The exocytosis of human blood platelets. A fast freezing and freeze-substitution analysis

Human platelets were stimulated with thrombin or collagen in order to induce the release of alpha-granules and dense bodies. The platelets were cryofixed in various stages of exocytosis and subsequently cryosubstituted in acetone containing 4% osmium tetroxide. The platelets embedded in araldite were analyzed in serial sections. The initial changes of the alpha-granules were characterized by an impressive swelling and a dispersal of the granular matrix. Swollen alpha-granules in different stages of exocytosis formed contacts. Between the attached membranes of the alpha-granules electron-dense connections were sometimes observed. In a later stage, the membranes formed a pentalaminar structure (apposition), typical for the prefusion state. After apposition, sequential fusion of single alpha-granules took place and fusions of single or of compound granules with the plasmalemma were observed. The formation of a pore on the platelet surface allowed the passage of granular constituents to the exterior. The dense bodies extruded their electron-dense contents in a similar way after fusion with the plasmalemma but, compared with the alpha-granules, after less extensive swelling. These findings suggest that swelling of the secretory organelles plays an important role for granule fusion and platelet exocytosis. There is some evidence that the characteristic "internal contraction" of cytoskeletal structures in stimulated platelets is not the driving force of the platelet release reaction. An involvement of membranes of the surface connected system in the secretory pathway could not be ascertained.     

Alpha-granule secretion from alpha-toxin permeabilized, MgATP-exposed platelets is induced independently by H+ and Ca2+

In order to better understand granule release from platelets, we developed an alpha-toxin permeabilized platelet model to study alpha-granule secretion. Secretion of alpha-granules was analyzed by flow cytometry using P-selectin as a marker for alpha-granule release. P-selectin surface expression occurred when platelets were permeabilized in the presence of Ca2+. Responsiveness to Ca2+ was lost 30 min after permeabilization but could be reconstituted with MgATP. Alpha-toxin-permeabilized, MgATP-exposed platelets also degranulated within a pH range of 5.4-5.9 without exposure to and independent of Ca2+. ATP, GTP, CTP, UTP, and ITP supported Ca2+-induced alpha-granule secretion, while H+-induced alpha-granule secretion occurred only with ATP and GTP. Both Ca2+- and H+-induced alpha-granule secretion required ATP hydrolysis. Kinase inhibitors blocked both Ca2+- and H+-induced secretion. These data suggest that alpha-granule secretion in this permeabilized platelet system shares many characteristics with granule secretion studied in other permeabilized cell models. Furthermore, these results show that H+ can trigger alpha-granule release independent of Ca2+.     

A distinct 2,5-di-(tert-butyl)-1,4-benzohydroquinone-sensitive calcium store in bovine adrenal chromaffin cells

The fluorescent Ca2+ indicator Fura 2 was used to monitor Ca2+ release induced by the Ins(1,4,5)P3-mobilizing agonist angiotensin II (Ag II), caffeine and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tuBHQ), in intact bovine adrenal chromaffin cells. Under low external Ca2+ conditions, tuBHQ, Ag II and caffeine elicited Ca2+ rises, indicating Ca2+ release from internal stores. Prior addition of Ag II had no noticeable effect on the extent of release of Ca2+ induced by tuBHQ. Stimulation of the cells with tuBHQ before either Ag II or caffeine, similarly had no effect on Ca2+ released by these two agonists. It was concluded, therefore, that there is a third intracellular Ca2+ store in bovine adrenal chromaffin cells, distinct and non-overlapping, from those sensitive to caffeine or Ins(1,4,5)P3-mobilizing agonists.     

Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.     

Functioning of the electromechanical connection in the course of the contracture contraction

The effects of calcium release blocker dantrolene was tested on electrically evoked twitches and on contractures induced by potassium depolarization, by acetylcholine or caffeine. It was shown that the first: developmental, stage of potassium or acetylcholine contracture is inhibited by dantrolene and is not influenced by calcium free medium, therefore we may interpret it as based on a "voltage-dependent Ca release" (VDCR) mechanism of activation, whereas depolarization directly opens the rhyanodin receptor calcium channels. On the contrary, the next stage: the long-lasting plateau of contracture, is directly dependent on external Ca2+ and inhibited by dantrolene, and therefore can be described as "calcium induced Ca-release" (CICR) activation mechanism. In this case stored calcium is also released by rhyanodine receptors, although by means of entering the extracellular Ca2+. Finally, the last stage of low amplitude is not influenced by dantrolene nor by calcium-free medium. Therefore the activation of contraction on this stage is not based on the Ca2+ release through the rhyanodin receptor calcium channels.     

The role of Ca2+ and Ca2+-activated phospholipid-dependent protein kinase in degranulation of human neutrophils

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.     

Studies on the Ca2+-induced lysis of platelet alpha-granules

Platelet alpha-granules have been reported to lyse upon addition of submillimolar Ca2+ (J. Van der Meulen and S. Grinstein, J. Biol. Chem. 257, 5190). Similar observations in parotid granules have been attributed to extensive lipid hydrolysis. Experiments were performed to assess the role of lipases and proteases in Ca2+-induced lysis of alpha-granules. No differences were detected between lipids of Ca2+-treated and control granules by two-dimensional thin-layer chromatography. Moreover, several phospholipase inhibitors were without effect on Ca2+-induced lysis. Similarly, the polypeptide patterns of control and treated granules were identical and protease inhibitors failed to prevent lysis. In contrast, lysis could be suppressed by increasing the osmolarity of the medium or by substitution with nonpermeating ions. Lysis was unaffected by quinine, amiloride, furosemide, or tetraethylammonium but was inhibited by 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), a powerful inhibitor of anion transport. The data suggest that Ca2+-induced lysis of alpha-granules does not result from wholesale hydrolysis of either lipids or proteins. Instead, the results are consistent with a Ca2+-mediated change in membrane permeability. In the presence of permeating ions, this leads to entry of salt and osmotically obliged water with consequent swelling and eventual lysis.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Effects of alloxan and streptozotocin on calcium transport in isolated mouse liver mitochondria

The effect of alloxan and streptozotocin on the fluxes of Ca2+ in isolated mouse liver mitochondria was studied with dual wave-length spectrophotometry, using antipyrylazo III as metallochromic indicator. Streptozotocin had no effect on Ca2+ uptake, whereas alloxan inhibited the initial rate and extent of Ca2+ influx in a way dependent on the duration of preincubation, and occurrence of Pi in the reaction mixture. A rapid release of Ca2+ followed upon addition of either FCCP or alloxan after the reaction had been started. When added to preloaded mitochondria, alloxan induced a concentration dependent release of Ca2+. The data suggest that alloxan induces an initial release of mitochondrial Ca2+, which is followed by inhibition of Ca2+ influx. The initial release may be due to uncoupler activity induced by alloxan, and the inhibition of Ca2+ influx may be a consequence of inhibited Pi transport.     

Angiotensin II Effects on the Cytosolic Free Ca2+ Concentration in N1E-115 Neuroblastoma Cells: Kinetic Properties of the Ca2+ Transient Measured in Single Fura-2-Loaded Cells

The effect of angiotensin II on the cytosolic free Ca2+ concentration was measured in single mouse neuroblastoma N1E-115 cells loaded with fura-2. Angiotensin II induced a transient concentration-dependent increase in Ca2+ and also increased the production of inositol polyphosphates. The Ca2+ increase did not require extracellular Ca2+ and was unaffected by pretreatment with pertussis toxin. These data suggest that angiotensin II increased Ca2+ by an inositol trisphosphate-mediated release of intracellular Ca2+ following activation of phospholipase C via a pertussis toxin-insensitive guanine nucleotide binding protein. Similar results were obtained with bradykinin. The angiotensin II- or bradykinin-induced increase in Ca2+ occurred after a concentration-dependent latent period. Low concentrations of agonist elicited a small increase in Ca2+ following a variable lag that sometimes exceeded 1 min, whereas at maximally effective angiotensin II concentrations a larger, more rapid increase in Ca2+ occurred without a measurable delay. In some cells, oscillatory increases in Ca2+ were induced by angiotensin II and bradykinin. Possible mechanisms to explain the concentration dependency of the latent period and the oscillatory nature of the increases of Ca2+ are discussed. These results indicate that the mouse neuroblastoma N1E-115 cell represents a useful model for studying the signal response transduction mechanisms regulating the effects of angiotensin II in neuronal cells.     

Coordination between Ca2+ release and subsequent re-uptake in the sarcoplasmic reticulum

We here report the results of our recent effort to produce, in the isolated sarcoplasmic reticulum (SR), a biphasic Ca2+ release and Ca2+ re-uptake transient and to resolve the kinetic relationship between Ca2+ release and re-uptake of the released Ca2+. Ca2+ release from the SR was induced by polylysine (the ryanodine receptor-specific Ca2+ release trigger) at various levels of calcium loading, or at various doses of the trigger. The changes in the Ca2+ concentration in the reaction solution and in the lumenal Ca2+ concentration were determined by stopped-flow spectroscopy using fluo-3 and mag-fura-2AM, respectively. At higher levels of calcium loading (>150 nmol/mg), polylysine induced monophasic Ca2+ release curves (without an appreciable re-uptake phase) as reported in most studies in the literature. However, lowering the calcium loading level to an intermediate range (100-150 nmol/mg) produced the desired biphasic transient curves consisting of Ca2+ release and Ca2+ re-uptake phases. Under these conditions, the increase in the polylysine concentration resulted in the increase of both the rate of Ca2+ release and that of re-uptake of the released Ca2+. The maximal rate of Ca2+ release and that of re-uptake showed a parallel relationship in the polylysine concentration range of 0-10 microM. This indicates that Ca2+ release from the SR and re-uptake of the released Ca2+ via the SR Ca2+ pump are well-coordinated processes. The changes in the lumenal Ca2+ concentration during the release and re-uptake reaction were monitored at an optimum level of calcium loading while clamping the extravesicular Ca2+ concentration at a constant value. There was again a tight correlation between Ca2+ release (decrease of the lumenal Ca2+ concentration) and re-uptake (increase of the lumenal Ca2+ concentration), indicating that acceleration of the re-uptake is controlled by the rate of decrease of the lumenal Ca2+ concentration. We propose that one of the mechanisms, by which the mode of coordination between the two components of the biphasic Ca2+ transient (viz. Ca2+ release via the ryanodine receptor and Ca2+ re-uptake via the SR Ca2+ pump) is controlled, is the change in the Ca2+ concentration gradient across the SR membrane.     

Sulfhydryl oxidation induces rapid calcium release from sarcoplasmic reticulum vesicles

Micromolar concentrations of cupric ion (Cu2+) and mercaptans such as cysteine, cysteamine, and homocysteine trigger large and rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles. At the concentrations used, Cu2+ alone does not induce Ca2+ release nor does cysteine alone; both are required to induce Ca2+ release from SR. Cu2+ is known to catalyze the autooxidation of cysteine to its disulfide form cystine; Cu2+/mercaptan-induced Ca2+ release appears to be caused by Cu2+-catalyzed formation of a mixed disulfide between the exogenous mercaptan and a critical sulfhydryl on a transmembrane protein. In the oxidized state the SR is highly permeable to Ca2+. Supporting evidence for this interpretation is as follows. The order of Ca2+-releasing reactivity of the mercaptans is the same as the order in which these compounds undergo oxidation to disulfide forms in the presence of Cu2+. Ca2+ efflux induced by cysteine and Cu2+ can be reversed by the addition of the disulfide reducing agent dithiothreitol. Hypochlorous acid and plumbagin, both potential sulfhydryl oxidants, induce rapid Ca2+ efflux from SR vesicles; in addition, Cu2+, which catalyzes H2O2 oxidation of cysteine, enhances H2O2-induced release. Oxidation-induced Ca2+ release from SR can be partially reversed or blocked by ruthenium red or the local anesthetics procaine and tetracaine. The Ca2+ efflux rates are strongly Mg2+ dependent and are significantly higher in heavy SR than in light SR. These data suggest that the Ca2+ efflux thus induced is via the "Ca2+ release channel" and that the oxidation state of a critical sulfhydryl group on this protein may be the principal means by which the Ca2+ permeability of the SR is regulated in vivo.     

Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2'dithiodipyridine (2,2' DTDP) and 4,4' dithiodipyridine (4,4' DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption of thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2(+)-induced Ca2+ release. (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mM Mg2+free for 2,2' DTDP and 4,4' DTDP, respectively. (ii) In the presence of adenine nucleotides (0.1-1 mM), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mM) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner. (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2(+)-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2(+)-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2(+)-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.     

Effects of compound 48/80 on the Ca2+ release by reversal of the Ca2+ pump and by the Ca2+ channel of sarcoplasmic reticulum membranes

The effect of the calmodulin antagonist, compound 48/80, on the Ca2+ release from skeletal muscle sarcoplasmic reticulum was investigated. Both the Ca2+ release by reversal of the Ca2+ pump and the Ca2+ release by the Mg2(+)-controlled Ca2+ channel were studied. It was observed that, when reversal of the pump is inoperative and Mg2+ is not present in the reaction medium, 48/80 stimulates Ca2+ release from the vesicles. In contrast, in the presence of Mg2+, which blocks the Ca2+ channel, 48/80 inhibits Ca2+ release induced by ADP and Pi. This effect is strong at low concentrations of Pi (approximately 1 mM), whereas high concentrations (approximately 15 mM) protect the system against the drug. Furthermore, it was observed that 48/80 has a maximum effect on the channel-mediated Ca2+ release at concentrations of about 20 micrograms/ml, whereas maximal inhibition of the pump-mediated Ca2+ release occurs at concentrations of about 60-80 micrograms/ml. The results indicate that both the Ca2+ channel complex and the Ca2(+)-ATPase may be target systems for the effects of 48/80 on the Ca2+ transport activity of sarcoplasmic reticulum. However, the Ca2+ channel is more sensitive to the drug, suggesting an involvement of calmodulin on this mechanism of Ca2+ release.     

Roles of Ca2+ on the inositol 1,4,5-trisphosphate-induced release of Ca2+ from saponin-permeabilized single cells of the porcine coronary artery

The release of Ca2+ from the intracellular store site, as induced by inositol 1,4,5-trisphosphate, was studied in relation to free Ca2+ concentrations or amounts of stored Ca2+ in smooth muscle cells. The maximal Ca2+ release induced by inositol 1,4,5-trisphosphate was observed when the amount of Ca2+ in the store site was about 50% of the maximal capacity of the Ca2+ storage, and when the extravesicular free Ca2+ concentration was less than 1.5 X 10(-6) M. The Ca2+ release induced by inositol 1,4,5-trisphosphate was accelerated by ATP and 5'-adenylylimidodiphosphate (AMPPNP), but not by ADP and AMP. This inositol 1,4,5-trisphosphate-induced Ca2+ release appeared to be specific for intracellular Ca2+ store sites (mainly sarcoplasmic reticulum), and this Ca2+ release was not apparent in the sarcolemmal fraction.     

Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores

Ram spermatozoa incubated in the presence of Ca2+ and the Ca2+-ionophore A23187 undergo a process which is known as the acrosome reaction. This reaction is characterized by fusion of the outer acrosomal membrane and the overlying plasma membrane to form mixed vesicles which can be seen in the electron microscope. As a result, the trypsin-like acrosin is released from the cells to the medium. The occurrence of the acrosome reaction was determined by following acrosin activity in the medium. After 2 h of incubation of the cells in the presence of ionophore and Ca2+, the released acrosin activity is related to the ionophores according to the sequence: A23187 greater than monensin greater than valinomycin greater than FCCP = without ionophore. The study of Ca2+ uptake by the cells revealed that Ca2+ enters the cell prior to the release of acrosin. Monensin can induce Ca2+ uptake and acrosin release only when Na+ is present in the incubation medium. There is no increase in Ca2+ uptake with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the Na+/H+ exchange induced by monensin causes an increase in intracellular Na which is the driving force for the Ca2+ entry via a Ca2+/Na+ antiporter. Since monensin can induce an increase in Ca2+ uptake only in the presence of Na+, FCCP enhances Ca2+ uptake in the presence of valinomycin, and A23187 is a Ca2+/2H+ exchanger, we suggest that alkalization of the intracellular space is involved in the acrosome reaction. Calcium uptake in the presence of monensin is not affected by the uncoupler FCCP, a result which indicates that Ca2+ is not accumulated in the mitochondria. Incubation of cells for 3 h in the absence of Ca2+ or ionophore caused a 3-fold increase in the rate of acrosin release when monensin and Ca2+ were added together. There was no change in this rate when A23187 was used. We suggest that during the preincubation time (known as capacitation) the permeability of the plasma membrane to Ca2+ is enhanced. This study shows that acrosin release and Ca2+ uptake can be used as a quantitative asay for the determination of the acrosome reaction.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Angiotensin II induces prostaglandin E(2) release in human gingival fibroblasts

We investigated the effect of angiotensin II on prostaglandin E(2) release in human gingival fibroblasts. Stimulation of human gingival fibroblasts with angiotensin II elicited prostaglandin E(2) release in a concentration- and time-dependent manner. Angiotensin III also induced prostaglandin E(2) release, but the effect was weaker than that of angiotensin II. Angiotensin II- and angiotensin III-induced prostaglandin E(2) release was inhibited by AT(1) receptor antagonist FR-130,739, but not AT(2) receptor antagonist PD-123,319. Angiotensin II evoked an increase in intracellular Ca(2+) in fura-2-loaded human gingival fibroblasts. These results suggest that angiotensin II functions as a physiological mediator via Ca(2+)-mobilizing AT(1) receptor activation in human gingival fibroblasts.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.