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.     

Influence of granulocyte elastase-like proteinase (ELP) on platelet functions

The influence of granulocyte elastase-like proteinase (ELP) on platelet functions was investigated. ELP inhibited the platelet aggregations induced by a wide variety of agonists. The inhibition was marked in the case of receptor-mediated agonists such as thrombin, ristocetin, etc. It was moderate with the pervading agonist, arachidonic acid, and mild with the bypassing agonist, Ca²⁺ ionophore A23187. ELP inhibited the release of thromboxane A2 from platelets in the case of the platelet aggregation induced by thrombin. On the other hand, ELP did not inhibit the release of thromboxane A2 from platelets in the platelet aggregation induced by arachidonic acid or Ca²⁺ ionophore A23187. ELP suppressed the release of serotonin from platelets induced by thrombin, while it did not markedly suppress the release of serotonin induced by Ca²⁺-ionophore A23187. Treatment of platelets with ELP resulted in a slight increase of intraplatelet cAMP levels. These results suggest that ELP acts on receptors and inhibits platelet functions. As a results, ELP markedly inhibits the platelet functions such as aggregation or release of serotonin or thromboxane A2 stimulated by receptor-mediated agonists. ELP slightly elevates the CAMP level in the platelets, resulting in the mild inhibition of the platelet functions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca²⁺-ionophore A23187.     

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.     

Influence of cations on the electrical activity of neuroblastoma × glioma hybrid cells

Electrical excitability is one of the various neuronal properties of neuroblastoma × glioma hybrid cells. At a Ca²⁺ concentration of 1.8mM the action potential is inhibited by tetrodotoxin, suggesting that the inward current is carried by Na⁺ ions. In contrast, at a Ca²⁺ concentration of 20–36mM and even in the absence of Na⁺, spikes (sometimes repetitive) with strong hyperpolarizing afterpotential occur, which are no longer affected by tetrodotoxin. They are, however, blocked by antagonists of Ca²⁺ like La³⁺, Co²⁺, Mn²⁺, and the synthetic compounds D-600 and BAY a-1040. This seems to indicate that at high concentrations of Ca²⁺, the inward current of the action potential is essentially carried by Ca²⁺. Sr²⁺, but not Mg²⁺ can effectively substitute for Ca²⁺. It shows down the time course of the action potential. Ba²⁺ depolarizes the membrane gradually. If Ca²⁺ is also present, Ba²⁺ causes a reduced depolarization and spontaneous action potentials with no hyperpolarizing after-potential are observed.     

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.     

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.     

Function of serotonin in physiologic secretion of growth hormone and prolactin: Action of 5,7-dihydroxytryptamine, fenfluramine and p-chlorophenylalanine

The effect of 4-aminopyridine (4-AP) on the release of labeled transmitters in mouse brain synatosomes was studied in a superfusion system. 4-AP at μM concentrations notably stimulated the spontaneous release of labeled GABA and glutamate, and of acetylcholine (ACh) derived from tritiated choline. No effects on the release of labeled -aminoisobutyric acid were observed. The stimulation of GABA and ACh release was dependent on the presence of Ca²⁺ in the superfusion media, whereas the effect on glutamate release was more variable and no clear Ca²⁺-dependence was observed. In contrast to these results, 4-AP did not have any effect on the release of the above transmitters by K⁺-depolarization in the presence of Ca²⁺. These results are discussed in terms of the possible participation of Ca²⁺ in the action of 4-AP on spontaneous transmitter release in isolated nerve endings.     

Aeguorin-detected calcium changes in stimulated thrombasthenic platelets. Aggregation - dependent calcium movement in response to ADP

Calcium changes in normal and thrombasthenic platelets were recorded using the PICA-apparatus. Aequorin was loaded in the presence of DMSO, EGTA and PGE1. Platelets of three patients with type I thrombasthenia stimulated with A-23,187, thrombin, PMA in the presence 1mM Ca⁺⁺ and 1mM Mg⁺⁺ were able to normally raise their calcium concentrations. The maximal values could be found below the normal range with collagen, ADP and PAF-acether. Calcium mobilization from internal stores in response to thrombin was normal. There were two calcium peaks in normal platelets stimulated with ADP. The second one was suppressed by omitting fibrinogen, stirring, or by adding aspirin, and was absent in thrombasthenic platelets. Thus the GP IIb-Illa complex is not a prerequisite for calcium fluxes but is involved, when weak agonists such ADP are used, through an aggregation-dependent reinforcement of 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.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

Antiplatelet effect of capsaicin

Capsaicin was found to be a potent inhibitor of platelet aggregation and release reaction. It inhibited the aggregation of rat platelet induced by collagen and thrombin, but only slightly reduced those of AA and A23187. The IC₅₀ on collagen-induced platelet aggregation was about 85 μg/ml. Less inhibition was observed in the aggregation of platelet-rich plasma. Increase of the calcium concentration could not overcome the inhibitory effect. Washing of the capsaicin-pretreated platelets only partially reversed the inhibition. Capsaicin also inhibited ATP release induced by thrombin and A23187 in the presence of EDTA. MDA and TXB₂ formation were markedly inhibited by capsaicin in platelets challenged by collagen, thrombin and A23187. In AA-stimulated platelets, MDA formation was slightly decreased and TXB₂ formation was not affected. Capsaicin showed more marked inhibition in the presence of CP/CPK, indomethacin or a combination of both. Capsaicin reduced the hemolysis of RBCs induced by hydrogen peroxide or hypotonicity. It was concluded that capsaicin had some membrane stabilizing property and this might lead to the interferance of the activation of phospholipase A₂.     

Effects of Cathepsin G Pretreatment of Platelets on their Subsequent Responses to Aggregating Agents

Cathepsin G, a proteolytic enzyme from activated leukocytes, can interact with platelets during inflammation and thrombosis. Platelets that have been exposed to cathepsin G in thrombi may recirculate if they are freed during fibrinolysis. To determine whether some of the subsequent functions of such platelets would be impaired, we investigated the responses of cathepsin G-pretreated platelets to agonists that they would encounter in the circulation. Suspensions of washed human platelets were labeled with [¹⁴C]serotonin and resuspended in Tyrode-albumin solution (with 2 mM Ca²⁺ and apyrase). After 15 minute incubation with 400 nM cathepsin G at 37°C, 52±3% of [¹⁴C]serotonin had been released, and glycoprotein Ib was degraded. The platelets were washed and resuspended in fresh medium to remove cathepsin G and released materials. Ristocetin-induced agglutination was abolished, indicating that the binding site for von Willebrand Factor on glycoprotein Ib had been removed. Aggregation and release of residual [¹⁴C]serotonin in response to 0.1–1.0 U/mL thrombin was blocked or greatly reduced by the cathepsin G pretreatment. This inhibition is probably largely due to cleavage by cathepsin G of some of the protease-activated receptors at the C-terminal side of Ser⁴² so that the tethered ligand is lost. Pretreatment with cathepsin G did not affect responses to ADP or a low concentration of platelet-activating factor in the presence of fibrinogen, indicating that receptors for these agonists were unaffected and that the function of the fibrinogen receptor, GPIIb/IIIa was unchanged. Responses to cathepsin G, the thrombin receptor-activating peptide SFLLRN, collagen, or the thromboxane A₂ mimetic U46619 were partially inhibited, even in the presence of added fibrinogen. Platelet adhesion to a collagen-coated surface was 51±7% inhibited, which may indicate cleavage of a collagen receptor or receptors; this may partly account for strong inhibition of collagen-induced aggregation and release of granule contents; additionally, as shown by inhibition of responses to U46619, the function of the thromboxane A₂ receptor may be compromised. Thus, although cathepsin G activates platelets, if they recirculate after interaction with it, their subsequent adhesion to damaged vessel walls, aggregation, and release of granule contents induced by thrombin and collagen will be diminished.     

Characterization of phospholipase A2 secretion from human platelets

Human platelets secreted phospholipase A₂ in a dose- and time-dependent manner when challenged with thrombin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or collagen. Enzyme release was maximal at concentrations of 0.1 units/ml of thrombin, 100 nM TPA, or 2 μg/ml of collagen; and complete by 2 min in platelets treated with thrombin or TPA. Cells challenged with collagen required up to 5 min for maximal secretion. Besides dose and time functions, phospholipase A₂ secretion was also dependent on platelet concentration and the levels of bovine serum albumin in the incubation medium. The secreted enzyme was soluble and exhibited substrate and Ca²⁺ requirements similar to a detergent-solubilized, partially purified phospholipase A₂ from whole platelets [Kramer et al., Biochim. Biophys. Acta (1988) 959, 269–279]. The pH optimum of the secreted enzyme, however, was 1–2 units lower than the pH optimum of the phospholipase A₂ from whole cells. Secreted phospholipase A₂ hydrolyzed phosphatidylethanolamine at 5–12 times the rate of phosphatidylcholine when the substrates were present in pure form. These apparent differences in activity were greatly diminished, though, when 1:1 molar mixtures of the two substrates were employed. Because phospholipase A₂ catalyzes a key reaction during the formation of bioactive arachidonate metabolites, the secretion of this enzyme from platelets may be important in the regulation of thrombosis.     

Effect of the concentration of Ca2+ in the suspending medium on the responses of human and rabbit platelets to aggregating agents

The effect of the concentration of Ca2+ in the suspending medium of human and rabbit platelets on aggregation, release of 14C-serotonin, and TXB2 formation in response to ADP, thrombin, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF), collagen and arachidonic acid was studied in either platelet-rich plasma anticoagulated with D-phenylalanyl-prolyl-arginyl chloromethylketone (PPACK) or citrate, or suspensions of washed platelets in modified Tyrode-albumin solutions containing 1 mM Mg2+ and concentrations of added Ca2+ ranging from 0 to 5 mM. In response to ADP, thrombin, or PAF, human platelets were stimulated to form TXA2 by close platelet contact in a low-Ca2+ medium; at physiological concentrations of Ca2+, TXB2 formation was much less and declined progressively as the concentration of Ca2+ was raised. When the formation of TXA2 was blocked with aspirin or indomethacin, aggregation and release by human platelets were strongest at physiological concentrations of Ca2+. Rabbit platelet responses differed markedly from those of human platelets because close contact of rabbit platelets in a low-Ca2+ medium did not promote TXA2 formation. Rabbit platelet responses were more strongly inhibited by the lack of added Ca2+ in the medium than the responses of human platelets, possibly because rabbit platelets do not contain releasable Ca2+. In all studies of human platelets in media with low concentrations of Ca2+, the additional contribution to platelet responses of TXA2 formed because of close platelet contact should be considered because TXA2 formation is not usually stimulated in this way at physiological concentrations of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Activation of protein kinase C by the action of 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, in human platelets

Incubation of human washed platelets with 9,11-epithio-11,12-methano-thromboxane A₂ (STA₂), a stable analogue of thromboxane A₂, caused the activation of protein kinase C and myosin light chain (MLC) kinase to the same extents as those induced by thrombin as judged by measuring the phosphorylation of a 40-kilodalton protein and MLC, respectively. However, STA₂ stimulated much less phosphoinositide turnover than thrombin. Furthermore, the doses of STA₂ necessary for protein kinase C activation and phosphoinositide turnover were higher than those necessary for MLC kinase activation, although the doses of thrombin necessary for these three reactions were nearly the same. These results suggest that protein kinase C may be activated at the Ca²⁺ concentrations higher than those required for MLC kinase activation by the action of STA₂, presumably due to the inability of this agonist to produce diacylglycerol in an amount enough to increase the affinity of the enzyme for Ca²⁺.     

Dopaminergic regulation of GABA release from the intact goldfish retina

The rate of release of [³H]GABA from intact goldfish retinas was studied using a modified superfusion technique. Small, significant increases in the rate of GABA release were observed when the retinas were exposed to dopamine (DA) (100–1000 μM); however, when free Ca²⁺ was removed from the medium, the basal rate of GABA release was increased and DA became inhibitory. Forskolin, a non-specific stimulator of adenylate cyclase in intact cells, also inhibited GABA release in the absence of Ca²⁺. There was no significant effect of forskolin in the presence of Ca²⁺; however, (+)-butaclamol, a dopamine antagonist, increased basal GABA release under these conditios. l-glutamic acid (l-Glu) (1–10 mM) causes up to a 10-fold increase in GABA release. In the presence of Ca²⁺, DA did not significantly alter the effects of l-Glu; however, in the absence of Ca²⁺ a significant inhibition of the effects of l-Glu by DA was observed. Forskolin, on the other hand, inhibited the effects of l-Glu both in the presence and absence of Ca²⁺. Finally, EGTA (0.3–1 mM) produced a large release of GABA: this release was inhibited by DA, forskolin, theophylline and 8-bromo cyclic AMP. These results suggest a model wherein DA stimulates Ca²⁺-dependent GABA release from one site and inhibits Ca²⁺-independent GABA release from another site via cyclic AMP-mediated event.     

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.     

Novel Ca currents in mammalian CNS neurons

Akaike, Norio, Hisashi Yamanaka and Mitsutoshi Munakata: Novel Ca²⁺ Currents in Mammalian CNS Neurons. Prog. Neuro-Psychopharmacol. & Biol. Psychiatry. 1992, 16(6): 943–957.

1. 1. Voltage-dependent Ca²⁺ currents (I_Ca) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly dissociated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca²⁺ current (termed ‘TTX-I_Ca’). The TTX-I_Ca showed a heterogeneous distribution, preferentially in the dorsal site of CA1 region.

2. 2. Activation and inactivation processes of the TTX-I_Ca were highly potential-dependent, and the latter was fitted by a double exponential function. The TTX-I_Ca was activated at a threshold potential of about −55 mV and reached full activation at −30 mV. The steady-state inactivation of TTX-I_Ca could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of −72.5 mV.

3. 3. When the peak amplitudes of TTX-I_Ca were plotted as a function of extracellular Ca²⁺ concentration [Ca²⁺]_o), the current amplitude increased linearly without showing any saturation.

4. 4. The ratio of peak amplitude in the individual I-V relationships of Ca²⁺, Sr²⁺ and Ba²⁺ currents assing through the TTX-sensitive Ca²⁺-conducting channel was 1 : 0.33 : 0.05, although the current kinetics were much the same.

5. 5. TTX inhibited the TTX-I_Ca in time- and concentration-dependent manner without affecting the current kinetics. Lignocaine inhibited the TTX-I_Ca in a second in a concentration-dependent manner, with accelerating the inactivation process. The concentrations of half-inhibition (IC₅₀) were 3.5 × 10⁻⁹ M for TTX and 3.6 × 10⁻⁴ M for lignocaine.

6. 6. Scorpion toxin prolonged the inactivation phase of TTX-I_Ca in a time- and concentration-dependent manner. In the toxin-treated neurons, both the slow time constant of inactivation (τ_is) and its functional contribution to the total current increased with increasing the toxin concentration.