Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]i) was studied in both ³²P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A₂ analogue (STA₂). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca²⁺]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca²⁺]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA₂. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca²⁺]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.     

The involvement of calcium in epinephrine or ADP potentiation of human platelet aggregation

The mechanism by which low doses of epinephrine or ADP potentiate primary platelet aggregation was investigated. Aspirin (lmg/ml)-treated human blood platelets were isolated by albumin density gradient centrifugation. Platelet ⁴⁵Ca uptake associated with epinephrine or ADP addition was determined over a 240 sec time course. Pretreatment of the platelets with ADP (0.5μM) significantly increased aggregation in response to epinephrine (0.1μM). This increased aggregation was associated with a substantially greater ⁴⁵Ca uptake than that which occurred in the presence of epinephrine (0.1μM) alone. The potentiated epinephrine response was inhibited by the Ca²⁺ antagonist verapamil (25μM). This inhibition could in turn be reduced by Ca²⁺ (1mM) addition. Pretreatment of platelets with epinephrine (0.1μM) also increased aggregation in response to ADP (0.5μM). Although this potentiated response was not associated with measurable ⁴⁵Ca uptake, it was nevertheless completely abolished by verapamil (25μM) treatment. These findings suggest that low doses of ADP promote the ability of epinephrine to stimulate an increase in membrane permeability to Ca²⁺.     

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.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

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.     

Impaired function of TXA2 and/or calcium in the platelets from SHRSP at hypertensive ages

Aggregation of washed platelets from stroke-prone spontaneously hypertensive rats(SHRSP) was markedly reduced with the development of hypertension in comparison with age-matched normotensive Wistar Kyoto rats(WKY) (Tomita et al. Stroke 15, 70-75, 1984). The mechanism of the hypoaggregability of SHRSP platelets was studied. Ca2+-dependence of thrombin-induced aggregation and MDA formation, and Ionophore A23187-induced aggregation of the platelets from SHRSP at a hypertensive age(16-weeks) was similar to that of the aggregation of platelets from age-matched WKY. Optimum Ca2+ concentration for aggregation and MDA formation was 1-2 mM. There was no difference in aggregation in Ca2+-free medium between the two strains. The enhancement by Ca2+ of both thrombin- and Ionophore A23187- induced aggregation, however, was markedly less in SHRSP than in WKY, whereas their MDA formation was equally enhanced by Ca2+. At a prehypertensive age (4-weeks) the degree of enhancement of aggregation by Ca2+ did not differ in the two strains. The magnitude of phospholipid(PI, PC, PE) degradation, and MDA formation IN SHRSP at early- and late- hypertensive ages(11- and 17-weeks) were either the same as or greater than that of age-matched WKY. A linear correlation line between the amount of MDA formed and the degree of platelet aggregation of SHRSP shifted to the right of WKY. In addition, thrombin-induced thromboxane B2 formation in SHRSP platelets was similar to that in WKY in the concentration range of 0.22 - 0.44 U/ml, and became significantly higher at 0.65 U/ml despite severe hypoaggregability of SHRSP platelets in all the concentrations examined. The overproduction of MDA or thromboxane B2 appears to be a compensatory mechanism. These results suggest that abnormalities of SHRSP platelets at hypertensive ages are due to an impaired function of thromboxane A2 and/or calcium concerned in aggregation and secretion but not due to a defect in cyclo-oxygenase and thromboxane synthetase pathway.     

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.     

Platelet aggregation and endogenous 5-ht secretion in presence of ca, sr and ba. effects of calcium antagonists

Washed rat platelets aggregation and endogenous serotonin release were studied after thrombin stimulation in the presence of different concentrations of Ca²⁺, Sr²⁺ or Ba²⁺. The extent of platelet aggregation and release was found to depend upon the external concentration of these cations. For all of them, an optimum concentration could be defined. Higher concentrations were shown to inhibit both aggregation and release. Efficiency to support thrombin-induced aggregation was in the order Ca²⁺>Sr²⁺>Ba²⁺. Complete inhibition of aggregation and release induced by thrombin was obtained after a 30 second preincubation with 38 uM nitrendipine, 1 nM Cd^2- or 1 mM Mn²⁺. Inhibition was obtained in the presence of Ca²⁺, Sr²⁺ or Ba²⁺. These results are consistent with the hypothesis that Sr²⁺ and Ba²⁺ are able to support platelet activation acting as Ca²⁺ substitutes. Following thrombin stimulation, they could penetrate the platelets and mimick a rise in cytoplasmic Ca²⁺.     

Dependence of the acid-sensitive ion channel, ASIC1a, on extracellular Ca(2+) ions

The early H⁺-induced current in the embryonic spinal cord neurone depends on extracellular Ca²⁺ for its function. We have studied the Ca²⁺-dependence of homo- and heteromultimeric acid-sensing ion channels (ASICs) expressed in Cos cells. It was found that single-channel conductance of both the ASIC2a and the ASIC1a channel is reduced at membrane potentials more negative than −40 mV by elevated extracellular Ca²⁺. Due to this effect on unitary currents, the macroscopic ASIC2a peak current at −60 mV decreases gradually with rising extracellular Ca²⁺ concentration. In addition, the macroscopic ASIC1a current is very small at low extracellular Ca²⁺ and increases with rising Ca²⁺ up to 5 mM before decreasing again at still higher concentrations of extracellular Ca²⁺.     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca²⁺ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130^Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130^Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca²⁺ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130^Cas involvement in platelets.     

Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages

The effects of the opioid peptide dynorphin A (DynA) on phagocytosis in peritoneal macrophages was examined by flow cytometry (FCM). DynA enhanced phagocytosis in a dose-dependent manner. Leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), β-neo-endorphin (βNeo-End), DynA(9–17) and DynA(13–17) had no such activity, -Neo-endorphin ( Neo-End), dynorphin B (DynB), DynA(l–13) and DynA(6–17) enhanced phagocytosis less effectively than DynA. Naloxone did not inhibit the enhancement of phagocytosis induced by DynA. Unstimulated control phagocytosis was partially suppressed in Ca²⁺-free EGTA-containing solution and even in this solution DynA enhanced phagocytosis. However, the enhancement by DynA was suppressed in EGTA- and BAPTA-AM-containing Ca²⁺-free solution. The present study showed that enhancement of phagocytosis by DynA was independent of extracellular Ca²⁺ ([Ca²⁺]_o) and dependent on intracellular Ca²⁺ ([Ca²⁺]_i). The present results support DynA being one of the mediators from the nervous system that modulates the immune system.     

Intracellular concentration of parvalbumin in nerve cells

Parvalbumin is supposed to function as an intracellular Ca²⁺ buffer in the brain, but its concentration in nerve cells is unknown. We combined radioimmunoassay of parvalbumin in rat brain extracts, together with image analysis of area fractions immunostained with the parvalbumin antibody on sections, to deduce the intracellular concentration of parvalbumin. We found that the average amount of parvalbumin varied between 6 μM in the caudatoputamen and 45 μM in the cerebellum. Although these values are only rough averages and do not demonstrate a Ca²⁺ buffering role for parvalbumin in neurons, the concentration of the protein is at least in the appropriate range for such a function.     

Effect of phorbol 12-myristate 13-acetate on collagen-induced signal transduction in rabbit platelet

Investigations were made on the inhibitory effect of phorbol 12-myristate 13-acetate (PMA), a powerful activator on protein kinase C, on collagen-induced signal transduction in washed rabbit platelets. Upon activation of the platelets with a low-dose of collagen (5 μg/ml), which was suppressed by 10 μM indomethacin, pretreatment of the platelets with 2 nM PMA caused prolongation of lag phase (2 min) before the onsets of the aggregation and ATP secretion as compared with PMA-untreated platelets (30 sec). Under this condition, appearance of the cell responses including the phosphatidic acid formation, thromboxane (Tx) generation and Ca²⁺-influx was similarly retarded for 2–3 min, whereas arachidonic acid liberation from the membrane phospholipids was not significantly affected by the PMA pretreatment. After such lag phase, every response appeared rapidly and reached almost the control value (without PMA). Upon activation of the same platelets with a high-dose of collagen (50 μg/ml), which was only half suppressible by indomethacin, PMA in the presence of indomethacin almost completely suppressed the phosphatidic acid formation as well as the aggregation and ATP secretion. Thus, our results suggest that collagen-platelet interaction may elicit direct activation of phospholipase A2 and C, and that the latter enzyme activation may be regulated by a negative effect of protein kinase C. However, the phospholipase A2 activation may be regulated by a mechanism independent of such effect. In PMA-pretreated platelets in response to a low-dose of collagen, the prolonged lag phase for aggregation appears to be due to impaired conversion of liberated arachidonic acid to TxA₂.     

Analysis of potential shifts associated with recurrent spreading depression and prolonged unstable spreading depression induced by microdialysis of elevated K in hippocampus of anesthetized rats

The potential shifts (ΔV_o) associated with spreading depression (SD) were analysed with the help of multiple extracellular recording and ion-selective microelectrodes in the CA1 region of the dorsal hippocampus of anesthetized rats. Recurrent waves of SD were induced by perfusing high K⁺ solution through microdialysis probes. SD-related ΔV_o had a composite wave shape, consisting of an early, rapidly shifting part (phase I) followed by a slower shift to a second negative maximum (phase II). ΔV_o shifts in stratum radiatum usually started earlier, always lasted longer and had lartger amplitude than those recorded in stratum pyramidale. The ΔV_o responses in stratum radiatum had an inverted saddle shape created by a transient relatively positive “hump” interposed between phases I and II. During this “hump”, the potentials in the two layers transiently approached one another. During continuous high K⁺ dialysis, successive ΔV_o waves episodes evolved according to a consistent pattern: while phase I remained unchanged, phase II increased in amplitude and duration with each episode. Eventually, a depressed state developed which lasted for many minutes, termed here prolonged unstable spreading depression. During phase I, ΔV_o and extracellular K ([K⁺]_o) changes were correlated. During phase II, [K⁺]_o decreased even as ΔV_o continued to increase. During SD, [Ca²⁺]_o decreased to <0.01 mM. During phases I and II, both [Ca²⁺]_o and [Na⁺]_o remained low. the recoverries of [Ca²⁺]_o and [Na⁺]_o had an initial fast and a later much slower phase and took several minutes longer than the recoveries of [K⁺]_o and ΔV_o. Depth profiles of ΔV_o and Δ[K⁺]_o revealed strikingly steep gradients early and late during a wave; but voltage and ion gradients were not precisely correlated either in time or in space. We conclude that ΔV_o of phases I and II are generated by different processes. Membrane ion currents cannot fully explain the ΔV_o responses. The possible contributions by ion diffusion and by active ion transport are discussed. The extremely low level to which [Ca²⁺]_o sinks during SD, and its two-phase recovery, indicate intracellular sequestration or binding of substantial amounts of Ca²⁺ ions. The residual deficit of [Ca²⁺_o following recovery of SP shifts may account for the persistent depression of synaptic transmission after repolarization of neurons.     

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.     

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.     

Hypoaggregability of washed platelets from stroke-prone spontaneously hypertensive rats (SHRSP)

The aggregation properties of washed SHRSP platelets were investigated in comparison with normotensive WKY platelets at prehypertensive (4 weeks), early hypertensive (11 weeks) and late hypertensive (17 weeks) ages in the absence of plasma factors. The number of platelets in SHRSP was markedly lower with the development of hypertension than that in WKY. The thrombin- and collagen-induced aggregation was markedly reduced in the platelets from 11 and 17 week old SHRSP compared with that of age-matched WKY, whereas the degree of platelet aggregation in 4 week old SHRSP showed a tendency to be even greater than that in WKY. The changes in blood pressure and platelet aggregability were correlated inversely. ADP did not induce aggregation in the same system used for thrombin and collagen stimulation but in another system it aggregated washed rat platelets. Aggregation responses to ADP and ionophore A23187 were also significantly lower in 14 week old SHRSP platelets than age-matched WKY platelets. Together with other evidence, these results suggest that defective Ca2+ function, rather than the presence of exhausted platelets, is responsible for hypoaggregability in SHRSP platelets.     

Defective calcium transport in platelets from stroke-prone spontaneously hypertensive rats

Aggregation and secretion of washed platelets from stroke-prone spontaneously hypertensive rats(SHRSP) were greatly reduced by the development of hypertension in comparison with age-matched normotensive WKY platelets. In an attempt to clarify the mechanism of the defective functions, Ca2+ transport in platelets from SHRSP and WKY were studied. Changes of cytoplasmic free Ca2+ concentration ([Ca2+]i) were examined by using Quin 2. [Ca2+]i increase in response to thrombin(0.028 - 0.11 U/ml) was significantly delayed in SHRSP platelets compared with that of age-matched WKY platelets. The time(sec) to peak in [Ca2+]i was about two times longer in SHRSP platelets than in WKY platelets. [Ca2+]i levels at resting state were significantly lower in SHRSP platelets while there was no difference in maximal [Ca2+]i level in response to thrombin (0.031 - 0.125 U/ml) between the two strains. In addition thrombin-induced 45Ca2+ uptake was significantly delayed in SHRSP platelets. This delay of [Ca2+]i increase following thrombin stimulation might be associated with the hypofunctions of SHRSP platelets.