Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca²⁺-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca²⁺-activated protease similar to myofibrillar Ca²⁺-activated protease has been described in platelets. To explore the role of Ca²⁺-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca²⁺-activated protease-like activity. The hydrolysis of actin-binding protein by Ca²⁺-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca²⁺-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca²⁺-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca²⁺-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E₁, prostaglandin I₂, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca²⁺-activated protease in platelets and indicate that the source of calcium important in Ca²⁺-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca²⁺-activated protease-like activity as measured by the present analytical technique.     

Cross-Talk between Reactive Oxygen Species and Calcium in Living Cells

The results of many investigations have shown that calcium is essential for production of reactive oxygen species (ROS). Elevation of intracellular calcium level is responsible for activation of ROS-generating enzymes and formation of free radicals by the mitochondria respiratory chain. On the other hand, an increase in intracellular calcium concentration may be stimulated by ROS. H₂O₂ has been recently shown to accelerate the overall channel opening process in voltage-dependent calcium channels in plant and animal cells. The 1,4,5-inositol-triphosphate-receptors as well as the ryanodine receptors of sarcoplasmic reticulum have also been demonstrated to be redox-regulated. Activity of Ca²⁺-ATPases and Na²⁺/Ca²⁺ exchangers of animal cells are modulated by the intracellular redox state. Simultaneously, Ca²⁺ may activate antioxidant enzymes, such as plant catalase and glutathione reductase, and increase the level of superoxide dismutase in animal cells. Reviewed data support the speculation that Ca²⁺ and ROS are two cross-talking messengers in various cellular processes.     

cADPR stimulates SERCA activity in Xenopus oocytes

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca²⁺ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca²⁺ following Ca²⁺ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP₃), and by Ca²⁺ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca²⁺ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH₂-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca²⁺ sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca²⁺.     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and induces production of leukotriene C₄ (LTC₄). This model was used to examine the role of Ca²⁺ in LTC₄ formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2×10⁻⁷M), selectively prevented the stimulatory effect of the antigen on LTC₄ production whereas the response to calcium inophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 μg/ml) did not overcome the inhibitory action of HC. An elevated [Ca²⁺]_i is known to be essential for the activation ob both 5-lipoxygenase and phospholipase A₂. The stimulatory effect of the antigen on LTC₄ production was abolished when the cells were incubated in Ca²⁺-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca²⁺. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca²⁺ concentration of 150 μM and 40 μM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca²⁺]_i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of ⁴⁵Ca²⁺ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca²⁺]_i, and this may be associated with the inhibitory action of HC on LTC₄ formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Effects of sphingosine-1-phosphate on pacemaker activity of interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P_1,3,4,5 agonist) and SEW 2871 (an S1P₁ agonist) had no effects on pacemaker activity. Suramin (an S1P₃ antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P₂ antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P₂ in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH₂-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca²⁺ ([Ca²⁺]_i) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca²⁺]_i oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P₂ via regulation of external and internal Ca²⁺ and mitogenactivated protein kinase activation.     

The Ca2+ influx induced by β-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation

Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca²⁺]_i elevations and electrical activity were enhanced by removal of extracellular Mg²⁺, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca²⁺ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca²⁺]_i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca²⁺]_i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca²⁺ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.     

Mechanism underlying the block of human Cav3.2 T-type Ca2+ channels by benidipine, a dihydropyridine Ca2+ channel blocker

AimsBenidipine, a dihydropyridine Ca²⁺ channel blocker, has been reported to block T-type Ca²⁺ channels; however, the mechanism underlying this effect was unclear. In this study, we characterized the mechanism responsible for this blocking activity. Furthermore, the blocking activity was compared between two enantiomers of benidipine, (S, S)- and (R, R)-benidipine.Main methodsHuman Ca_v3.2 (hCa_v3.2) T-type Ca²⁺ channels stably expressed in the human embryonic kidney cell line, HEK-293, were studied in whole-cell patch-clamp recordings and Ca²⁺ mobilization assay.Key findingsIn whole-cell patch-clamp recordings, benidipine blocked hCa_v3.2 T-type Ca²⁺ currents elicited by depolarization to a comparable extent as efonidipine. The block was dependent on stimulation frequency and holding potential, but not test potential. Benidipine significantly shifted the steady-state inactivation curve to the hyperpolarizing direction, but had no effect on the activation curve. Benidipine prolonged the recovery from inactivation of hCa_v3.2 T-type Ca²⁺ channels without any effect on the kinetics of activation, inactivation, or deactivation. In the Ca²⁺ mobilization assay, benidipine was more potent than efonidipine in blocking Ca²⁺ influx through hCa_v3.2 T-type Ca²⁺ channels. (S, S)-Benidipine was more potent than (R, R)-benidipine in blocking hCa_v3.2 T-type Ca²⁺ currents, but there was no difference in blocking the Ca²⁺ influx.SignificanceWe have characterized the blocking activity of benidipine against hCa_v3.2 Ca²⁺ channels and revealed the difference between the two enantiomers of benidipine. The blocking action of benidipine could be mediated by stabilizing hCa_v3.2 Ca²⁺ channels in an inactivated state.     

Regulation of ovarian ornithine decarboxylase: Role of calcium ions in enzyme induction in isolated swine granulosa cells in vitro

When swine granulosa cells were cultured in chemically defined medium selectively deficient in Ca²⁺, the dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in response to prostaglandin E₂, l-epinephrine or the somatomedin, multiplication-stimulating activity, was attenuated markedly. Putative calcium influx blockers, verapamil and diltiazem, also inhibited hormone-stimulated enzymic activity. Similar inhibitory effects were exerted by divalent (cobalt) or trivalent (lanthanum) cations believed to compete with calcium for extracellular binding sites. The suppressive effects of extracellular calcium deprivation were time-dependent (suggesting gradual depletion of intracellular calcium stores), and could be mimicked by the intracellular antagonist of calcium action, trifluoperazine. The mechanism(s) subserving diminished hormonal induction of enzyme activity could not be accounted for by alterations in cell viability, general protein synthesis, half-life of decay of enzyme activity (measured in the presence of cycloheximide), or apparent K_m of ornithine decarboxylase. Ca²⁺ and/or calcium antagonists did not modify enzyme activity in cell-free preparations. These observations implicate Ca²⁺ in the hormonal induction of a discrete cytosolic enzyme in isolated intact ovarian cells.     

Prostaglandin synthetase activity of goat vesicular microsomes: Co-factor requirement and effect of calcium ions

The effects of several co-factors and bivalent cations on the activity of prostaglandin synthetase isolated from goat seminal vesicles were studied. Ca²⁺ appears to play a regulatory role in the biosynthesis of prostaglandin E₂ by goat vesicular microsomes as the normal parabolic time course of synthesis changed to a sigmoid curve in the presence of 4 mM Ca²⁺ and to nearly a hyperbolic pattern when the microsomes were preincubated with the metal ions. The Ca²⁺ modulated reaction showed increased rate of prostaglandin E₂ synthesis only when the period of incubation was extended beyond 30 min. The co-factor requirement of the goat enzyme was similar to that of the bovine and ovine prostaglandin synthetase systems.     

Receptor-Induced Diacylglycerol Formation in Permeabilized Platelets; Possible Role for a Gtp-Binding Protein

Abstract

Exposure of human platelets to 10 discharges from a 4.5 μF capacitor charged at 3 kV permitted isolation of a stable preparation of permeabilized platelets that, after equilibration with Ca²⁺ buffers (pCa < 6) for 15 min at 0°C, secreted 5-hydroxytryptamine (5-HT) at 25°C. Thrombin enhanced the sensitivity to Ca²⁺ of the secretion of 5-HT by about 10-fold, whereas Arg -vasopressin and the prostaglandin endoperoxide analogue, U-46619, increased sensitivity to Ca²⁺ by 3 to 4-fold. This action of thrombin was associated with stimulation of diacylglycerol formation, a marked increase in phosphorylation of protein P47 and a smaller increase in phosphorylation of the P-light chain of myosin. Thrombin exerted these effects at a [Ca²⁺ _free] of 0.1 μM, suggesting that the receptor-activated breakdown of platelet phosphoinositides to diacylglycerol may not require prior Ca²⁺ mobilization in intact platelets. In both the presence and absence of thrombin, a higher [Ca²⁺ _free] was required for optimal secretion than for maximal phosphorylation of P47 and myosin light-chain, indicating that Ca²⁺ and possibly diacylglycerol have roles in the secretory mechanism additional to activation of the enzymes that phosphorylate these proteins. Stable GTP analogues such as guanosine-5′-0-(3-thiotriphosphate) (GTPγS), and to a lesser extent GTP itself, enhanced the Ca²⁺ sensitivity of the secretion of 5-HT from permeabilized platelets. Moreover, GTP potentiated the stimulatory action of thrombin. These effects of GTPγS and GTP were associated with increased diacylglycerol formation and were inhibited by guanosine-5′-0-(2-thiodiphosphate) (GDPβS) suggesting that a GTP-binding protein may play a role in the receptor-activated breakdown of phosphoinositides. However, as GDPβS did not inhibit the potentiation of secretion caused by thrombin alone, a GTP-independent pathway of platelet activation may also exist.     

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the Gq/PLC/Ca pathway in a lipid raft-dependent manner

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G_i/o-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca²⁺-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP₃ formation and intracellular Ca²⁺ mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca²⁺ ionophore and the activation of a typical G_q-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G_q/phospholipase C activation and intracellular Ca²⁺ mobilization in a lipid raft microdomain-dependent manner.     

Functional Expression of T-Type Ca2+ Channels in Spinal Motoneurons of the Adult Turtle

Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising three subfamilies named Ca_V1, Ca_V2 and Ca_V3. The Ca_V3 channel subfamily groups the low-voltage activated Ca²⁺ channels (LVA or T-type) a significant role in regulating neuronal excitability. Ca_V3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, Ca_V3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of Ca_V3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni²⁺ and NNC55-0396, two antagonists of Ca_V3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the Ca_V3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of Ca_V3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.     

Elevations of intracellular calcium reflect normal voltage-dependent behavior,and not constitutive activity,of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca²⁺ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca²⁺ influx and determining the bulk average cytoplasmic Ca²⁺ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca²⁺ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca²⁺ current (I_Ca) and evoked a rise in [Ca²⁺] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca²⁺] increase ([Ca²⁺]_c) was approximately uniform, whereas that of the subplasma membrane space ([Ca²⁺]_PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca²⁺ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca²⁺ channels were not normally constitutively active. The repetitive localized [Ca²⁺]_PM rises (“persistent Ca²⁺ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca²⁺ channels regulate the bulk average [Ca²⁺]_c. A dihydropyridine blocker of Ca²⁺ channels, nimodipine, which blocked I_Ca and accompanying [Ca²⁺]_c rise, reduced neither the resting bulk average [Ca²⁺]_c (at −70 mV) nor the rise in [Ca²⁺]_c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca²⁺ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca²⁺]_c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca²⁺].     

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252–257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca²⁺ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca²⁺-ATP on the association of the ATPase inhibitor peptide to F₁-ATPase. This action of Ca²⁺ may explain why mitochondria utilize respiratory energy for the transport of Ca²⁺ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.     

cAMP and Ca signaling in secretory epithelia: Crosstalk and synergism

The Ca²⁺ and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca²⁺ and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca²⁺ and cAMP signaling operate at 5–10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca²⁺ and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca²⁺ and cAMP signaling and the function of IRBIT (IP₃ receptors binding protein release with IP₃) as a third messenger that mediates the synergistic action of the Ca²⁺ and cAMP signaling.     

Quantitative aspects of drug-receptor interactions. I. Ca2+ and cholinergic receptor activation in smooth muscle: a basic model for drug-receptor interactions

An attempt has been made to devise a general model of drug-receptor interactions as it relates to the initiation of mechanical responses. A key feature of this model is the regulatory role played by membrane-bound Ca²⁺ (Ca_mem²⁺).The effects on the mechanical responsiveness of guinea pig ileal longitudinal muscle of four muscarinic agonists derived from and including the highly active cis-2-methyl-4-dimethylaminomethyl-1, 3-dioxolane methiodide have been studied. The concentration-response (isotonic contraction) curves of these four agonists at normal Ca_ext²⁺-levels show evidence of cooperativity (n_H > 1) and this was found to increase dramatically with decreasing [Ca_ext²⁺]. A three step model has been proposed, based on that previously advanced by Hurwitz &; Suria (1971), in which activation of the acetylcholine receptor initiates a Ca²⁺ translocation mechanism supplying the contractile machinery with Ca²⁺. Arguments are advanced to suggest that two sources of Ca²⁺ are thus utilized: membrane-bound (Ca_mem²⁺) and free extracellular (Ca_ext²⁺), the former being responsible for the initial phasic contraction and the latter for the slower phase of contractile development.Analysis of the theoretical model shows that the cooperativity of the concentration-response relationships derives not from the initial agonist-receptor interaction but from the subsequently initiated Ca²⁺ translocation step so that [Ca_int²⁺] ∝ [Ca_ext²⁺]ⁿ. The limiting value of n is found to be 6 and to be the same for agonists and partial agonists. According to this model intrinsic activity is determined by the linkage between the agonist-receptor complex and the Ca²⁺ translocation process.The general findings of this work are discussed in terms of an equilibrium between Ca²⁺-associated and Ca²⁺-dissociated membrane states. The similarities to other Ca²⁺ dependent processes are emphasized.     

Evidence that cyclic AMP may regulate Ca2+-mobilization and phospholipases in thrombin-stimulated human platelets

The regulation of human platelet responses by cyclic AMP (cAMP) has been investigated by measuring thrombin-stimulated serotonin release, Ca²⁺ uptake and phospholipase activity. Thrombin-induced 1,2-diacylglycerol (DG) formation as a result of phospholipase C activation was inhibited by pretreatment with dibutyryl cAMP (dbcAMP) in a dose-dependent manner. Subsequent failure to produce phosphatidic acid (PA), which is converted from 1,2-DG by phosphorylation and would serve as intracellular Ca²⁺ ionophore, appeared to parallel the decrease in Ca²⁺ uptake activity. Phospholipase A₂ activity, monitored by the production of [³H]lysophosphatidylcholine and [³H]lysophosphatidylethanolamine, was also suppressed by dbcAMP. These data indicate that the intracellular cAMP level may be closely associated with Ca²⁺ uptake and phospholipases activation. In addition, it is suggested that alteration of intracellular cAMP regulates phospholipase activation and consequently platelet responses, perhaps by controlling available Ca²⁺ content.     

Platelet calcium pump activity in essential hypertensives and their first-degree relatives

Intracellular free Ca²⁺ concentration has been shown to be elevated in platelets of patients with essential hypertension. This study was designed to characterize Ca²⁺-pump activity of the platelet membranes (surface and intracellular) in these patients. A double-blind study was carried out. Untreated and treated (on R-blockers) essential hypertensives were studied in comparison with normotensive control subjects. First degree blood relatives of essential hypertensives were also studied. The Ca²⁺-activation kinetics of the enzyme showed a significant decrease in the V_max. (for the plasma- and intracellular membranes) and Km (for the intracellular membranes) in the essential hypertensive patients. Increased platelet membrane cholesterol content was observed in these patients. Lowered Ca²⁺-efflux by Ca²⁺-ATPase may lead to elevated intracellular free Ca²⁺-levels in platelets of essential hypertensives. A lowered Ca²⁺-ATPase activity may emerge as a marker for essential hypertension.     

Second-messenger-induced signalling events in pollen tubes of Papaver rhoeas

A role for cytosolic free Ca²⁺ (Ca²⁺_i) in the regulation of growth of Papaver rhoeas pollen tubes during the self-incompatibility response has recently been demonstrated [Franklin-Tong et al. Plant J. 4:163–177 (1993); Franklin-Tong et al. Plant J. 8:299–307 (1995); Franklin-Tong et al. submitted to Plant J.]. We have investigated the possibility that Ca²⁺_i is more generally involved in the regulation of pollen tube growth using confocal laser scanning microscopy (CLSM). Data obtained using Ca²⁺ imaging, in conjunction with photolytic release of caged inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], point to a central role of the phosphoinositide signal transduction pathway in the control of Ca²⁺ fluxes and control of pollen tube growth. These experiments further revealed that increases in cytosolic levels of Ins(1,4,5)P₃ resulted in the formation of distinct Ca²⁺ waves. Experiments using the pharmacological agents heparin, neomycin and mastoparan further indicated that Ca²⁺ waves are propagated, at least in part, by Ins(1,4,5)P₃-induced Ca²⁺ release rather than by simple diffusion or by “classic” Ca²⁺-induced Ca²⁺ release mechanisms. We also have data which suggest that Ca²⁺ waves and oscillations may be induced by photolytic release of caged Ca²⁺. Ratio-imaging has enabled us to identify an apical oscillating Ca²⁺ gradient in growing pollen tubes, which may regulate normal pollen tube growth. We also present evidence for the involvement of Ca²⁺ waves in mediating the self-incompatibility response. Our data suggest that changes in Ca²⁺_i and alterations in growth rate/patterns are likely to be closely correlated and may be causally linked to events such as Ca²⁺-induced, or Ins(1,4,5)P₃-induced wave formation and apical Ca²⁺ oscillations.Presented at the 1997 SEB Annual Meeting: Interactive MultiMedia Biology - Experimental Biology Online Symposium, Canterbury, 7-11 April     

Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca²⁺ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca²⁺]_i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca²⁺]_i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca²⁺ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca²⁺]_i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca²⁺]_i may be attributed to a release of Ca²⁺ from intracellular stores. Quisqualate (QA) stimulates Ca²⁺ release from an intracellular store that is independent of Ca²⁺ influx; presumably this store is activated by inositol phosphates. The increase in [Ca²⁺]_i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca²⁺ release from internal stores. Only one of the latter stores is dependent on Ca²⁺ influx with regard to release of Ca²⁺, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca²⁺ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca²⁺ pool(s). By using this drug it has been possible to show to what extent these Ca²⁺ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca²⁺ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca²⁺ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.