Inhibition of platelet secretion of ATP by phalloidin

The involvement of actin in the secretion of ATP by platelets was studied using two stimulants, ADP and A23187, and two actin-mediating reagents, cytochalasin B and phalloidin. The degree of actin polymerization was determined using DNase I. Preincubation of platelets with cytochalasin B suppressed the polymerization of actin and ATP secretion induced by stimulants. In the absence of the stimulant, phalloidin-treated platelets exhibited time-dependent actin polymerization and the maximum level was reached at 5 min. No secretion of ATP was observed. The polymerization was enhanced by phalloidin when the platelets were preincubated for 3 to 5 min with the stimulants, but little ATP was secreted. After a 30-min preincubation, the amount of polymerized actin was lower than that after a 5-min incubation, and no ATP was secreted.     

Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).     

Inhibition of growth of Chlamydia trachomatis by the calcium antagonist verapamil

Treatment of BGM (African Green Monkey kidney) cells with the calcium antagonist Verapamil resulted in a reduced yield of chlamydial infectious particles. The inhibitory effect was concentration-dependent, the maximal effect being achieved at 200 microM-Verapamil, which produced a 99.99% reduction of infectious particle yield. Electron microscopy showed that control Chlamydia trachomatis-infected BGM cells contained typical large inclusions in which most of the particles were elementary bodies, whereas Verapamil-treated infected cells contained small inclusions consisting predominantly of reticulate bodies. The findings indicate a possible therapeutic use of this calcium antagonist as an anti-chlamydial drug.     

Alterations in intracellular calcium homeostasis and platelet aggregation induced by ethanol

The in vitro effects of ethanol on intracellular Ca(2+) homeostasis and tyrosine phosphorylation have been investigated in human platelets in order to clarify the cellular mechanisms underlying its described anti-aggregant effects. Ethanol (1-50 mM) reduced, in a dose-dependent manner, the rate and amplitude of aggregation and attenuated the phosphotyrosine content both induced by 0.1U/ml of the physiological ligand, thrombin. Thrombin-induced Ca(2+) entry to the cytosol was significantly reduced, and capacitative Ca(2+) entry (CCE) significantly altered, by 50 mM ethanol, so that ethanol reduces CCE mediated by depletion of the 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive store but enhances CCE induced by the TBHQ-insensitive pool. In conclusion, we provide considerable evidence that ethanol reduces thrombin-induced aggregation, which is likely a result of a significant inhibition of Ca(2+) entry, as well as a reduction in the activity of protein tyrosine kinases.     

Spontaneous oscillations of intracellular calcium and growth hormone secretion

A novel combination of two single cell assays allowed the simultaneous measurement of intracellular calcium concentration and hormone secretion in normal pituitary cells. [Ca2+]i was recorded using the fluorescent Ca2+ indicator fura-2 and digital imaging microscopy. This technique was combined with a reverse hemolytic plaque assay for growth hormone in order to identify somatotropes and quantitate the amount of hormone released. A dynamic profile of rhythmic calcium oscillations was found in spontaneously secreting somatotropes. Each somatotrope displayed a distinct frequency (one pulse every 5-30 s) and amplitude (range 50-450 nM) generated asynchronously from cell to cell. The amount of growth hormone (GH) released correlated directly with both the frequency and amplitude of calcium oscillations at the level of single GH cells. Furthermore, calcium excursions in somatotropes were rapidly suppressed by either (i) removal of extracellular calcium, (ii) somatostatin (1 mM), or (iii) the calcium channel blockers cobalt (2 mM) and verapamil (100 microM). These observations demonstrate that spontaneous calcium oscillations are characteristic for normal somatotropes. These oscillations are related to spontaneous hormone secretion and due to influx through calcium channels in the membrane. Somatostatin, the physiologic inhibitor of GH secretion, suppresses calcium transients. These findings suggest that the intracellular signaling information may be encoded both in the frequency and amplitude of calcium oscillations.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Inhibition of receptor-stimulated guanylyl cyclase by intracellular calcium ions in Dictyostelium cells.

In Dictyostelium discoideum extracellular cAMP stimulates guanylyl cyclase and phospholipase C; the latter enzyme produces Ins(1,4,5)P3 which releases Ca2+ from internal stores. The following data indicate that intracellular Ca2+ ions inhibit guanylyl cyclase activity. 1) In vitro, Ca2+ inhibits guanylyl cyclase with IC50 = 41 nM Ca2+ and Hill-coefficient of 2.1. 2) Extracellular Ca2+ does not affect basal cGMP levels of intact cells. In electro-permeabilized cells, however, cGMP levels are reduced by 85% within 45 s after addition of 10(-6) M Ca2+ to the medium; halfmaximal reduction occurs at 200 nM extracellular Ca2+. 3) Receptor-stimulated activation of guanylyl cyclase in electro-permeabilized cells is also inhibited by extracellular Ca2+ with half-maximal effect at 200 nM Ca2+. 4) In several mutants an inverse correlation exists between receptor-stimulated Ins(1,4,5)P3 production and cGMP formation. We conclude that receptor-stimulated cytosolic Ca2+ elevation is a negative regulator of receptor-stimulated guanylyl cyclase.     

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.     

Vanadate stimulates rat pancreatic enzyme secretion through the release of calcium from an intracellular store

Orthovanadate accelerates 45Ca efflux and enzyme secretion from the rat pancreas incubated in either control (2.5 mM Ca) or nominally Ca-free buffers. Secretion induced by vanadate does not appear to be mediated by changes in either adenylate cyclase or sodium pump activity. Instead, vanadate appears to act at an intracellular site to cause the release of calcium from the same pool mobilised by acetylcholine. Vanadate action is not inhibited by DIDS. The effect of pH on vanadate action may be accounted for by changes in the distribution of the vanadates. Vanadyl sulphate inhibits secretion evoked by acetylcholine. This suggests that intracellular reduction of vanadate (+5 oxidation state) to the +4 oxidation state may account for an inhibitory component observed during stimulation with vanadate.     

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.     

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.     

Absence of blocking effects on cardiac slow calcium channels by the intracellular calcium antagonist 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene

Extensive pharmacological evidence supports the contention that 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene hydrochloride (pr-MDI) is a calcium antagonist with a predominantly intracellular site of action. On the other hand, electro-physiological evidence points to a possible membrane slow inward calcium channel blocking property of this agent. To gain further insight as to the site of action of pr-MDI, the interactions between the negative inotropic action of this agent and the positive inotropic actions of excess extracellular calcium (which directly penetrates the myocardial cells through the slow calcium channels), isoproterenol (which indirectly augments calcium influx through the slow calcium channels), and ouabain (which enhances calcium influx through membrane calcium entry routes distinct from the slow calcium channels) were investigated in the isolated, electrically drive guinea pig left atrium. Although excess extracellular calcium, isoproterenol, and ouabain reversed the negative inotropic effect of pr-MDI, an analysis of the concentration-response relationships to all three positive inotropic agents in the presence and the absence of pr-MDI demonstrated that this agent did not significantly inhibit the contractile effects of calcium, isoproterenol, or ouabain, at pr-MDI concentrations which exhibit intrinsic negative inotropic effects. It is concluded that pr-MDI does not block the membrane slow inward calcium channel nor other presumptive membrane routes of calcium entry into myocardial cells at concentrations of 10(-4) M or less. At very high concentrations (3 X 10(-4) M) some inhibition of slow channel calcium influx may occur.     

The calcium channel antagonist omega-conotoxin inhibits secretion from peptidergic nerve terminals

The binding of omega-conotoxin to isolated rat neurohypophysial nerve terminals, its effect on the depolarization-induced increase of cytoplasmic Ca2+ and on the potassium and electrically-induced release of vasopressin (AVP) have been studied. The results show that isolated neurosecretory nerve endings have calcium channels with a high affinity for omega-CgTx and that this toxin inhibits neurohormone release at very low concentration (IC50 = 0. 1nM). Although secretion of vasopressin is inhibited to a great extent by the toxin it is shown that a small but significant amount of the depolarization-induced AVP release is insensitive to omega-CgTx and to the dihydropyridine molecule nicardipine.     

Inhibition of orexin-induced wakefulness by pyrilamine,an H1 receptor antagonist

Sleep and Biological Rhythms -     

Type IVB secretion by intracellular pathogens

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. One class, called type IV, are defined as having homology to the conjugal transfer systems of naturally occurring plasmids. It has been proposed that pathogens with type IV secretion systems have acquired and adapted the conjugal transfer systems of plasmids and now use them to export toxins. Several well-characterized intracellular pathogens, including Legionella pneumophila , Coxiella burnetii , Brucella abortus , and Rickettsia prowazekii , contain type IV systems which are known or suspected to be of critical importance in their ability to cause disease. Specifically, these systems are believed to be the key factors determining intracellular fate, and thus the ability to replicate and cause disease.