Elevation of intracellular calcium levels contributes to the inhibition of nitric oxide production by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) attenuates LPS-induced inducible nitric oxide synthase (iNOS) expression in murine macrophages by destabilizing iNOS mRNA. Because elevated intracellular free Ca2+ levels [Ca2+]i reduce iNOS mRNA stability, the aim of the present study was to determine whether inhibition of iNOS by ANP is due to alterations in intracellular calcium. As determined by fluorescence photometry, ANP (10(-7) and 10(-6) mol/L) was shown to elevate intracellular calcium levels in bone marrow-derived macrophages. This effect seemed to be mediated via the guanylate cyclase-coupled A receptor, because dibutyryl-cGMP mimicked and the A-receptor antagonist HS-142-1 partially abrogated the effect of ANP. Because the Ca2+ increase was also observed in Ca2+-free buffer, it is suggested that the liberation of intracellular calcium pools contributes to the elevation of [Ca2+]i by ANP. The B-receptor ligand C-type natriuretic peptide (CNP), which does not alter iNOS expression, had no effect on [Ca2+]i. The Ca2+-ionophore 4-Br-A23187 and thapsigargin, a compound known to liberate Ca2+ from intracellular stores, were further demonstrated to reduce LPS-induced NO production in macrophages (Griess assay), confirming a functional link for elevated [Ca2+]i and iNOS inhibition. These effects were abrogated by coincubation with extra- as well as intracellular Ca2+ chelators (EGTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)). The inhibitory effect of ANP on NO production was also abrogated by Ca2+ chelation. These findings support a causal relationship between reduced iNOS induction and elevation of [Ca2+]i. Taken together, the data indicate that intracellular Ca2+ elevation by ANP is involved in the inhibition of LPS-induced nitric oxide production in macrophages.     

Synthesis of the mitogenic S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylpentapeptide from Escherichia coli lipoprotein

The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid. The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids. S-[2,3-Bis(palmitoyloxy)propyl]-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid. The compounds were characterized unequivocally by 13C-NMR and mass spectra. The diastereomers of S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column.     

Cl-]i modulation of Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells of guinea pig

The effects of intracellular Cl- concentration ([Cl-]i) on acetylcholine (ACh)-stimulated exocytosis were studied in guinea pig antral mucous cells by video microscopy. ACh activated Ca2+-regulated exocytosis (an initial phase followed by a sustained phase). Bumetanide (20 microM) or a Cl- -free (NO3-) solution enhanced it; in contrast, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, a Cl- channel blocker) decreased it and eliminated the enhancement induced by bumetanide or NO3- solution. ACh and Ca2+ dose-response studies demonstrated that NO3- solution does not shift their dose-response curves, and ATP depletion studies by dinitrophenol or anoxia demonstrated that exposure of NO3- solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3- solution after ATP depletion induced only a sustained phase. Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that bumetanide and NO3- solution enhanced the ACh-stimulated [Ca2+]i increase. Measurements of [Cl-]i revealed that ACh decreases [Cl-]i and that bumetanide and NO3- solution decreased [Cl-]i and enhanced the ACh-evoked [Cl-]i decrease; in contrast, NPPB increased [Cl-]i and inhibited the [Cl-]i decrease induced by ACh, bumetanide, or NO3- solution. These suggest that [Cl-]i modulates [Ca2+]i increase and ATP-dependent priming. In conclusion, a decrease in [Cl-]i accelerates ATP-dependent priming and [Ca2+]i increase, which enhance Ca2+-regulated exocytosis in ACh-stimulated antral mucous cells.     

A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium.

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.     

Modifications of Ca2+ mobilization and noradrenaline release by S-nitroso-cysteine in PC12 cells

The effects of nitrogen monoxide (NO)-related compounds on cytosolic free Ca2+ concentrations ([Ca2+]i) and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. The addition of S-nitroso-cysteine (SNC) stimulated [Ca2+]i increases from an intracellular Ca2+ pool continuously in a concentration-dependent manner. Other NO donors, which stimulate cyclic GMP accumulation, did not cause [Ca2+]i increases. After treatment with 0.2 mM SNC, transient increases in [Ca2+]i from the Ca2+ pool induced by caffeine were completely abolished. The addition of N-ethylmaleimide (NEM) caused sustained [Ca2+]i increases from the intracellular Ca2+ pool. Furthermore, caffeine did not stimulate further [Ca2+]i increases in PC12 cells pretreated with NEM. These findings suggest that SNC and NEM predominantly interact with a caffeine-sensitive Ca2+ pool. The addition of dithiothreitol (DTT) to 0.4 mM SNC-stimulated cells reduced [Ca2+]i to basal levels, and the addition of DTT to NEM-stimulated cells locked [Ca2+]i at high levels. The stimulatory effects of SNC but not NEM were not abolished by pretreatment with DTT. These findings suggest that modification of the oxidation status of the sulfhydryl groups on the caffeine-sensitive receptors by SNC or NEM regulates Ca2+ channel activity in a reversible manner. SNC did not stimulate NA release by itself but did inhibit ionomycin-stimulated NA release. In contrast, NEM stimulated NA release in the absence of extracellular CaCl2 and further enhanced ionomycin-stimulated NA release. Ca2+ mobilization by SNC from the caffeine-sensitive pool was not a sufficient factor, and other factors stimulating NA release may be negatively regulated by SNC.     

Ca2+-sensitivity and cGMP-independent effects of NO in vascular smooth muscle.

The effects of NO on Ca2+-sensitivity of vascular smooth muscle (VSM) myofilaments have been the focus of this study. Simultaneous measurements of [Ca2+]i and force were carried out in rat tail artery segments. NO, 10(-7) M, evoked a transient decrease in [Ca2+]i accompanied by sustained relaxation (45.3+/-6.3 vs. 69.45+/-7.2%, P<0.05, respectively) of VSM precontracted with K+ (70 mM), suggesting a decrease in Ca2+-sensitivity of VSM. This decrease in Ca2+-sensitivity was completely abolished by preincubation of VSM with ODQ (10(-6) M) (63.9+/-7.8% for [Ca2+]i vs. 20.5+/-8.4% for relaxation, P<0.05). Ca2+-presensitization of VSM myofilaments with PE (10(-6) M) decreased the efficacy of NO to relax VSM (44.25+/-6.9% vs. 69.45+/-7.2%, P<0.05), but increased its ability to lower [Ca2+]i (70.5+/-6.8% vs. 45.3+/-6.3%, P<0.05). Application of DTT (10(-3) M) together with ODQ (10(-6) M) to subtract possible cGMP-independent effects revealed the total suppression of both the relaxant responses and [Ca2+]i of VSM under high-K+ preactivation of VSM. The data indicate that NO not only relaxes VSM and lowers [Ca2+]i in K+-preactivated VSM, but also decreases Ca2+-sensitivity of VSM myofilaments and these effects are strongly cGMP-dependent. In PE-induced contractions of VSM, NO relaxed VSM of rat tail artery and lowered [Ca2+]i, but failed to reverse Ca2+-presensitized myofilaments. We suggest that alternative cGMP-independent effects of NO are primarily manifested via activation of K+-channels and inhibition of Ca2+ current rather than to affect relaxation. An importance of reduced SH-groups within VSM myoplasm for both relaxation and [Ca2+]i disposal evoked by NO is evident whatever Ca2+-mobilization pathways are involved.     

Expression of iNOS gene in macrophages stimulated with 17beta-estradiol is regulated by free intracellular Ca2+.

17Beta-estradiol has potent Ca2+ ionophore capability and its signaling in macrophages is mediated through binding to surface and genomic receptors, resulting in transient nitric oxide (NO) elaboration. We decided to examine if the transient release of NO is due to Ca2+ influx pattern or the quenching effect of superoxide (*O2-) through peroxynitrite formation. Differential chelation of intracellular Ca2+ ([Ca2+]i) showed that NO generation was favored by [Ca2+]i concentration of 237 nM. Application of an estrogen receptor antagonist ICI 182 780 resulted in attenuation of estradiol mediated NO release. Studies directed at identifying the possible role of *O2-; in the attenuation of NO showed no supportive evidence. Inhibition of extracellular Ca2+ channel or extracellular and intracellular Ca2+ channels showed data consistent with a case for optimum Ca2+ influx signal favoring iNOS gene expression, accompanied by an elevation in iNOS protein. These data show that Ca2+ influx pattern determines macrophage NO elaboration.     

Synthesis of phosphonate and ether analogs of rac-phosphatidyl-L-serine

The chemical synthesis of four phosphonate-containing phosphatidylserine analogs namely, L-serine (±)-[2,3-bis(hexadecyloxy) and 2,3-bis(Palmitoyloxy)-propyl] phosphonates, and L-serine (±)-[3,4-bis(hexadecyloxy and 3,4-bis(palmitoyloxy)-butyl]phosphonates is described. (±)-2,3-Bis(hexadecyloxy) and 2,3-bis(palmitoyloxy)-propylphosphonic acids and (±)-3,4-bis (hexadecyloxy)butylphosphonic acid were prepared by reaction of tris(trimethylsilyl) phosphite on the corresponding haloalkane. Condensation of the above phosphonic acids or (±)-3,4-bis (palmitoyloxy)butylphosphonic acid with N-carboxy-L-serine dibenzyl ester in the presence of trichloroacetonitrile or triisopropylbenzenesulfonyl chloride yielded the protected serine intermediates, which on hydrogenolysis gave the desired L-serine analogs. By a similar route, 1,2-dihexadecyl-rac-glycero-3-phosphoric acid was converted to 1,2-dihexadecyl-rac-glycerophospho-L-serine [L-serine (±)-2,3-bis(hexadecyloxy)propyl hydrogen phosphate(ester)].     

Endostatin uncouples NO and Ca2+ response to bradykinin through enhanced O2*- production in the intact coronary endothelium

The present study tested the hypothesis that endostatin stimulates superoxide (O2*-) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca2+]i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca2+]i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca2+]i was unchanged. With the conversion rate of L-[3H]arginine to L-[3H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O2*-. production was enhanced in endothelial cells. O2*-. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O2*-. production through activation of NAD(P)H oxidase. This ceramide-O2*-. signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.     

Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.     

Presynaptic Glutamate/Quisqualate Receptors: Effects on Synaptosomal Free Calcium Concentrations

Intracellular free [Ca2+]i was measured using fura-2 in synaptosomes prepared from cerebral cortices of adult male rats (12 weeks). L-(+)-Glutamate, D-(-)-glutamate, and quisqualate produced similar dose-dependent increases in [Ca2+]i, with EC50 values of 0.38 microM, 0.74 microM, and 0.1 microM, respectively, and maximum increases of approximately 40%. Ibotenate showed less affinity (EC50 4.4 microM) but had a greater maximum effect (57%). N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) did not increase [Ca2+]i. The increases in [Ca2+]i induced by quisqualate and ibotenate were not diminished in the absence of extrasynaptosomal Ca2+. L-2-Amino-4-phosphonobutyrate (L-AP4) (1 microM) completely blocked the changes in [Ca2+]i induced by L-(+)-glutamate, D-(-)-glutamate, quisqualate, or ibotenate. The effects of quisqualate and ibotenate on [Ca2+]i were also blocked by coincubation of synaptosomes with L-(+)-serine-O-phosphate (L-SP) (1 mM) (which, like L-AP4, blocks the effects of quisqualate and ibotenate on inositol phospholipid metabolism). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) had no effect on agonist-mediated increases in [Ca2+]i when coincubated with either quisqualate or ibotenate. These data are consistent with the existence of presynaptic glutamate receptors (of the excitatory amino acid metabotropic type) which activate phospholipase C leading to the elevation of inositol 1,4,5-trisphosphate and release of Ca2+ from intracellular stores.     

Feedback inhibition of Ca2+ release by Ca2+ is the underlying mechanism of agonist-evoked intracellular Ca2+ oscillations in pancreatic acinar cells.

Oscillations of free intracellular Ca2+ concentration ([Ca2+]i) are known to occur in many cell types during physiological cell signaling. To identify the basis for the oscillations, we measured both [Ca2+]i and extracellular Ca2+ concentration ([Ca2+]o) to follow the fate of Ca2+ during stimulation of [Ca2+]i oscillations in pancreatic acinar cells. [Ca2+]i oscillations were initiated by either t-butyloxycarbonyl-Tyr(SO3)-Nle-Gly-Tyr-Nle-Asp-2-phenylethyl ester (CCK-J), which mobilized Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-insensitive pool, or low concentration of cholecystokinin octapeptide (CCK-OP), which mobilized Ca2+ from the IP3-sensitive internal pool. Little Ca2+ efflux occurred during the oscillations triggered by CCK-J or CCK-OP in spite of a large average increase in [Ca2+]i. When internal store Ca2+ pumps were inhibited with thapsigargin (Tg) during [Ca2+]i oscillations, a rapid Ca2+ efflux occurred similar to that measured in intensely stimulated, nonoscillatory cells. Tg also stimulated 45Ca efflux from internal pools of cells stimulated with CCK-J or a low concentration of CCK-OP. Hence, a large fraction of the Ca2+ released during each spike is reincorporated by the internal store Ca2+ pumps. Surprisingly, when the increase in [Ca2+]i during stimulation of oscillations was prevented by loading the cells with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, a persistent activation of Ca2+ release and Ca2+ efflux occurred. This was reflected as a persistent increase in [Ca2+]o in cells suspended at low [Ca2+]o or persistent efflux of 45Ca from internal stores of cells maintained at high [Ca2+]o. Since agonist-stimulated Ca2+ release evidently remains activated when [Ca2+]i is highly buffered, the primary mechanism determining Ca2+ oscillations must include an inhibition of Ca2+ release by [Ca2+]i. Loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no apparent effect on the levels or kinetics of IP3 formation in agonist-stimulated cells. This suggests that [Ca2+]i regulated the oscillation by inhibition of Ca2+ release independent of its possible effects on cellular levels of IP3.     

Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.     

Possible mechanisms of the potentiation of blood-platelet activation by adrenaline.

Vasopressin and adrenaline in combination exert synergistic effects on platelet activity. This study investigated the effects of sub-threshold concentrations of adrenaline (0.1-1 microM) on vasopressin (10 nM-1 microM)-induced platelet aggregation, ATP secretion, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Potentiation of vasopressin-induced aggregation and ATP secretion by adrenaline was accompanied by enhanced elevation of [Ca2+]i and [32P]phosphatidic acid formation. The stimulatory effects of adrenaline on vasopressin-induced platelet activation were mimicked by the combination of the Ca2+ ionophore, ionomycin, and the protein kinase C activator, phorbol 12-myristate 13-acetate, but not by either of these agents alone. These results suggest that the potentiation of vasopressin-induced platelet activation by adrenaline is mediated via enhancement of inositol phospholipid hydrolysis and elevation of [Ca2+]i.     

Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization.

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.     

Conjugates of synthetic lymphocyte-activating lipopeptides with segments from HIV proteins induce protein-specific antibody formation.

Lipopeptide analogues of bacterial lipoprotein activate macrophages and B lymphocytes. The products formed by coupling these lipopeptides to low molecular mass antigens can be used to induce antigen-specific antibodies in mice. In the present work, it is shown that HIV-1 gp160-derived synthetic oligopeptides coupled to the synthetic lipodipeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-s eryl- serine (P3CSS) induce peptide-specific antibodies in mice without adding further adjuvants. Depending on the peptides applied, the conjugates exhibited different lymphocyte stimulatory activity, immunoglobulin isotype patterns, and boost reactions; lipopeptide conjugates inducing a pronounced secondary immune response are considered to possess both B- and T-cell epitopes. Antibodies induced by the lipopeptide-HIV-1-peptide conjugates were also reactive against the recombinant gp160 of HIV-1.     

Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.