Intracellular and plasma membrane-initiated pathways involved in the [Ca2+]i elevations induced by iodothyronines (T3 and T2) in pituitary GH3 cells

The role of 3,5,3'-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca(2+) concentration ([Ca(2+)](i)) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH(3) cells in the absence or presence of extracellular Ca(2+). When applied in Ca(2+)-free solution, T2 and T3 increased [Ca(2+)](i), in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N(G)-nitro-l-arginine methyl ester and l-n(5)-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca(2+)](i) increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca(2+) stores did not interfere with the T2- and T3-induced [Ca(2+)](i) increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca(2+)](i) and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca(2+)](i) increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na(+)/Ca(2+) exchanger by CGP37157 significantly reduced the [Ca(2+)](i) increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca(2+)](i) and intracellular Na(+) concentration; nimodipine reduced the [Ca(2+)](i) increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na(+)/H(+) pump by 5-(N-ethyl-N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na(+)/Ca(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.     

Nitric oxide activation of soluble guanylyl cyclase reveals high and low affinity sites that mediate allosteric inhibition by calcium

Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.     

Differential effects of soluble and particulate guanylyl cyclase on Ca(2+) sensitivity in airway smooth muscle.

Maximal relaxation of airway smooth muscle (ASM) in response to atrial natriuretic peptide (ANP), which stimulates particulate guanylyl cyclase (pGC), is less than that produced by nitric oxide (NO) and other compounds that stimulate soluble guanylyl cyclase (sGC). We hypothesized that stimulation of pGC relaxes ASM only by decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), whereas stimulation of sGC decreases both [Ca(2+)](i) and the force developed for a given [Ca(2+)](i) (i.e., the Ca(2+) sensitivity) during muscarinic stimulation. We measured the relationship between force and [Ca(2+)](i) (using fura 2) under control conditions (using diltiazem to change [Ca(2+)](i)) and during exposure to ANP, diethylamine-NO (DEA-NO), sodium nitroprusside (SNP), and the Sp diastereoisomer of beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Sp-8-Br-PET-cGMPS), a cell-permeant analog of cGMP. Addition of DEA-NO, SNP, or Sp-8-Br-PET-cGMPS decreased both [Ca(2+)](i) and force, causing a significant rightward shift of the force-[Ca(2+)](i) relationship. In contrast, with ANP exposure, the force-[Ca(2+)](i) relationship was identical to control, such that ANP produced relaxation solely by decreasing [Ca(2+)](i). Thus, during muscarinic stimulation, stimulation of pGC relaxes ASM exclusively by decreasing [Ca(2+)](i), whereas stimulation of sGC decreases both [Ca(2+)](i) and Ca(2+) sensitivity.     

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.     

Platelet-activating factor increases endothelial [Ca2+]i and NO production in individually perfused intact microvessels

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor N(omega)-monomethyl-l-arginine (l-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 286: H195-H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is the initiating event and Ca(2+)-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca(2+)](i) and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca(2+)](i) increased from 69 +/- 8 nM to a peak value of 374 +/- 26 nM within 3 min and then declined to a sustained level at 190 +/- 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca(2+)](i). PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 +/- 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 +/- 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with l-NMMA and attenuated in the absence of extracellular Ca(2+). These results provide direct evidence from intact microvessels that increased endothelial [Ca(2+)](i) is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.     

Simultaneous in situ monitoring of intracellular Ca2+ and NO in endothelium of coronary arteries

We developed an in situ assay system to simultaneously monitor intracellular Ca(2+) concentration ([Ca(2+)](i), fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 microM) stimulated a rapid increase in [Ca(2+)](i) followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 microM) induced a gradual, small increase in [Ca(2+)](i) and a slow increase in intracellular NO levels. Removal of extracellular Ca(2+) and depletion of Ca(2+) stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca(2+)](i) by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca(2+)](i) and intracellular NO production in the intact endothelium is a powerful tool to study Ca(2+)-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca(2+) in tyrosine phosphorylation-induced NO production.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

Regulation of pulmonary venous tone in response to muscarinic receptor activation

We investigated cellular mechanisms that mediate or modulate the vascular response to muscarinic receptor activation (ACh) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E-). Tension and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured simultaneously in fura-2-loaded E- PV strips. In the absence of preconstriction, ACh (0.01-10 microM) caused dose-dependent contraction in E+ and E- rings. ACh contraction was potentiated by removing the endothelium or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methyl ester, P = 0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E- PV rings (P = 0.013 and P = 0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated Ca(2+) channels (nifedipine, P < 0.001), inositol-1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release (2-aminoethoxydiphenyl borate, P = 0.001), PKC (bisindolylmaleimide I, P = 0.001), Rho-kinase (Y-27632, P = 0.002), and tyrosine kinase (TK; tyrphostin 47, P = 0.015) in E- PV rings. ACh (1 microM) caused a leftward shift in the [Ca(2+)](i)-tension relationship (P = 0.015), i.e., ACh increased myofilament Ca(2+) sensitivity. Inhibition of PKC, Rho-kinase, and TK attenuated the ACh-induced increase in myofilament Ca(2+) sensitivity (P < 0.001, P < 0.001, and P = 0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway and involves Ca(2+) influx through voltage-operated Ca(2+) channels and IP(3)-mediated Ca(2+) release. In addition, ACh induces increased myofilament Ca(2+) sensitivity, which requires the PKC, Rho-kinase, and TK pathways.     

Dopamine-induced graded intracellular Ca2+ elevation via the Na+Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts

Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca(2+) influx from the extracellular space. Additionally, dopamine induces a massive [Na(+)](i) elevation via the Na(+)K(+)2Cl(-) cotransporter (NKCC). We have reasoned that Ca(2+)-entry is mediated by the Na(+)Ca(2+) exchanger (NCE) operating in the Ca(2+)-entry mode. To test this hypothesis, [Ca(2+)](i) and [Na(+)](i) were measured by using the fluorescent dyes Fura-2, Fluo-3, and SBFI. Inhibition of Na(+)-entry from the extracellular space by removal of extracellular Na(+) or inhibition of the NKCC by 10 microM bumetanide did not influence resting [Ca(2+)](i) but completely abolished the dopamine-induced [Ca(2+)](i) elevation. Simultaneous recordings of [Ca(2+)](i) and [Na(+)](i) revealed that the dopamine-induced [Na(+)](i) elevation preceded the [Ca(2+)](i) elevation. During dopamine stimulation, the generation of an outward Na(+) concentration gradient by removal of extracellular Na(+) boosted the [Ca(2+)](i) elevation. Furthermore, prolonging the dopamine-induced [Na(+)](i) rise by blocking the Na(+)/K(+)-ATPase reduced the recovery from [Ca(2+)](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na(+)](i), which reverses the NCE activity into the reverse mode causing a graded [Ca(2+)](i) elevation in the duct cells.     

Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca(2+) concentration ([Ca(2+)](o)) regulates the functions of many cell types through a G protein-coupled [Ca(2+)](o)-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca(2+)](o), neomycin, and gadolinium) failed to increase intracellular Ca(2+) concentration ([Ca(2+)](i)), the CaR agonist spermine stimulated an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) and was abolished after depletion of an intracellular Ca(2+) pool with thapsigargin or after blocking IP(3)- and ryanodine receptor-mediated Ca(2+) release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca(2+)](i) and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca(2+)](i), primarily due to release of IP(3)- and ryanodine-sensitive intracellular Ca(2+) stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Mechanisms of the lysophosphatidic acid-induced increase in [Ca(2+)](i) in skeletal muscle cells.

Although lysophosphatidic acid (LPA) is known to increase intracellularfree calcium concentration ([Ca(2+)](i)) in different cell types, the effect of LPA on the skeletal muscle cells is not known. The present study was therefore undertaken to examine the effect of LPA on the [Ca(2+)](i) in C2C12 cells. LPA induced a concentration and time dependent increase in [Ca(2+)](i), which was inhibited by VPC12249, VPC 32183 and dioctanoyl glycerol pyrophosphate, LPA1/3 receptor antagonists. Pertussis toxin, a G(i) protein inhibitor, also inhibited the LPA-induced increase in [Ca(2+)](i). Inhibition of tyrosine kinase activities with tyrphostin A9 and genistein also prevented the increase in [Ca(2+)](i) due to LPA. Likewise, wortmannin and LY 294002, phosphatidylinositol 3-kinase (PI3-K) inhibitors, inhibited [Ca(2+)](i) response to LPA. The LPA effect was also attenuated by ethylene glycolbis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), an extracellular Ca(2+) chelator, Ni(2+) and KB-R7943, inhibitors of the Na(+)-Ca(2+) exchanger; the receptor operated Ca(2+) channel (ROC) blockers, 2-aminoethoxydiphenyl borate and SK&F 96365. However, the L-type Ca(2+) channel blockers, verapamil and diltiazem; the store operated Ca(2+) channel blockers, La(3+) and Gd(3+); a sarcoplasmic reticulum calcium pump inhibitor, thapsigargin; an inositol trisphosphate receptor antagonist, xestospongin and a phospholipase C inhibitor, U73122, did not prevent the increase [Ca(2+)](i) due to LPA. Our data suggest that the LPA-induced increase in [Ca(2+)](i) might occur through G(i)-protein coupled LPA(1/3) receptors that may be linked to tyrosine kinase and PI3-K, and may also involve the Na(+)-Ca(2+) exchanger as well as the ROC. In addition, LPA stimulated C2C12 cell proliferation via PI3-K. Thus, LPA may be an important phospholipid in the regulation of [Ca(2+)](i) and growth of skeletal muscle cells.     

17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3

We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca(2+) ([Ca(2+)](i)) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca(2+)](i), however exposure of cells to E2 in the presence of the microsomal Ca(2+) ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca(2+)](i) compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca(2+)](i). The E2-induced increase in [Ca(2+)](i) was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100muM). The estrogen receptor antagonist ICI 182,780 (1muM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca(2+)](i) from ryanodine-receptor-gated stores. The E2-induced effect on [Ca(2+)](i) could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10muM), the protein kinase A (PKA) inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate (200muM) and the MEK inhibitor PD98059 (10muM). We established E2 rapidly activates the novel PKC isoform PKCvarepsilon, PKA and Erk 1/2 MAPK in a PKCdelta and estrogen-receptor-dependent manner. The E2-induced effect was specific to 17beta-estradiol, as other steroids had no effect on [Ca(2+)](i). We have demonstrated a novel mechanism by which E2 rapidly modulates [Ca(2+)](i) release from ryanodine-receptor-gated intracellular Ca(2+) stores. The signal transduction pathway involves the estrogen receptor coupled to a PKC-PKA-Erk 1/2 signalling pathway.     

Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells

Galectin-1 (gal-1) a member of the mammalian beta-galactoside-binding proteins recognizes preferentially Galbeta1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. In the present work, gal-1 has been identified to be a ligand for the CD3-complex as well as for CD2 as detected by affinity chromatography of Jurkat T-cell lysates on gal-1 agarose and by binding of the biotinylated lectin to CD3 and CD2 immunoprecipitates on blots. In CD45(+)Jurkat E6.1 cells, the lectin stimulates a sustained increase in the intracytoplasmic calcium concentration ([Ca(2+)](i)) consisting of both the release of calcium from intracellular stores and the calcium influx from the extracellular space. This effect of gal-1 on [Ca(2+)](i)is completely inhibited by lactose at 10 mM and was absent in CD45(-)Jurkat J45.01 cells. Preincubation of Jurkat E6.1 cells with cholera toxin or with the protein tyrosine kinase inhibitor herbimycin A reduced the gal-1 induced calcium response whereas the increase in [Ca(2+)](i)stimulated by CD2 or CD3 monoclonal antibodies (mAbs) was completely inhibited. Depolarization of E6.1 cells in a high-potassium buffer, a standard method to activate voltage-operated calcium channels, was without effect on [Ca(2+)](i). Membrane depolarization with gramicidin or by a high-potassium buffer was without effects on the lectin-mediated calcium release from intracellular stores but inhibited the gal-1 induced receptor-operated calcium influx. In Jurkat E6.1 cells the lectin stimulates the transient generation of inositol-1,4,5-trisphosphate and the tyrosine phosphorylation of phospholipase Cgamma1. The results suggest that the ligation of CD2 and CD3 by gal-1 induces early events in T-cell activation comparable with that elicited by CD2 or CD3 mAbs.     

Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE(2), which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in hepatic parenchymal cells by agonists released during stress, such as with PGE(2) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca(2+)](i) in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE(2) caused a dose-dependent increase in [Ca(2+)](i) from basal values of 130 +/- 10 to maximal levels of 434 +/- 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca(2+) dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca(2+) from intracellular stores, also partially blocked the increase in [Ca(2+)](i) caused by PGE(2), suggesting that intracellular Ca(2+) pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca(2+) pools are involved in the increase in [Ca(2+)](i) caused by PGE(2). Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca(2+)](i) due to PGE(2) in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl(-) was omitted, glycine was much less effective in preventing the increase in [Ca(2+)](i) due to PGE(2). Phenylephrine, an alpha(1)-type adrenergic receptor agonist, also increased [Ca(2+)](i), as expected, from 159 +/- 20 to 432 +/- 43 nM. Glycine also blocked the increase in [Ca(2+)](i) due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca(2+)](i) in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.     

Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.     

The effect of exposure to high flux density static and pulsed magnetic fields on lymphocyte function

We investigated whether a combination of static electromagnetic field (EMF) at a flux density of 4.75 T together with pulsed EMF at a flux density of 0.7 mT generated by an NMR apparatus (NMRF), could promote movements of Ca(2+), cell proliferation, and the eventual production of proinflammatory cytokines in human lymphocytes as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 micro g/ml phytohaemagglutinin (PHA) immediately before the exposure period. Our results clearly demonstrate that NMRF exposure increases the [Ca(2+)](i), without any proliferative, or activating, or proinflammatory effect on both normal and PHA stimulated lymphocytes. Accordingly, the levels of interferon gamma, tumor necrosis factor alpha, interleukin-1beta, interleukin-2, and interleukin-6 remained unvaried after exposure. Exposure of Jurkat cells statistically decreased the [Ca(2+)](i) and the proliferation. This is consistent with the low levels of IL-2 measured in supernatants of these cells after exposure. On the whole our data suggest that static and pulsed NMRF exposure contribute synergistically in the increase of the [Ca(2+)](i) without any activating or proinflammatory effect either in normal or in PHA challenged lymphocytes. In Jurkat cells, by changing the properties of cell membranes, NMRF exposure can influence Ca(2+) transport processes and hence Ca(2+) homeostasis, causing a marked decrease of proliferation.     

Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.     

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²⁺]i, nitric oxide, and gap formation in intact venules

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.