Concentration Of Human Platelets Research Articles

Thrombin regulates intracellular cyclic AMP concentration in human platelets through phosphorylation/activation of phosphodiesterase 3A

AbstractThrombin-induced cyclic AMP (cAMP) reduction potentates several steps in platelet activation, including Ca++ mobilization, cytoskeletal reorganization, and fibrinogen receptor conformation. We now reinvestigate the signaling pathways by which intracellular cAMP content is controlled after platelet activation by thrombin. When washed human platelets were stimulated with thrombin, cAMP-dependent phosphodiesterase (PDE3A) activity was significantly increased. A nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the PDE3 selective inhibitors milrinone and cilostazol each suppressed thrombin-induced cAMP-dependent PDE responses, but not 2 different PDE2 inhibitors. Selective inhibition of PDE3A resulted in reversal of thrombin-induced cAMP reduction, indicating that thrombin activated PDE3A. In synergy with inhibition of adenylate cyclase by thrombin, activated PDE3A accelerates cAMP hydrolysis and maximally reduces the cAMP content. Thrombin-induced PDE3A activation was diminished concomitantly with dephosphorylation of PDE3A by protein phosphatase 1 (PP1). An Akt inhibitor blocked PDE3A activation and constrained thrombin-induced cAMP reduction. A P2Y12 inhibitor also reduced thrombin-induced cAMP reduction. The combination of both reversed cAMP decrease by thrombin. Thrombin-mediated phosphorylated PDE3A was isolated by liquid chromatography, detected by a monoclonal antibody against Akt-phosphorylated substrate, and verified by immunoprecipitation study. The predominant isoform phosphorylated by Akt was the 136-kDa species. We suggest that activation/phosphorylation of PDE3A via Akt signaling pathway participates in regulating cAMP during thrombin activation of platelets.

Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac K(ATP) channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the K(ATP) channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2delta C26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac K(ATP) channel, 5 microM 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2delta C26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.

Carbon monoxide inhibits capacitative calcium entry in human platelets

The cytosolic calcium concentration in human platelets is elevated by several agonists via receptor-operated mechanisms involving both Ca 2+ release from intracellular stores and Ca 2+ entry. In order to get a mechanistic insight in the effect of carbon monoxide (CO)-containing solutions, this work examines the changes in [Ca 2+] i induced by 100 μM adenosine 5′diphosphate (ADP), 0.1 IU/ ml thrombin, 0.5 μM thapsigargin or 0.5 μM ionomycin in human platelets. In a saline solution bubbled with CO, the increase of [Ca 2+] i produced by thrombin was 72±4% of the response evoked in the control solution (CO-free) and the response elicited by ADP was 64±8% of the control. When a mixture of 5% CO/95% N 2 was used, the responses were 70±7% of control for thrombin and 79±6% of control for ADP. The mobilization of stored calcium produced by thrombin in a calcium-free solution and the increase of [Ca 2+] i produced by subsequent introduction of 1 mM extracellular calcium were both reduced in the presence of CO (82±6% and 78±5% of control, respectively). Similar reductions in the presence of CO were found when platelets were stimulated by ADP (62±8% and 60±8% for mobilization in calcium-free media and calcium entry, respectively). Although the change in [Ca 2+] i induced by ionomycin in the presence of extracellular calcium was almost the same in the absence or presence of CO (97±5% of control), the entry induced by depletion of reservoirs with the ionophore undergoes a significant reduction in a solution bubbled with CO (84±5% of control). In agreement with the concept that CO has a direct inhibitory effect on capacitative calcium entry, a reduction to 47±6% of control was obtained when sarco/endoplasmic reticulum ATPase was blocked by thapsigargin. Diverse mechanisms could be responsible for the effect of CO on calcium entry. On the one hand, a decrease in the calcium release from intracellular stores or an increase in the rate of its back-sequestration could occur, being the reduction of capacitative calcium entry an indirect consequence of a diminished emptying of reservoirs. On the other hand, CO could have a direct inhibitory effect on the pathway that produces the calcium entry. The decrease in the Ca 2+ signal in the presence of CO evoked by receptor-independent emptying of reservoirs indicates that a direct effect of CO on capacitative calcium entry participates in the antiaggregatory properties of CO. The proposal that CO inhibits directly store-operated calcium influx widens the potential mechanisms by which heme oxygenase regulates cell functions.

Cyclic Nucleotides Modulate Store-mediated Calcium Entry through the Activation of Protein-tyrosine Phosphatases and Altered Actin Polymerization in Human Platelets

Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.

Effect of non-saponin fraction from Panax ginseng on cGMP and thromboxane A 2 in human platelet aggregation

The non-saponin fraction (NSF; lipophilic fraction) from the roots of Panax ginseng inhibited the aggregation of human platelets induced by thrombin (0.1 units/ml) in a dose-dependent manner. NSF induced the elevation of cGMP concentration in human platelets in a similar manner to molsidomine, a known vasodilator. NSF also inhibited Ca 2+-influx into platelets. While verapamil, a Ca 2+-antagonist, increased the cAMP level in platelets stimulated by thrombin, NSF had little effect on cAMP formation. Instead, NSF potently inhibited the thromboxane A 2 (TXA 2) production. The results suggest that NSF may regulate the levels of cGMP and TXA 2 to inhibit platelet aggregation induced by thrombin.

Effect of the transmembrane gradient of magnesium and sodium on the regulation of cytosolic free magnesium concentration in human platelets.

1. To clarify the mechanism by which cytosolic free Mg2+ concentrations ([Mg2+]i) are regulated in human platelets, we investigated platelet membrane permeability to Mg2+ by altering extracellular Mg2+ concentrations, and tested the existence of a Na(+)-Mg2+ exchanger by manipulating the transmembrane Na+ gradient. 2. Platelet [Mg2+]i was 421 +/- 52 mumol/l in healthy men. [Mg2+]i remained constant during a 120 min exposure to nominally zero (low) or 5 mmol/l (high) external Mg2+ concentrations. 3. Preincubation of platelets with 10(-4) mol/l ouabain effectively decreased the transmembrane Na+ gradient. The ouabain-induced increase in [Mg2+]i was statistically significant after 30 min of exposure (14.6 +/- 2.0%) and reached 24.6 +/- 4.5% after 60 min. Similarly, the replacement of extracellular Na+ with N-methyl-D-glucamine significantly increased [Mg2+]i by 48.5 +/- 3.9% and 78.8 +/- 12.5%, respectively. 4. These results suggest that [Mg2+]i is well controlled in the presence of large transmembrane Mg2+ concentration gradients, and a Na(+)-Mg2+ exchanger may be involved in the regulation of [Mg2+]i in human platelets.

Role of Protein Kinase C in the Anti-Aggregatory Effects of Endothelin-1 on Human Platelets

1. Endothelin-1 has anti-aggregatory properties, but the mechanism underlying this inhibitory action is unknown. This in vitro study investigates effects of endothelin-1 on thrombin-stimulated aggregation and intracellular free calcium concentration in human platelets and assesses the role of protein kinase C in the interactions between endothelin-1 and thrombin. Aggregation was measured turbidometrically and the intracellular free calcium concentration was determined with the fluorescent indicator fura 2-acetoxymethyl ester. 2. Endothelin-1 at concentrations from 10(-11) to 10(-6) mol/l had no effect on platelet aggregation or intracellular free calcium concentration but inhibited in a dose-dependent manner aggregation induced by 0.05 unit/ml thrombin (pD2 for inhibition by endothelin = 8.1 +/- 0.12). 3. Endothelin-1 at 10(-9) mol/l significantly decreased (P < 0.01) thrombin-stimulated aggregation from 81.4 +/- 1.5% (in the absence of endothelin-1) to 53.5 +/- 1.1% (in the presence of endothelin) and thrombin-stimulated intracellular free calcium concentration from 179 +/- 1.7 nmol/l to 140 +/- 1.8 nmol/l. 4. Preincubation of platelets with 10(-7) mol/l staurosporine (protein kinase C inhibitor), calphostin C (highly selective protein kinase C inhibitor) or 5-(N,N-hexamethylene) amiloride (highly selective Na(+)-H+ exchange blocker) significantly inhibited (P < 0.01) thrombin-stimulated platelet responses and suppressed the inhibitory effect of endothelin-1 on thrombin-induced aggregation and intracellular free calcium concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of α- and β-Adrenergic Antagonist on Epinephrine-Induced Aggregation and Intracellular Free Calcium Concentration in Human Platelets

Recently, it has been reported that an α-adrenergic antagonist phentolamine inhibits epinephrine-induced platelet aggregation but a β-adrenergic antagonist propranolol did not. In this study, we examined the effects of α- and β- adrenergic antagonist on cytosolic free calcium movements induced by epinephrine. An α-adrenergic antagonist phentolamine was found to inhibit the elevation of cytosolic free calcium concentration induced by epinephrine, while a β-adrenergic antagonist propranolol had no effect on cytosolic free calcium concentration induced by epinephrine.

Na(+)-Ca2+ exchange modulates Ca2+ handling of human platelets by altering intracellular Ca2+ store size.

In order to elucidate the role of Na(+)-Ca2+ exchange in regulating cytosolic free Ca2+ concentration in human platelets, we investigated the relationship between cytosolic free Na+ and Ca2+ concentrations in human platelets. Sodium-binding benzofuran isophthalate and fura-2 were used to monitor cytosolic free Na+ and Ca2+ concentrations, respectively. Ouabain at a concentration of 100 mumol/l induced an increase in cytosolic free Na+ concentration within 5 min, followed by increases in resting cytosolic free Ca2+ concentration and intracellular Ca2+ store. These three parameters were increased in a time-dependent manner significantly above the timed control over a period of 60 min. Pre-incubation of platelets with 100 mumol/l ouabain for 30 min significantly enhanced the cytosolic free Ca2+ response to thrombin and arginine vasopressin in the absence of extracellular Ca2+. The decrease from peak cytosolic free Ca2+ concentration in response to ionomycin in the absence of extracellular Ca2+ was significantly slower in low-Na+ buffer than in standard buffer. In addition, 5 mumol/ionomycin increased the cytosolic free Na+ concentration markedly in the presence of 0.1 mmol/l extracellular Ca2+, but the rise in cytosolic free Na+ concentration was suppressed by 2 mmol/l Ni2+ (NiCl2) or by removal of extracellular Ca2+. These results suggest that Na(+)-Ca2+ exchange is important in extruding Ca2+ from the cytosol in human platelets, and inhibition of this exchange leads to the accumulation of intracellular Ca2+ store, which may be responsible for the enhanced responsiveness of cytosolic free Ca2+ to agonists.

Wheat Germ Agglutinin-Induced Intracellular Calcium Mobilization in Human Platelets: Suppression by Staurosporine and Resistance to Cyclic AMP Inhibition

The lectin wheat germ agglutinin (WGA) elicited a prompt and sharp increase in intracellular Ca 2+ concentration in human platelets. The WGA- induced Ca 2+ mobilization was markedly inhibited by a protein kinase inhibitor staurosporine, whereas Ca 2+ mobilization by receptor-mediated agonists, including thrombin, platelet-activating factor, and arginine-vasopressin, was not. In contrast, the lectin-induced Ca 2+ mobilization was resistant to cyclic AMP inhibition, compared with that induced by receptor-mediated agonists. These findings indicate that the mechanism of intracellular Ca 2+ mobilization, or possibly phospholipase C activation, induced by WGA is different from that induced by receptor-mediated agonists in human platelets.

Cytosolic free magnesium concentration in human platelets

Although the magnesium ion (Mg 2+) is considered to play an important role in cell activation, information is limited by the lack of suitable methods for measuring cytosolic free Mg 2+ concentration ([Mg 2+]i). We measured [Mg 2+]i in resting and activated human platelets using a new fluorescent Mg 2+-indicator, mag-fura-2. [Mg 2+]i was 0.54±0.14 mM in resting platelets from 15 healthy volunteers. [Mg 2+]i was elevated to 1.33±0.44 mM and 0.92±0.37 mM in platelets stimulated with thrombin and collagen, respectively. Increased Mg 2+ was considered to be derived chiefly from intracellular Mg 2+ mobilization. These results suggest that platelet activation is associated with the increase in [Mg 2+]i. The estimation of [Mg 2+]i using this method is usefull for the investigation of mechanism for various cell activation.

Toxicodynamics of tumour promoters of mouse skin. III. Specific binding of the tumour promoter thapsigargin as measured by the cold-acetone filter assay.

A method is described for measuring rapid, specific, and saturable binding of the skin irritant and tumour-promoting secretagogue thapsigargin (sesquiterpene lactone) to the microsomal fraction from mouse brain. Employing the tritium-labelled compound its apparent dissociation constant, Kd, and the maximal amount of binding Bmax are shown to be 9.8 nM and 1.9 pmol/mg protein respectively. Such a Kd for thapsigargin is similar to (a) its IC50 value for inhibiting Ca2+ uptake in the microsomal fraction from rat brain and (b) its EC50 values for inducing a rise in the cytoplasmic Ca2+ concentration of human platelets and histamine release from rat peritoneal mast cells. A positive correlation is found between the binding affinities of thapsigargin, thapsitranstagin, and trilobolide, their potencies as secretagogues and their lipophilicities. This correlation does not extend to the skin-irritant activities of the compounds thus emphasizing that their mechanism of action is unlike that of 12-O-tetradecanoylphorbol 13-acetate.

Phosphorylation of the inhibitory guanine-nucleotide-binding protein as a possible mechanism of inhibition by protein kinase C of agonist-induced Ca2+ mobilization in human platelet.

Increases in the intracellular Ca2+ concentration of human platelets caused by receptor agonists, such as thrombin, 9,11-epithio-11,12-methanothromboxane A2 (STA2), platelet-activating factor (PAF) and arginine-vasopressin, were inhibited by prior addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) in time-dependent and concentration-dependent manners. The inhibitions were mostly reversed by staurosporine, and inhibitor of protein kinase C, added 1 min before TPA. Prior treatment of platelets with thrombin or STA2, the efficacious Ca2+ mobilizer, suppressed the increase in the intracellular Ca2+ concentration of the cells to other agonists, but treatment with less efficacious PAF or vasopressin did not. The heterologous receptor desensitizations were also reversed by staurosporine. The antibody, directed against the carboxy-terminal region of the alpha subunits 1 and 2 of the inhibitory guanine-nucleotide-binding proteins (Gi1 alpha and Gi2 alpha), was raised in rabbit and was used to immunoprecipitate Gi alpha in 32P-labeled platelets. The radioactivity was detected in Gi alpha after incubation of 32P-labeled platelets with TPA, thrombin or STA2, but not in the cells incubated with PAF or vasopressin. The time-dependency or concentration-dependency of TPA-induced phosphorylation of Gi alpha was similar to the dependency of its inhibitory action on agonist-induced Ca2+ mobilization. Thus, strong activation of Ca2+/phospholipid-dependent protein kinase C by phorbol ester or agonists of certain Ca(2+)-mobilizing receptors leads to phosphorylation of the alpha subunit of guanine-nucleotide-binding protein, thereby impairing the coupling of the G protein to receptors as a feedback regulatory component of the receptor-triggered intracellular Ca(2+)-mobilizing system.

Effect of nitroglycerin on intracellular calcium concentration in human platelets

Effect of nitroglycerin on intracellular calcium concentration in human platelets

Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets.

Most platelet agonists activate and elevate the cytosolic free calcium concentration in human platelets through receptor-dependent mechanisms that are antagonized by cAMP- and cGMP-elevating agents. Nitrovasodilators such as nitroprusside and endothelium-derived relaxing factor are potent cGMP-elevating platelet inhibitors. In the present study, the role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of ADP- and thrombin-evoked calcium elevation and activation of human platelets was investigated. Preincubation of platelets with 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP; a membrane-permeant selective activator of the cGMP-dependent protein kinase that does not significantly affect cGMP-regulated phosphodiesterases) inhibited the thrombin-induced phosphorylation mediated by myosin light chain kinase and protein kinase C. Nitrovasodilator-induced protein phosphorylation in human platelets was distinct from that induced by cAMP-elevating prostaglandins and could be mimicked by 8-pCPT-cGMP. Preincubation of human platelets with nitrovasodilators or 8-pCPT-cGMP inhibited the ADP- and thrombin-evoked calcium elevation in the presence and absence of external calcium. Nitrovasodilators and 8-pCPT-cGMP also inhibited the agonist-induced Mn2+ influx, but stopped-flow experiments indicated that the ADP receptor-operated cation channel was not significantly inhibited. These results suggest that in human platelets nitrovasodilators inhibit the agonist-induced calcium mobilization from intracellular stores and the secondary store-related calcium influx but not the ADP receptor-operated cation channel. The results also suggest that these nitrovasodilator effects are mediated by cGMP and the cGMP-dependent protein kinase.

Inhibition of Human Platelet Aggregation by Ibudilast (3-isobutyryl-2-isopropylpyrazolo [1, 5-a] pyridine, KC-404)

The effect of a novel compound, 3-isobutyryl-2-isopropylpyrazolo [1, 5-a] pyridine (ibudilast, KC-404), on human platelet aggregation and its mechanism of action were investigated.In vitro, KC-404 inhibited human platelet aggregation induced by ADP, collagen, adrenalin, platelet activating factor and arachidonic acid but not by ristocetin. Together, KC-404 and agents which increased cAMP (prostaglandin I2, prostaglandin E1 (PGE1), forskolin) or cGMP (3-morpholinosydnonimine (SIN-1)) produced synergistic inhibitory effects on platelet aggregation.KC-404 inhibited human platelet cAMP phosphodiesterase (PDE) (IC50: 50μM) and cGMP-PDE (IC50: 5.2μM) activities. cAMP and cGMP concentration of human platelets were not increased by KC-404 itself. PGE1, an adenylate cyclase stimulator, increased cAMP content; KC-404 enhanced the effect of PGE1 on cAMP accumulation. SIN-1, which stimulates guanylate cyclase, increased cGMP content; KC-404 enhanced the effect of SIN-1 on cGMP accumulation.These results suggest that effects of KC-404 on accumulation of cyclic nucleotides and inhibition of platelet aggregation are mediated via inhibition of platelet cyclic nucleotide phosphodiesterase activities.

Resting and ADP‐evoked changes in cytosolic free sodium concentration in human platelets loaded with the indicator SBFI.

1. Cytosolic free Na+ concentration, [Na+]i, was investigated in human platelets loaded with the fluorescent indicator SBFI (sodium-binding benzofuran isophthalate). 2. SBFI fluorescence from platelet suspensions was measured at excitation wavelengths of 340 and 385 nm and the 340/385 nm fluorescence ratio was calibrated in terms of [Na+]i in situ. [Na+]i was set to known values by resuspending cells in media with various [Na+], in the presence of the Na(+)-K+ ionophore, gramicidin. 3. Basal free [Na+]i was 5.5 +/- 0.3 mM (n = 50). This is considerably lower than estimates of total platelet Na+, suggesting that much intracellular Na+ is sequestered or bound. 4. ADP (40 microM) evoked a rise in [Na+]i from 6.4 +/- 0.7 to 18.3 +/- 1.1 mM (n = 8). The ADP-evoked rise in [Na+]i was abolished when external Na+ was replaced with N-methyl-D-glucamine. This indicates that the rise in [Na+]i was due to Na+ entry. 5. In platelets loaded with the fluorescent pH indicator, BCECF, 40 microM-ADP was shown to evoke a fall in cytosolic pH (pHi) from 7.21 +/- 0.03 to 7.12 +/- 0.03 (n = 10). Three minutes after ADP addition pHi had only recovered to 7.15 +/- 0.03. The recovery was dependent on external Na+, suggesting it was mediated by Na(+)-H+ exchange. However, this would only account for an increase in [Na+]i of approximately 0.5 mM, indicating most of the ADP-evoked Na+ entry occurred by other mechanisms. 6. Stopped-flow fluorimetry showed that the ADP-evoked rise in [Na+]i commenced without measurable delay and peaked within 1 s. The initial kinetics were thus similar to those reported for ADP-evoked rises in [Ca2+]i. 7. Cell-attached patch-clamp recordings showed that ADP evoked single-channel inward currents when included in the pipette-filling solution. The currents were similar whether Ca2+ was present or absent from the pipette. The slope conductance was 11 pS in the presence of external Ca2+ and 10 pS in its absence. Current-voltage relationships were similar and the reversal potentials were close to 0 mV under both conditions. 8. SK & F 96,365 (20 microM), a blocker of receptor-mediated Ca2+ entry in several non-excitable cells, blocked the ADP-evoked rise in [Na+]i. This compound has been shown to only partly block the biphasic ADP-evoked rise in [Ca2+]i, being selective for the fast, receptor-operated phase of entry. 9. These data suggest that ADP rapidly activates a channel in that platelet plasma membrane which is permeable to Na+ and divalent cations.

Stimulation by thrombin increases the cytosolic free Na+ concentration in human platelets. Studies with the novel fluorescent cytosolic Na+ indicator sodium-binding benzofuran isophthalate.

The new fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) was used for determination of the cytosolic free Na+ concentration, [Na+]i, in human platelets. The dye could be loaded into platelets in the form of its acetoxymethyl ester (SBFI-AM). Calibration of the fluorescence in terms of [Na+]i was done by measuring the 345/385 nm excitation ratio (emission 490 nm) at various extracellular Na+ concentrations, [Na+]o, in the presence of gramicidin D. The 345/385 intensity ratio increased almost linearly when [Na+]i was stepwise raised from 20 to 60 mM. The basal value for [Na+]i was found to be 26.0 +/- 4.5 mM (n = 15). Incubation of platelets in Na(+)-free buffer decreased [Na+]i, whereas inhibition of the (Na+ + K+)-ATPase by 0.5 mM ouabain increased [Na+]i to 56 +/- 4 mM (n = 4) within 60 min. Activation of Na+/H+ exchange by exposing platelets to propionic acid also raised [Na+]i, and a comparable effect was produced by the Na+/H+ ionophore monensin. Activation of platelets with thrombin (0.1-0.5 unit/ml) also increased the 345/385 nm intensity ratio, an effect that was not seen in Na(+)-free buffer or after raising intracellular cAMP by treatment of platelets with prostaglandin E1. On the average, [Na+]i was raised to 59.5 +/- 5.3 mM (n = 15) at 10 min after addition of thrombin without a significant decrease for further 10 min. An increase in [Na+]i was also seen when platelets were challenged with the Ca2+ ionophore ionomycin, an effect that did not occur in the absence of Na+o. Our findings confirm earlier reports which demonstrated a rise in [Na+]i in stimulated platelets and show that SBFI is a useful tool for determination of [Na+]i in resting and stimulated platelets.

Relationship between the inhibition of receptor-induced increase in cytosolic free calcium concentration and the vasodilator effects of nitrates in patients with congestive heart failure

Nitroglycerin, isosorbide dinitrate and sodium nitroprusside, like nifedipine, were found to inhibit the receptor-provoked increase of cytosolic free calcium concentration in human platelets loaded with 2-[(2-amino-5-methylphenoxy)methyl]-6-methoxy-8-aminoquinoline- N,N,N′,N′-tetraacetate. Sodium nitroprus-side and nitroglycerin induced elevation of cyclic guanosine 3′,5′-monophosphate content in platelets which correlated with their calcium-blocking activity. Methylene blue and epinephrine decreased the calcium-blocking effect and the influence of nitroglycerin on cyclic guanosine 3′-5′-monophosphate content, but failed to suppress the inhibitory effect of sodium nitroprusside. Ascorbic acid increased the calcium blocking effect of sodium nitroprusside and its influence on cyclic guanosine 3′,5′-monophosphate content, but did not alter the inhibitory effect of nitroglycerin. In order to evaluate the relationship between the mode of action of nitrates at cellular level and their vasodilatory effectiveness, we studied the circulatory response of the forearm to isosorbide dinitrate and the influence of nitroglycerin on free calcium concentration in the platelets in 10 patients with chronic heart failure. We established a significant positive correlation between the basal values for venous tone and its peak decrease after administration of the 10-mg dose of isosorbide dinitrate. A correlation was also found between the deviation of maximal decrease of venous tone by this dose of isosorbide dinitrate from the regression line (the relationship between the basal venous tone and its lowering by the drug) and mean inhibitory concentration values for nitroglycerin in blocking that proportion of the rise of calcium ion concentration in platelets due to blocking of the platelet-activating factor. Thus, nitrates, like calcium antagonists, inhibit the receptor-provoked calcium supply to the contractile system of the cells so neutralizing the effects of increased concentrations of vasoconstrictors. This suggests that the effectiveness of nitrates appears to be positively related to the contribution of receptor-induced increase of cytosolic free calcium concentration in vasoconstriction together with their capacity to raise cyclic guanosine 3′,5′-monophosphate.

Agonist‐evoked changes in cytosolic pH and calcium concentration in human platelets: studies in physiological bicarbonate.

1. Cytosolic pH (pHi) and calcium concentration ([Ca2+]i) have been investigated in the presence and absence of physiological HCO3- in human platelets co-loaded with the fluorescent indicators BCECF and Fura-2. Basal pHi and changes evoked by butyrate, thrombin, platelet activating factor (PAF), ADP and phorbol ester were investigated, as were the effects of removing external Na+. 2. In the presence of physiological HCO3- and CO2, basal pHi was 7.02 +/- 0.04 compared with 7.15 +/- 0.05 in the absence of HCO3-. Estimated cytosolic buffering power was reduced from 35.6 +/- 3.0 to 14.5 +/- 0.4 mM/pH unit by the omission of HCO3-. 3. Thrombin evoked an immediate acidification of 0.03 +/- 0.01 pH units in the presence of HCO3- and 0.07 +/- 0.01 pH units in its absence. The acidifications were followed by a slow alkalinization. The final pHi was 0.10 +/- 0.01 units above basal in the presence of HCO3- and 0.08 +/- 0.02 units above basal in the absence of HCO3-. The initial acidification was significantly greater in the absence of HCO3-. The subsequent increase in pHi was similar in the presence and absence of this ion, but the calculated loss of proton equivalents was greater in the presence of HCO3-. 4. Replacement of extracellular Na+ with N-methyl-D-glucamine resulted in a fall in basal pHi and abolished recovery from thrombin-evoked acidification in both the presence and absence of HCO3-. 5. In the presence of HCO3-, PAF and ADP evoked an intracellular acidification similar to that caused by thrombin. However, with PAF and ADP, the subsequent recovery in pHi was slow and did not rise above basal levels. Phorbol dibutyrate, an activator of protein kinase C, evoked a similar elevation in pHi of 0.04 +/- 0.01 units over 3 min in the presence and absence of HCO3-. 6. Stopped-flow fluorimetric measurements were made of both BCECF and Fura-2 fluorescence in the presence of HCO3-. In the presence and absence of external Ca2+, thrombin-evoked rises in [Ca2+]i peaked before any cytoplasmic alkalinization occurred. ADP evoked rapid elevations in [Ca2+]i, but caused no alkalinization.(ABSTRACT TRUNCATED AT 400 WORDS)