Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

Reduced functions of intracellular Ca in aggregation, secretion and protein phosphorylation of permeabilized platelets from stroke-prone spontaneously hypertensive rats

Aggregation, secretion and 47kDa protein (P47) phosphorylation by various agonists such as thrombin, ADP and ionophore A23187 were markedly reduced in platelets from stroke-prone spontaneously hypertensive rats (SHRSP) compared with those of age-matched Wistar Kyoto rat (WKY) platelets, suggesting defective functions of intracellular Ca²⁺ in SHRSP platelets (Tomita et al. Hypertension 1989: 14: 304–315). To clarify the mechanism of the platelet hypofunctions, saponin permeabilized platelets were prepared to compare the responses of platelets from both rats in varying concentrations of extracellular Ca²⁺. The leakage of lactate dehydrogenase from saponin (15 μg/ml)-treated platelets was approx. 5 % of total activity; the degree of the leakage in both platelets did not differ. In saponin-treated platelets, extracellular Ca²⁺ alone did not induce either aggregation or secretion in both strains. However, in the presence of 1-oleoyl-2-acetylglycerol (10 μg/ml), Ca²⁺ dose dependently stimulated both aggregation and secretion. Under this condition, Ca²⁺ sensitivity of aggregation, secretion and P47 phosphorylation in SHRSP platelets were significantly reduced compared with those in WKY platelets. These results strongly suggest that intracellular Ca²⁺ functions are impaired in SHRSP platelets.     

Endothelin and platelet function

The effects of newly discovered vasoconstrictor peptide endothelin was studied on human, rabbit and canine platelet function. Endothelin (0.01 nM - lμM) did not promote platelet aggregation. In human platelets, endothelin (0.1 μM) did not significantly affect aggregation responses to ADP, collagen, epinephrine, arachidonic acid, PGH₂ or thrombin. Endothelin did not promote the mobilization of intracellular calcium in Fura2 loaded human platelets. In rabbit and canine platelets endothelin produced signficant potentiation of platelet aggregation mediated by low concentrations of ADP. Aggregation responses to higher concentration of ADP (5 μM) were unaffected by endothelin. These data reveal that under certain circumstances endothelin may potentiate rabbit and canine platelet aggregation responses to ADP, however endothelin does not produce direct effects on human platelet function.     

Decreased platelet vitamin C in diabetes mellitus: Possible role in hyperaggregation

To determine whether the handling of Vitamin C in the diabetic might be altered and might relate to the increased platelet sensitivity, we have investigated levels of platelet Vitamin C in the diabetic and determined the effects of Vitamin C or on platelet aggregation.

Levels of ascorbic acid, as tested by a lingual method, were significantly lower in diabetics than in normals (p < . 01). Ascorbic acid levels in washed platelets from diabetics were significantly lower than from normals (45. 2±3 μg/10¹⁰ platelets vs. 25. 5±2 μg/10¹⁰ platelets, p < . 001). The effects of ascorbic acid on platelet aggregation were studied by adding ascorbic acid in buffered solution (pH 7. 35) prior to aggregating agents. Ascorbic acid (1000 μg/ml) in platelet-rich plasma consistently inhibited platelet aggregation with threshold concentrations of ADP, epinephrine, and collagen, but enhanced aggregation with arachidonic acid. With washed platelets, ascorbic acid inhibited arachidonic acid-induced aggregation. To rule out an interaction of ascorbi acid and arachidonic acid in the medium, platelets were incubated at 37°C for 10 minutes with varying concentrations of ascorbic acid, rewashed, and aggregation with arachidonic acid tested. Aggregation was inhibited in a linear dose-dependent fashion. Oral ingestion of ascorbic acid (2 gm/day) for seven days by normal non-smoking males produced a marked inhibition of aggregation. In a similar study, platelets from an insulin-dependent diabetic showed no change in aggregation. These results suggest that platelet levels of ascorbic acid may relate to the hyper-aggregation of platelets from diabetics.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Effects of trifluoperazine on platelet activation

Previous reports of the inhibitory effects of trifluoperazine on platelet responses to different aggregating agents have been conflicting, and the mechanism of action remains unclear. We have found that aggregation by minimum concentrations of collagen and arachidonic acid, and second phase aggregation by minimum concentrations of ADP, thrombin, epinephrine and the calcium ionophore A23187 were inhibited by 40–60μM trifluoperazine. The first phase of aggregation by a minimum concentration of epinephrine was completely inhibited by 100μM trifluoperazine, and the first phase of aggregation induced by ADP, thrombin or A23187 was decreased by 300μM trifluoperazine. The platelet shape change caused by collagen, but by no other aggregating agent examined, was inhibited by 300μM trifluoperazine. Secretion of ³H-5 hydroxytryptamine by minimum concentrations of ADP, collagen, epinephrine and arachidonic acid was completely suppressed by 50μM trifluoperazine. Secretion by thrombin and A23187 was incompletely inhibited by 300μM trifluoperazine. Thromboxane B₂ formation caused by all aggregating agents, except epinephrine, was incompletely suppressed by 50μM trifluoperazine, and 300μM trifluoperazine only caused complete inhibition of thromboxane B₂ formation by ADP, collagen and epinephrine. The phorbol ester, TPA, which mimics diacylglycerol by activating protein kinase C, caused aggregation and secretion. Aggregation, but not secretion, by low concentrations of TPA was inhibited by concentrations of trifluoperazine as low as 50μM. However, aggregation by a combination of TPA and A23187 was only inhibited by concentrations of trifluoperazine in excess of 100 μM. Secretion by TPA was inhibited by concentrations of trifluoperazine in excess of 200μM. Our findings suggest that low concentrations of trifluoperazine inhibit platelet activation by inhibiting phospholipase A₂, and that higher concentrations inhibit platelet responses by interfering with protein kinase C.     

Mechanisms of vascular graft thrombosis: Role of altered canine platelet sensitivity to thromboxane

Using the standard turbidimetric method of platelet aggregation and quantitation of platelet secretion with ¹⁴C-Serotonin, we have examined the responsiveness of the platelets of mongrel dogs to arachidonic acid (AA), and the thromboxane agonist U46619 in the presence and absence of a subthreshold concentration of epinephrine. In response to stimulation with 750 μM AA, the platelets of 18 dogs produced irreversible aggregation (Group I), the platelets of 22 dogs showed, at most, reversible aggregation (Group II), while the platelets of 8 dogs demonstrated no aggregatory response (Group III). In the presence of AA and a subthreshold concentration of epinephrine (0.5 μM), the platelets of all three groups demonstrated enhanced aggregatory and secretory responses although the extent of ¹⁴C-Serotonin secretion differed significantly between all three groups. These differences in platelet aggregation correlate with the deposition of platelets onto synthetic vascular grafts and the maintenance of graft patency. When stimulated with 0.5 μM U46619 and a subthreshold concentration of epinephrine, the platelets of 97% Group I dogs and 75% of Group II dogs exhibited irreversible aggregation, while the platelets of all Group III dogs showed only reversible aggregation. In addition, significant differences in the extent of ¹⁴C-Serotonin secretion to this combination of agonists were observed between groups. Further examination of the specific effects of U46619 on canine platelets revealed that although the aggregatory and secretory responses to U46619 vary between the different canine platelet populations, the threshold concentration of U46619 required to produce platelet shape change is identical among all groups. Quantitation of the stable metabolite of AA produced via the cyclooxygenase pathway, thromboxane B₂(TxB₂), revealed no significant differences in the production of TxB₂ by the platelets of these different populations upon stimulation with AA. Our results suggest that the mechanisms underlying the differences in responsiveness of canine platelets to AA, are likely due to differences in sensitivity of canine platelets to TxA₂, and may be localized to the mechanism responsible for mediating platelet aggregation and secretion in response to TxA₂.     

Cooperative effect of GNB3 825C>T and GPIIIa PI(A) polymorphisms in enhanced platelet aggregation

Introduction: Platelet aggregation contributes to various thrombembolic disorders. Environmental factors affect platelet aggregability but only partially explain the interindividual variability in aggregation. While the platelet glycoprotein IIb/IIIa is involved in the pathogenesis of acute coronary syndromes whereas most platelet activating stimuli act via G Protein coupled receptors we investigated whether the 825C>T polymorphism of the gene GNB3 encoding the G protein β3 subunit together with the platelet glycoprotein (GP) IIIa Pl(A) polymorphism are predictive of platelet aggregability on stimulation with various agonists acting via GPCRs. Materials and methods: Platelet aggregation was measured by turbidometry in 150 non-smoking individuals aged 18–40 years at a density of 2×10⁵ platelets/μl with various agonists according to the method of Born. Genotypes of the GNB3 825C>T and glycoprotein IIb/IIIa PI(A) polymorphisms were determined using Pyrosequencing technology and restriction analysis. All functional studies were completed within 3 h. The data were analysed by Student's t-test for paired data. Results: Low concentrations of agonists resulted in enhanced platelet aggregation in subjects with the GNB3 CC-genotype compared to carriers of a 825T-allele. This effect was further enhanced in carriers of the GPIIIa Pl(A2) allele (2 μM ADP: 42% vs. 19%, p=0.017; 1 μM U-46619: 51% vs. 30%, p=0.03; 5 μM epinephrine: 69% vs. 53%, p=0.025). No significant pattern of aggregation was observed on stratification by GPIIIa genotypes alone. Conclusions: Our findings indicate that two genetic markers contribute synergistically to increased platelet aggregation. This will help to identify patients at increased risk for thrombosis.     

Independent regulation of activation and inactivation phases in non-contractile Ca transients by nicotinic receptor at the mouse neuromuscular junction

Non-contractile Ca²⁺ mobilization (not accompanied by muscle contraction) occurs by the prolonged activation of nicotinic acetylcholine receptor in mouse diaphragm muscles treated with anticholinesterase. To elucidate the regulation properties of non-contractile Ca²⁺ mobilization by nicotinic receptor, the modes of action of competitive and depolarizing neurmuscular blockers were investigated. (+)-Tubocurarine (0.07–0.1 μM), pancuronium (0.05 μM) and -bungarotoxin (0.03–0.06 μM) decreased decay time (T₂, duration of inactivation phase) without changes in rise time (T₁, duration of activation phase) of non-contractile Ca²⁺ transients. These competitive antagonists also suppressed their peak amplitude at higher concentrations than those affectingT₂. Contractile Ca²⁺ transients were not inhibited by these antagonists at the concentrations used. Decamethonium (1 μM), a depolarizing blocker, suppressed the peak amplitude of non-contractile Ca²⁺ transients without affecting their duration. In contrast, succinylcholine (0.3 μM) suppressed both peak amplitude andT₁ without changingT₂, presumably via the receptor desentization. Succinylcholine but not decamthonium inhibited contractile Ca²⁺ transients at the concentrations used. These results demonstrate that the activation and inactivation phase in non-contractile Ca²⁺ transients are independently regulated by nicotinic acetylcholine receptor.     

The primary wave of epinephrine-induced platelet aggregation represents alpha 2-adrenoceptor status

We examined relationships between epinephrine-induced slope of primary wave of aggregation and the alpha₂-adrenoceptor status on platelets. A concentration (10^-9 to 10^-6 )-dependent increase in slope of primary wave with EC50 of epinephrine at 4.5 ± 0.4 × 10^-7 was observed. In studies on epinephrine binding to alpha₂adrenoceptors in competition with ³H-yohimbine to platelets, (IC₅₀) of epinephrine was 4.8 ± 3.4 x 10^-7 M. There was a significant (P<0.02) correlation between EC₅₀ of epinephrine to evoke biological response and IC₅₀ of epinephrine to bind to alpha₂-adrenoceptors (r-0.75). There was no relationship between number of receptor sites or dissociation constant of ³H-yohimbine binding and primary wave of platelet aggregation. These data show that the slope of primary wave in response to epinephrine reflects alpha₂-adrenoceptor binding of the agonist.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

THE EFFECT OF CHOLESTEROL OXIDATION PRODUCTS ON HUMAN PLATELET AGGREGATION

The cholesterol oxidation products (oxysterols) cholest-3,5-diene-7-one, cholestan-5, 6-epoxy-3β-ol (cholesterol 5-epoxide), cholestan-5β,6β-epoxy-3β-ol (cholesterol 5β-epoxide), cholest-5-ene-3β-ol-7-one (7-ketocholesterol), cholest-5-ene-3β,7-diol (7-hydroxycholesterol), cholestan-3β,5,6β-triol (choestane triol), and cholest-5-ene-3β,26-diol (27-hydroxycholesterol) potentiated platelet aggregation and increased thromboxane A₂ formation in platelets challenged with thrombin, ADP or collagen. These effects were observed at oxysterol concentrations in the range 5–100 μM. Cholesterol 5β-epoxide and 7-ketocholesterol increased the mobilization of ³H-arachidonic acid from prelabelled platelet phospholipids in response to thrombin and collagen.     

Chronic depolarization induced by veratridine increases the survival of rat retinal ganglion cells ‘in vitro’

During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na⁺ channels opened) induces an increase in neuronal survival. In the present work we show that 3 μM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 μM amiloride (an inhibitor of Ca²⁺ channels), 25 μM benzamil (an inhibitor of Na⁺ channels), 30 μM dantrolene and 7.5 μM caffeine (both inhibitors of Ca²⁺ release from the endoplasmatic reticulum) and 10 μM BAPTA-AM (an intracellular Ca²⁺ chelator). However, 5 μM nifedipine (a selective inhibitor of voltage-dependent -type Ca²⁺ channels) and 100 μM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 μM genistein (an inhibitor of tyrosine kinase enzymes), 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 μM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na⁺ influx, intracellular Ca²⁺ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.     

Epinephrine-induced aggregation of rabbit platelets refractory to ADP

The mechanisms involved in platelet aggregation induced by epinephrine are unclear. Although epinephrine does not aggregate washed rabbit platelets, platelets made refractory to ADP will aggregate in response to epinephrine in the presence of ADP. We have examined whether the mechanism(s) by which epinephrine induces aggregation of refractory platelets involves fibrinogen binding and Ca2+ association. With normal platelets, ADP causes aggregation, fibrinogen binding and Ca2+ association in a medium containing 0.2 mM 45Ca2+. After 3 min of incubation with ADP, fibrinogen dissociates from platelets, but 45Ca2+ does not. Epinephrine alone does not cause aggregation, fibrinogen binding or 45Ca2+ association. Platelets that are refractory to ADP do not aggregate and bind fibrinogen upon addition of ADP, but aggregate and bind fibrinogen in response to epinephrine, provided ADP is still present. These effects of epinephrine are mediated by the alpha-adrenergic receptor since they are blocked by phentolamine or verapamil and potentiated by propranolol. However, epinephrine-induced aggregation of platelets refractory to ADP does not involve further detectable increase in the amount of 45Ca2+ associated with the platelets.     

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E₁ (PGE₁) on platelets is mediated through the PGE₁ receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE₁ and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 gm. The time course of the extension length (Dp, in μg) of the platelets in response to aspiration with a negative pressure (ΔP) of 5 cm H₂ O (ΔP × Rp = 0.15 dynes/cm) was analyzed. PGE₁ treatment (0.1 μM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE₁ -treated) / Dp/Rp (control), was significantly reduced to 0.90 ± 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE₁ -treated platelets had a higher [cAMP]_i than controls. Platelets treated with PGE₁ or dbcAMP showed a reduced [Ca²⁺]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE₁ and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]_i and low [Ca²⁺]_i after PGE₁ treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.     

Inhibitory effects of sulphated flavonoids isolated from Flaveria bidentis on platelet aggregation

Flaveria bidentis is a plant species that has as major constituents sulphated flavonoids in the highest degree of sulphatation. Among them, quercetin 3,7,3′,4′-tetrasulphate (QTS) and quercetin 3-acetyl-7,3′,4′-trisulphate (ATS) are the most important constituents. Both showed anticoagulant properties. The objective of the present study was to evaluate the effects of these flavonoids on human platelet aggregation in comparison with the well-known inhibitor quercetin (Qc) by using several agonists. Platelet-rich plasma (PRP) or washed human platelets (WP) were incubated with different concentrations of the flavonoids to be tested (1 to 1000 μM, final concentration), and the platelet aggregation was induced by using adenosine 5′-diphosphate (ADP), epinephrine (EP), collagen, arachidonic acid (AA) and ristocetin as agonists. QTS (500 μM) and Qc (250 μM) markedly inhibited platelet aggregation with all the aggregant agents, except ristocetin, whereas ATS (1000 μM) showed only slight antiplatelet effects. In addition, QTS and Qc antagonized the aggregation of PRP or WP induced by U-46619, a mimetic thromboxane A2 (TxA₂) receptor agonist. Challenged with collagen or arachidonic acid, the thromboxane B₂ (TxB₂) formation was also inhibited by the flavonoids, mainly by QTS and Qc, in WP. These results demonstrate that QTS and in minor extension ATS induce a deleterious effect on the production of TxA₂, as judged by TxB₂ formation, in stimulated WP and a marked interference on the TxA₂ receptor according to the profile of inhibition of the agonist-induced platelet aggregation when using ADP, EP, AA and collagen and confirmed with U-46619.     

Mn and Mg influxes through Ca channels of motor nerve terminals are prevented by verapamil in frogs

At frog neuromuscular junctions immersed in solutions containing 0.5 mM Mn²⁺, verapamil (40 μM) reduced the increase in miniature end-plate potential (MEPP) frequency produced by tetanic stimulation (50 Hz, 2 min) of the motor nerve to 5% of that in the absence of verapamil. In solutions containing 5 mM Mg²⁺, verapamil reduced the tetanic increase in MEPP frequency to 8% of that in the absence of verapamil. Verapamil added to solutions containing 0.15 mM Ca²⁺ decreased the tetanic rise in MEPP frequency to 6% of the control value. In low Ca²⁺ (nominally Ca²⁺-free) solutions, verapamil decreased the tetanic rise to 70% of the control value. The present results suggest that Mn²⁺ and Mg²⁺, as well as Ca²⁺, enter the nerve terminal through Ca²⁺ channels during nerve stimulation and promote transmitter release. In addition to its effect on the Ca²⁺ channel, verapamil at higher concentrations appears to have inhibitory effects on the acetylcholine-gated end-plate channel and on the Na⁺ channel as suggested by its depressive effects on the amplitudes of MEPPs, end-plate potentials and nerve terminal action potentials.     

Inhibition of platelet aggregation and the release of P-selectin from platelets by cilostazol

To evaluate the in vitro effects of cilostazol, a phosphodiesterase III inhibitor, on platelet responses, we measured platelet aggregation and the levels of soluble P-selectin, a glycoprotein present on the -granule membrane in resting platelets, and cAMP. Platelet-rich plasma and washed platelets from healthy human volunteers were treated with cilostazol (5, 25 and 50 μM). Platelet-rich plasma was stimulated by ADP (1 and 5 μM) or collagen (5 μg/ml). Washed platelets were stimulated by thrombin (4 U/ml) in the presence or absence of 1 μM forskolin. In vehicle-treated samples, soluble P-selectin levels in response to 1 μM ADP-induced primary aggregation were similar to those of circulating levels of healthy volunteers but the levels in response to 5 μM ADP-induced secondary aggregation and collagen-induced aggregation increased markedly compared to those in response to primary aggregation. This result suggests that P-selectin is released from platelets according to the extent of platelet aggregation. Cilostazol inhibited platelet aggregation as well as P-selectin release in a concentration-dependent manner. Cilostazol inhibited completely thrombin-induced aggregation in the presence of 1 μM forskolin, when cAMP levels were two-fold higher than those in the absence of forskolin. Cilostazol, which increases intracellular cAMP in platelets, may be useful in the treatment of arterial occlusive diseases.     

Properties of PAF-acether-induced platelet aggregation and secretion. Studies in gel-filtered human platelets

Human platelets rapidly lose their responsiveness to PAF-acether after blood collection. We collected blood from fasting donors and prepared gel-filtered platelets that remained responsive to PAF-acether for about 6 hours. Log-dose response studies showed bi-phasic aggregation between 20 and 100 nM PAF-acether with secretion of dense-, - and lysosomal granule contents during the second wave of aggregation. Between 0.2 and 10 nM PAF-acether aggregation was weak and no secretion occurred whereas 300 nM PAF-acether or more induced maximal aggregation and secretion. Secretion, however, was never more than 70, 55, and 30% of maximal secretable amount of 5HT, βTG and βN, respectively. Aggregation and secretion were enhanced by fibrinogen (optimal concentration 0.3–0.7 g.l⁻¹), required Ca²⁺ or Mg²⁺ but were inhibited when Mg²⁺ or Ca²⁺ were present at a concentration of 2 mM or more. These date show that human platelets are almost equally sensitive to PAF-acether as rabbit platelets, and respond with incomplete secretion of dense-, - and lysosomal granule contents.