Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP.

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).     

Small GTPase Rab4 regulates Ca2+-induced alpha-granule secretion in platelets

Upon activation, platelets release many active substances stored in alpha- and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca(2+)-induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca(2+)-induced secretion of von Willebrand factor (vWF) stored in alpha-granules, but not that of [(3)H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 co-fractionated with vWF and P-selectin (an alpha-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [(3)H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing alpha- and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca(2+)-induced exocytosis of alpha-granules.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Ca2+-dependent synaptotagmin binding to SNAP-25 is essential for Ca2+-triggered exocytosis

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.     

Ca2+ homeostasis in unstimulated platelets

Unstimulated platelets maintain a low cytosolic free Ca2+ concentration and a steep plasma membrane Ca2+ gradient. The mechanisms that are required have not been completely defined. In the present studies, 45Ca2+ was used to examine the kinetics of Ca2+ exchange in intact unstimulated platelets. Quin2 was used to measure the cytosolic free Ca2+ concentration. Under steady-state conditions, the maximum rate of Ca2+ exchange across the platelet plasma membrane, 2 pmol/10(8) platelets/min, was observed at extracellular free Ca2+ concentrations 20-fold less than in plasma. Two intracellular exchangeable Ca2+ pools were identified. The size of the more rapidly exchanging pool (t 1/2, 17 min) and the cytosolic free Ca2+ concentration were relatively unaffected by large changes in the extracellular Ca2+ concentration. In contrast, the size of the more slowly exchanging Ca2+ pool (t 1/2, 300 min) varied with the extracellular Ca2+ concentration, which suggests that it is physically as well as kinetically distinct from the rapidly exchangeable Ca2+ pool. The locations of the Ca2+ pools were determined by differential permeabilization of 45Ca2+-loaded platelets with digitonin. 45Ca2+ in the rapidly exchanging pool was released with lactate dehydrogenase, which suggests that it is located in the cytosol. 45Ca2+ in the slowly exchanging pool was released with markers for both the dense tubular system and mitochondria, but inhibition of mitochondrial Ca2+ uptake with carbonyl cyanide m-chlorophenylhydrazone had no effect on the size of the slowly exchangeable Ca2+ pool or the cytosolic free Ca2+ concentration. In contrast, addition of metabolic inhibitors (KCN plus carbonyl cyanide m-chlorophenylhydrazone plus deoxyglucose) or trifluoperazine caused a decrease in the size of the slowly exchangeable Ca2+ pool and an increase in the cytosolic free Ca2+ concentration. These observations suggest that Ca2+ homeostasis in unstimulated platelets is maintained by limiting Ca2+ influx from plasma, actively promoting Ca2+ efflux, and sequestering Ca2+ within an internal site, which is most likely the dense tubular system and not mitochondria.     

Structural transitions in the synaptic SNARE complex during Ca2+-triggered exocytosis

The synaptic SNARE complex is a highly stable four-helix bundle that links the vesicle and plasma membranes and plays an essential role in the Ca(2+)-triggered release of neurotransmitters and hormones. An understanding has yet to be achieved of how this complex assembles and undergoes structural transitions during exocytosis. To investigate this question, we have mutated residues within the hydrophobic core of the SNARE complex along the entire length of all four chains and examined the consequences using amperometry to measure fusion pore opening and dilation. Mutations throughout the SNARE complex reduced two distinct rate processes before fusion pore opening to different degrees. These results suggest that two distinct, fully assembled conformations of the SNARE complex drive transitions leading to open fusion pores. In contrast, a smaller number of mutations that were scattered through the SNARE complex but were somewhat concentrated in the membrane-distal half stabilized open fusion pores. These results suggest that a structural transition within a partially disassembled complex drives the dilation of open fusion pores. The dependence of these three rate processes on position within the SNARE complex does not support vectorial SNARE complex zipping during exocytosis.     

Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+-triggered neurotransmitter release

Synaptotagmin 2 resembles synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among synaptotagmins 1 and 2. Our data suggest that synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of synaptotagmins in controlling 'release slots' for synaptic vesicles at the active zone.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes.

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca2+-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca2+-influx. With purified lumisomes it has been shown that Ca2+-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with 22Na and 42D indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Cofilin activation during Ca(2+)-triggered secretion from adrenal chromaffin cells

Cofilin is one of the major actin depolymerizing proteins in eukaryotic cells and involved in many membrane modulating activities, such as cell growth and motility. Here we examined whether cofilin is activated upon Ca(2+) regulated noradrenalin secretion from bovine adrenal chromaffin cells. We found that triggering exocytosis by nicotine causes a dephosphorylation and thereby activation of cofilin. Furthermore, in permeabilized chromaffin cells the addition of Ca(2+) alone is sufficient to trigger both, regulated exocytosis and cofilin activation. This is consistent with cofilin activation being required for actin reorganization during exocytosis.     

Ca2+-triggered simultaneous membrane penetration of the tandem C2-domains of synaptotagmin I

Synaptotagmin I (syt), a transmembrane protein localized to secretory vesicles, functions as a Ca2+ sensor that facilitates SNARE-mediated membrane fusion. The cytoplasmic domain of syt harbors two C2-domains designated C2A and C2B. Upon binding Ca2+, C2A and C2B partially penetrate into membranes that contain anionic phospholipids. However, it is unknown whether these tandem C2-domains engage membranes at the same time, in a sequential manner, or in a mutually exclusive manner. We have used site-directed fluorescent probes to monitor the penetration of syt's C2-domains into phosphatidylserine-harboring lipid bilayers. We report that, in response to Ca2+, C2A and C2B copenetrate into these bilayers with diffusion-limited kinetics. Membrane penetration was more efficient when synthetic rather than natural phospholipids were used to prepare bilayers. The membrane penetration activity of the intact cytoplasmic domain of syt (C2A-C2B) exhibits significant resistance to changes in ionic strength. In contrast, the ability of isolated C2B to bind membranes in response to Ca2+ can be disrupted by subtle changes in ionic strength. Tethering C2B to a mutant version of C2A that does not bind Ca2+ or membranes significantly increases the stability of Ca2+.C2B.membrane complexes, confirming that C2A affects the membrane-binding properties of the adjacent C2B domain.     

Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease

Chicken gizzard smooth muscle contains large amounts of Ca2+-activated protease activity. Approximately 15 mg of purified enzyme can be obtained from 1 kg of fresh muscle. The enzyme consists of two subunits (Mr = 80,000 and 30,000) present in a 1:1 molar ratio. In the presence of CaCl2, the 80,000/30,000-dalton heterodimer (form I) is rapidly converted by limited autolysis to a 76,000/18,000-dalton species (form II). Both the 80,000- and 30,000-dalton subunits are degraded simultaneously. Moreover, the Ca2+ dependence for autolysis (K0.5 = 300 microM) is identical for both subunits. Neither the time course nor the Ca2+ dependence of the autolytic conversion reaction is altered by 10- and 20-fold molar excesses of substrate. Limited autolysis markedly reduces the Ca2+ requirement for substrate degradation. Using N-[ethyl-2-3H]maleimide-labeled 27,000-dalton cardiac myosin light chains as substrate, the Ca2+ requirement of form I was found to be quite high (K0.5 = 150 microM). Under similar conditions, the Ca2+ requirement of form II was 30-fold lower (K0.5 = 5 microM). Limited autolysis did not alter the specific activity of the enzyme. Our results demonstrate that smooth muscle contains an abundant amount of Ca2+-activated protease. Moreover, autolysis of this enzyme may play an important regulatory role by converting the native form to a species that is fully active at physiological levels of intracellular calcium ion.     

Trifluoperazine and the rapid, Ca2+-triggered damage of skeletal and cardiac muscle

[Ca2+]i was raised experimentally in mammalian and amphibian skeletal and cardiac muscles by A23187, DNP, anoxia or the Ca2+ -paradox. Trifluoperazine (TFP) at 10(-5) M failed to protect against the characteristic and rapid damage triggered by elevated [Ca2+]i in any of the preparations. It is concluded that calmodulin is not implicated in this rapid ultrastructural damage. TFP alone also causes identical patterns of damage. It may be acting to raise [Ca2+]i in skeletal and cardiac muscle cells.     

Characteristics of collagen-induced Ca2+ mobilization in bovine platelets

We characterized the collagen-induced increase in cytosolic Ca2+ ([Ca2+]i) of bovine platelets loaded with the Ca2+ indicator Fura-PE3/AM. Collagen (10 micrograms/ml)-induced increase in [Ca2+]i was only partially inhibited by aspirin, a cyclooxygenase inhibitor, or adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS, a P2Y1 receptor antagonist), while in human platelets it was almost completely suppressed by aspirin. Collagen-induced increase in [Ca2+]i of bovine platelets was inhibited by U73122 (0.3-5 microM), a phospholipase C inhibitor. Collagen (10 micrograms/ml) increased production of inositol 1,4,5-trisphosphate, which was prevented by pretreatment with U73122 (5 microM). Collagen (10 micrograms/ml) accelerated Mn2+ entry, since the rate of Fura-PE3 quenching by Mn2+ was enhanced by 13-fold following stimulation with collagen. U73122 inhibited the acceleration of Mn2+ entry induced by collagen. PGE1 (2.5 microM) partially inhibited the collagen (50 micrograms/ml)-induced increase in [Ca2+]i in bovine platelets but not in human platelets. The data suggest that collagen-induced Ca2+ mobilization in bovine platelets is mediated by phospholipase C. The Ca2+ mobilization in bovine platelets is different from that in human ones as to the dependency on arachidonic acid metabolites and sensitivity to PGE1.     

Cyclopiazonic acid reduces the coupling factor of the Ca2+-ATPase acting on Ca2+ binding

The mycotoxin cyclopiazonic acid (CPA) is a potent inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. The compound decreases the affinity of the Ca2+-ATPase for Ca2+ and reduces the maximum specific activity of the enzyme. Furthermore, CPA abolishes the cooperativity of Ca2+ transport, showing a Ca2+/ATP ratio approximately 1 at any extent of Ca2+ saturation. There is also an effect on the Ca2+-binding mechanism, where the addition of CPA results in binding of only half-maximal amount of Ca2+ observed in its absence. The experimental data suggest that in the presence of CPA, only a single Ca2+ ion binds to the Ca2+-ATPase.     

Alkalinization stimulates the purified plasma-membrane Ca2+ pump by increasing its Ca2+ affinity.

The finding that negatively charged phospholipids activate the plasma-membrane (Ca2+ + Mg2+)-ATPase and that polycations counteract this stimulation suggest that negative charges in the environment of the ATPase protein could be important for its function. The aim of the present work was to investigate whether changing the charges on the ATPase protein itself by modifying the pH within the physiological range affects the activity of the purified plasma-membrane Ca2+ pump from stomach smooth muscle. Increasing the pH from 6.9 to 7.4 and using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) as a Ca2+ buffer, doubled the ATPase activity at 0.3 microM-Ca2+ in the presence of 100% phosphatidylcholine (PC) or after substituting 20% of the PC by negatively charged phospholipids PtdIns, PtdIns4P, phosphatidylserine and phosphatidic acid. This stimulatory effect was due to an increased affinity of the enzyme for Ca2+, while the Vmax. remained unaffected. In the case of PtdIns(4,5)P2, a stimulatory effect upon alkalinization was only observed at a PtdIns(4,5)P2 concentration of 10%. When a concentration of 20% was used, alkalinization decreased the Vmax. and no stimulatory effect on the ATPase at 0.3 microM-Ca2+ could be observed. Alkalinization not only stimulated the purified Ca2+ pump, but it also increased the activity of the enzyme in a plasma-membrane-enriched fraction from stomach smooth muscle by a factor of 2.06. The ionophore A23187-induced Ca2+ uptake in closed inside-out vesicles also increased by a factor of 2.54 if the pH was changed from 6.9 to 7.4. This finding indicates that the effect of pH is most likely to be exerted at the cytoplasmic site of the Ca2+ pump protein.     

The dopamine-D2-receptor agonist ropinirole dose-dependently blocks the Ca2+-triggered permeability transition of mitochondria

Ropinirole, an agonist of the post-synaptic dopamine D2-receptor, exerts neuroprotective activity. The mechanism is still under discussion. Assuming that this neuroprotection might be associated with inhibition of the apoptotic cascade underlying cell death, we examined a possible effect of ropinirole on the permeability transition pore (mtPTP) in the mitochondrial inner membrane. Using isolated rat liver mitochondria, the effect of ropinirole was studied on Ca²⁺-triggered large amplitude swelling, membrane depolarization and cytochrome c release. In addition, the effect of ropinirole on oxidation of added, membrane-impermeable NADH was investigated. The results revealed doubtlessly, that ropinirole can inhibit permeability transition. In patch-clamp experiments on mitoplasts, we show directly that ropinirole interacts with the mtPTP. Thus, ropinirole reversibly inhibits the opening of mtPTP with an IC₅₀ of 3.4 µM and a Hill coefficient of 1.3. In both systems (i.e. energized mitochondria and mitoplasts) the inhibitory effect on permeability transition was attenuated by increasing concentrations of inorganic phosphate. In addition, we showed with antimycin A-treated mitochondria that ropinirole failed to suppress respiratory chain-linked reactive oxygen species release. In conclusion, our data suggest that the neuroprotective activity of ropinirole is due to the blockade of the Ca²⁺-triggered permeability transition.