mM) extracellular Ca2+ concentration. At low Ca2+ in the medium (1 mM EGTA), such platelets responded to agonists with a decreased rise in [Ca2+]i compared to the controls. These studies indicate that selective removal of glycosaminoglycans may sensitize platelets to the action of platelet aggregating agents. In addition, glycosaminoglycans may have a calcium storage function.     

Beta-adrenergic inhibition of AGEPC-stimulated Na+/Ca2+ exchange and AGEPC-induced platelet activation

Recently, AGEPC (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was found to initiate contraction of ileal smooth muscle strips and to enhance Na+/Ca2+ exchange in ileal plasmalemmal vesicles. In the present study, the effects of the smooth muscle relaxant, isoproterenol, on Na+/Ca2+ exchange in rat ileal plasmalemmal vesicles was examined. In this preparation, Na+/Ca2+ exchange was stimulated 131 +/- 8% and 264 +/- 19% by addition of 50 nM and 100 nM AGEPC, respectively. Isoproterenol, a beta-adrenergic agonist, inhibited AGEPC stimulation of Na+/Ca2+ exchange in a dose- and time-dependent manner but had no effect on basal rates of Na+/Ca2+ antiport. At 1 microM, isoproterenol inhibited 86% of the Na+/Ca2+ exchange stimulated by 50 nM AGEPC. Vesicular cAMP levels were increased over 100% following the addition of 1 microM isoproterenol for 30 s. Inhibition of AGEPC-stimulated vesicular Na+/Ca2+ exchange and elevation of vesicular cAMP levels by isoproterenol was prevented by the beta-receptor antagonist propranolol (5 microM), demonstrating that these effects of isoproterenol were mediated by interaction with vesicular beta-adrenergic receptors. Additional studies with washed rabbit platelets demonstrated that isoproterenol inhibited AGEPC-induced aggregation and serotonin release. These effects of isoproterenol were dose- and time-dependent and were antagonized by propranolol. Isoproterenol had no effect on thrombin-induced aggregation and did not change appreciably platelet cAMP levels. Moreover, dibutyryl cAMP could not mimic the effect of isoproterenol to inhibit an AGEPC-induced aggregation. On a molar basis, the inhibitory effects of isoproterenol toward AGEPC action were greater in the ileal preparation than in the platelets. It is suggested that beta-adrenergic agonists may modulate AGEPC-induced ileal Na+/Ca2+ exchange and AGEPC-induced platelet aggregation through cAMP-dependent and-independent mechanisms, respectively.     

Erythropoietin induced transmembrane calcium influx in essential hypertension.

The effects of erythropoietin (EPO) on cytosolic free calcium concentration ([Ca2+]i) in platelets of 20 essential hypertensive patients (HT) and of 25 normotensive subjects (NT) were investigated using the fura2 technique. In resting platelets [Ca2+]i were not significantly higher in HT compared to NT (74.3 +/- 7.8 nM vs 59.8 +/- 7.0 nM, mean +/- SEM). Addition of EPO significantly increased [Ca2+]i in HT compared to NT (13.8 +/- 5.3 nM vs 0.9 +/- 1.9 nM, p less than 0.01). EPO increased the amount of calcium in intracellular stores. This was confirmed independently using thrombin-induced changes of [Ca2+]i in a calcium-free medium and using chlorotetracycline as a marker of stored calcium. After preincubation with EPO thrombin-induced changes of [Ca2+]i were significantly lower in HT compared to NT (306.1 +/- 30.0 nM vs 407.7 +/- 35.7 nM, p less than 0.05). In a calcium-free medium after preincubation with EPO thrombin-induced changes of [Ca2+]i were significantly lower in HT compared to NT (54.7 +/- 11.8 nM vs 100.9 +/- 10.5 nM, p less than 0.05) indicating lower storage capacity in HT. It is concluded that elevated response to EPO may provide a powerful tool to evaluate diagnosis and underlying pathophysiological mechanisms in essential hypertension.     

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. I. Human platelets.

The effect of halothane on the regulation of blood platelet free cytosolic calcium was investigated in Quin-2-loaded cells from patients susceptible to Malignant Hyperthermia (MH) and healthy controls, respectively. The resting level of free cytosolic calcium was slightly, but statistically significantly, enhanced in platelets from patients (90 +/- 10 nM vs 110 +/- 35 nM). Halothane induced a dose-dependent, rapid Ca2+ release from intracellular stores both in normal and in MH derived cells, but the resulting increase in cytosolic calcium was significantly higher in the latter (2 mM halothane: [Ca2+]i = 117 +/- 12 nM vs 218 +/- 117 nM; 4 mM halothane: 225 +/- 35 nM vs. 417 +/- 201 nM). Whereas in platelets from healthy donors a complete reversibility of the halothane effect could be observed within 30-45 min, the cytosolic Ca2+ transients in platelets from patients were different from those in normals either in a higher initial peak or in a diminished decline velocity or in both. The basal Ca2+ permeability of the platelet plasma membrane was very low. Generally, halothane caused a dose-dependent increase in Ca2+ permeability. However, the influx of external calcium was significantly higher in platelets from patients than in controls (2 mM halothane: delta [Ca2+]i = 69 +/- 12 nM vs 135 +/- 63 nM; 4 mM halothane: 127 +/- 33 nM vs. 258 +/- 111 nM). Combining the results, the suggestion can be made that susceptibility to MH is characterized by a generalized membrane defect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of phospholipid platelet-activating factor by human platelets

The binding of the phospholipid platelet-activating factor 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) to washed human platelets was more than 80% complete within 2 min, which coincided with the time of initiation of platelet aggregation by AGEPC. Scatchard plot analysis of the binding of [3H]AGEPC to platelets without and with an excess of unlabeled AGEPC revealed two distinct types of binding sites. One platelet site for AGEPC exhibited a high affinity (KD = 37 +/- 13 nM, mean +/- SD), was saturable, and had a low maximal capacity of 1399 +/- 498 (mean +/- SD) molecules of AGEPC/platelet. The other platelet site demonstrated a nearly infinite binding capacity, consistent with nonreceptor uptake of AGEPC into cellular structures. The specificity of the high-affinity binding site for AGEPC was assessed by comparing the capacity of several analogues of AGEPC to inhibit the binding of [3H]AGEPC to platelets and to induce platelet aggregation. An ether linkage in position 1, a short-chain fatty acid in position 2, and a choline moiety in the polar head group proved to be critical both for the binding of [3H]AGEPC to platelets and for the initiation of platelet aggregation. Exposure of platelets to AGEPC for 5 min at 37 degrees C functionally deactivated the exposed platelets to subsequent stimulation by AGEPC, as assessed by diminished aggregation, and concomitantly reduced the specific binding of [3H]AGEPC. Evaluation of the time course of the events of deactivation revealed the loss of an aggregation response to AGEPC after 90 sec at 37 degrees C, despite the retention of up to 50% of the specific binding sites for AGEPC.     

Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

[Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.     

Increased internal calcium mobilization in platelets of patients with chronic renal failure.

Platelet free calcium concentrations ([Ca2+]i) were measured with Fura-2 to elucidate the intracellular calcium kinetics in patients with renal disease. There were no significant differences of the resting [Ca2+]i among the control subjects (C) (n = 12), patients with chronic glomerulonephritis (CGN) (n = 8), and patients with chronic renal failure (CRF) (n = 12). In all groups, platelets [Ca2+]i were significantly increased by agonists (thrombin, adenosine diphosphate) compared with their respective basal level. Thrombin-induced [Ca2+]i rise was significantly higher in CRF (840 +/- 265 nM) than in C (600 +/- 163) and CGN (562 +/- 137). Also adenosine diphosphate elicited similar responses. In the presence of calcium chelator in the incubation buffer, the elevation of [Ca2+]i after thrombin stimulation was statistically higher in CRF (469 +/- 85 nM) than in C (275 +/- 60) and CGN (301 +/- 41). These findings suggest that platelets of CRF were capable of increasing [Ca2+]i in response to agonists, through further mobilization of calcium from the intracellular pool rather than the elevation of transmembrane calcium influx.     

Secondary signals mediated by GPIIb/IIIa in thrombin-activated platelets

We have previously found that stimulation of aequorin-loaded platelets by thrombin produced a two-peaked increase in intracellular free calcium concentration ([Ca2+]i), and the development of the second peak of [Ca2+]i was closely related with the aggregation. In this report, we studied the interrelationship between the GPIIb/IIIa complex, aggregation, cytoskeletons and [Ca2+]i of platelets. The pretreatment of the platelets with dihydrocytochalasin B (4 microM), an actin polymerization inhibitor, did not inhibit aggregation and TXB2 production, but did inhibit both actin polymerization and the second peak of [Ca2+]i increase induced by thrombin, suggesting that actin polymerization and the second peak of [Ca2+]i are interrelated. GRGDSP (100 microM), a synthetic anti-adhesive peptide, has already been reported to inhibit platelet aggregation and the second peak of [Ca2+]i induced by thrombin. It also inhibited actin polymerization and TXB2 production, suggesting that aggregation was important for not only the generation of the second peak of [Ca2+]i but also for actin polymerization and TXB2 production. PGI2 (5 nM) did not abolish but only delayed aggregation, TXB2 production, actin polymerization and the second peak of [Ca2+]i increase. These findings suggest that the secondary signals are caused by aggregation (fibrinogen-binding to the GPIIb/IIIa) in thrombin-aggregated platelets, which results in the TXA2 production and the secondary peak of [Ca2+]i increase, and the latter was dependent on actin polymerization.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Inhibition of macrophage secretion by tetanus toxin is not directly linked to cytosolic calcium homeostasis

Tetanus toxin (TT) inhibits secretion of neurotransmitters from neurons and lysozyme from human macrophages (Mphi). Because these secretory events are associated with changes in cytosolic free calcium [Ca2+]i, we examined the effect of TT on Mphi calcium homeostasis and secretion in response to ionomycin and phorbol myristate acetate (PMA). Using Quin 2 to report [Ca2+]i, basal [Ca2]i was similar for control cells (133 nM) and Mphi treated with TT (127 nM). In response to ionomycin (50 nM) [Ca2+]i increased to 548 +/- 74 nM in control cells and to 357 +/- 36 nM in TT-treated Mphi (p less than 0.02, N = 12). Despite this rise in [Ca2+]i, neither control Mphi nor TT-treated Mphis secreted the lysosomal enzyme lysozyme in response to this concentration of ionomycin (50 nM). In both control and TT-treated Mphi, stimulation with a higher concentration of ionomycin (1000 nM) caused saturation of the quin 2 fluorescence signal. However, lysozyme secretion from TT-Mphi was inhibited. In response to the phorbol ester, PMA (3 uM), [Ca2+]i did not increase in either control Mphi or TT-treated Mphi. However, secretion of lysozyme from TT-treated Mphi was also inhibited in response to this stimulus (70.8% of control, p less than 0.02, N = 3). These data indicate that the ability of TT to inhibit secretion from Mphi is not directly linked to alterations of cytosolic calcium homeostasis.     

Phosphoinositide signalling system in platelets of schizophrenic patients and the effect of neuroleptic therapy.

Alterations in the phosphoinositide signalling system have been proposed as a possible biological marker of schizophrenia. We studied the levels of inositol 1,4,5-trisphosphate (IP3), cytosolic Ca2+ concentrations ([Ca2+]i), and the incorporation of [32P]-orthophosphate into inositol phospholipids and phosphatidic acid (PA) in blood platelets of neuroleptic-treated schizophrenics in comparison with controls. The [Ca2+]i was significantly higher in platelets of one month neuroleptic-treated patients (155+/-5.8 nM) in comparison with controls (95+/-5.4 nM). Neuroleptic therapy decreased the [Ca2+]i, but even after long-term therapy it remained significantly higher (114+/-5.7 nM) than in controls. Differences were also found in the level of IP3 between controls (30+/-4.0 pmol/10(9) platelets), drug-free schizophrenics (52+/-9.0 pmol/10(9) platelets) and treated patients (50+/-6.0 pmol/10(9) platelets). The increased turnover of PA was observed in platelets of neuroleptic-treated schizophrenic patients. The study suggests that the regulation of calcium homeostasis and pathways involved in the phosphoinositide signalling system are altered in the platelets of schizophrenics. Neuroleptic therapy did not remove the observed changes in [Ca2+]i and IP3 levels.     

High concentrations of exogenous arachidonate inhibit calcium mobilization in platelets by stimulation of adenylate cyclase.

1. Exposure of platelets to exogenous arachidonic acid results in aggregation and secretion, which are inhibited at high arachidonate concentrations. The mechanisms for this have not been elucidated fully. In our studies in platelet suspensions, peak aggregation and secretion occurred at 2-5 microM-sodium arachidonate, with complete inhibition around 25 microM. 2. In platelets loaded with quin2 or fura-2, the cytoplasmic Ca2+ concentration, [Ca2+]i, rose in the presence of 1 mM-CaCl2 from 60-80 nM to 300-500 nM at 2-5 microM-arachidonate, followed by inhibition to basal values at 25-50 microM. Thromboxane production was not inhibited at 25 microM-arachidonate. Cyclic AMP increased in the presence of theophylline, from 3.5 pmol/10(8) platelets in unexposed platelets to 8 pmol/10(8) platelets at 50 microM-arachidonate; all platelet responses were inhibited with doubling of cyclic AMP contents. 3. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine attenuated the inhibitory effect of arachidonate, suggesting that it is mediated by increased platelet cyclic AMP and that it is unlikely to be due to irreversible damage to platelets. 4. Aspirin or the combined lipoxygenase/cyclo-oxygenase inhibitor BW 755C did not prevent the inhibition by arachidonate of either [Ca2+]i signals or aggregation induced by U46619. 5. Thus high arachidonate concentrations inhibit Ca2+ mobilization in platelets, and this is mediated by stimulation of adenylate cyclase. High arachidonate concentrations influence platelet responses by modulating intracellular concentrations of two key messenger molecules, cyclic AMP and Ca2+.     

LY294002, but not wortmannin,increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.     

A 14 amino acid peptide derived from the amino terminus of the cleaved thrombin receptor elevates intracellular calcium and stimulates prostacyclin production in human endothelial cells.

Cleavage by thrombin of the platelet thrombin receptor exposes a new N-terminal segment SFLLRNPNDKYEPF (SFLL) which acts as a tethered ligand. The free peptide activates platelets and induces platelet aggregation. We now show that SFLL can also activate human umbilical vein endothelial cells (HUVEC) and induce rises in both cytosolic free calcium ([Ca2+]i) and prostacyclin (PGI2) production. These responses were time- and concentration-dependent and were similar to those for native thrombin except that they were not blocked by hirudin. Initial activation of HUVEC with thrombin desensitized the subsequent response to SFLL for both rises in [Ca2+]i and PGI2 production. Thus, SFLL alone can activate HUVEC and elevate [Ca2+]i and induce PGI2 production suggesting that the thrombin receptors on platelet and endothelial cells are functionally and structurally similar.     

Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets

The kinetics of ATP-driven Ca2+ uptake by the dense tubules were studied in digitonin-permeabilized human blood platelets. Digitonin at 3 micrograms/ml was shown capable of permeabilizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca2+. Experimentation was carried out with platelets treated with 3 micrograms/ml digitonin reisolated and resuspended in detergent-free medium ('digitonin-permeabilized' platelets). Active Ca2+ accumulation, which occurs over a period of minutes, was monitored by the increase in the fluorescence of chlorotetracycline after the addition of Mg-ATP (37 degrees C). The active uptake is inhibited by 15 microM trifluoperazine. The process is saturable with respect to external [Ca2+], with a Km of 180 +/- 5 nM and a Hill coefficient (n) of 1.40 +/- 0.05. Analysis of the maximal uptake in steady state gave similar results (Km = 160 +/- 5 nM, n = 1.50 +/- 0.05). The rate of uptake at [Ca2+] approximately Km is increased when the digitonin-permeabilized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca2+ is rapidly released (less than 1 min) by addition of D-myo-inositol trisphosphate (IP3). The maximal extent of release is 50%; the EC50 for IP3 is approx. 12 microM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet.     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Na+/Ca2+ exchange in cardiac myocytes. Effect of ouabain on voltage dependence.

Sarcolemmal sodium/calcium exchange activity was examined in individual chick embryonic myocardial cell aggregates that were loaded with quin 2. The baseline [Ca2+]i was 68 +/- 4 nM (n = 29). Abrupt superfusion with sodium-free lithium solution produced a fourfold increase in steady-state [Ca2+]i to 290 +/- 19 nM, which was reversible upon sodium restitution. Other methods of increasing [Ca2+]i such as KCl-depolarization or caffeine produced a dose-dependent increase in quin 2 fluorescence, accompanied by sustained contracture. The [Ca2+]i increase in zero sodium was linear, and its half-time (t1/2) of 15.1 +/- 0.1 s was similar to that of the sodium-free contracture (t1/2 = 14.4 +/- 0.5 s) under the same conditions. The sodium-dependent [Ca2+]i increase was not significantly greater when potassium served as the sodium substitute instead of lithium. This suggests that sodium/calcium exchange has little voltage dependence in this situation. However, in aggregates pretreated with ouabain (2.5 microM), the [Ca2+]i increase was almost threefold greater with potassium than with lithium (P less than 0.007). Ouabain therefore potentiated the effect of membrane potential on calcium influx. We propose that elevation of [Na2+]i is a prerequisite for voltage dependence of the sodium/calcium exchange under the conditions studied. Sodium loading will then drastically increase calcium influx during the action potential while inducing an outward membrane current that could accelerate repolarization.