Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.     

Multiple P2 receptors contribute to a transient increase in intracellular Ca2+ concentration in ATP-stimulated rat brown adipocytes

Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca(2+) concentration ([Ca(2+)](i)), which arises primarily from a release of Ca(2+) from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca(2+)](i) elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 microM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca(2+)](i) elevation mediated through store-operated Ca(2+) entry. The inhibitory effect of ATP was antagonized by PPADS with IC(50) of 0.7 microM. In the presence of PPADS at concentrations of more than 5 microM, the ATP-induced [Ca(2+)](i) elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca(2+)](i) elevation was reduced in a concentration-dependent manner by PPADS with an IC(50) of 200 microM. In contrast, the ATP-induced [Ca(2+)](i) elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca(2+)](i) responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca(2+) stores, which is probably followed by the activation of store-operated Ca(2+) entry. Extracellular ATP, however, would inhibit this Ca(2+) entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca(2+)](i) elevation in response to extracellular ATP.     

Rapid,non-genomic actions of progesterone and estradiol on steady-state calcium and resting calcium influx in lens epithelial cells

The effects of steroids on the steady-state intracellular [Ca(2+)] ([Ca(2+)](i)) and resting Ca(2+) influx in Fura-2-loaded bovine lens epithelial cells were examined to identify potential rapid, non-genomic actions. When administered in the presence of 1-2 mM extracellular Ca(2+) ([Ca(2+)](o)), 100 micro M progesterone produced large (up to 12-fold) and transient (5 min) increases in [Ca(2+)](i). These effects were abolished in EGTA-containing solutions, and were associated with large increases in the rate at which extracellularly administered Mn(2+) quenched the intracellular Fura signal. Lower concentrations of progesterone (10-100 micro M) produced smaller increases in [Ca(2+)](i) that were concentration dependent, and 17beta-estradiol induced large, rapid and brief increases in [Ca(2+)](i) at 100 nM and smaller oscillations in [Ca(2+)](i) at 10 nM. In cells pretreated with thapsigargin, 100 micro M progesterone produced slower increases in [Ca(2+)](i) that were maintained for several minutes. These results demonstrate rapid non-genomic actions of progesterone and estradiol on resting Ca(2+) influx and [Ca(2+)](i) that may involve specific interactions with a recently discovered steroid-binding protein in the plasma membrane of lens epithelial cells.     

In airways ATP refills sarcoplasmic reticulum via P2X smooth muscle receptors and induces contraction through P2Y epithelial receptors

In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca(2+)](i). Cytosolic Ca(2+) is extruded to the extracellular space by the Na(+)/Ca(2+) exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCX(REV)), inducing Ca(2+) entry. We investigated whether ATP, via P2X receptors, activates the NCX(REV) and whether the increment in [Ca(2+)](i) is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca(2+)](i) not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca(2+)](i), (a Ca(2+) peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca(2+) peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca(2+)](i) increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCX(REV) and this effect was abolished by SKF 96365 and TNP-ATP (P2X(1) and P2X(3) receptors antagonist). P2X(1) and P2X(3) receptors were corroborated by immunoblotting of ASM. NCX(REV) activation and ATP in the presence of RB2 favor the SR Ca(2+) refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca(2+)](i) through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X(1) and P2X(3) receptors and the NCX(REV) which refills the SR.     

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.     

NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca(2+) concentrations in MDCK renal tubular cells

The effect of NPC-14686 (Fmoc-L-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca(2+) dye. At concentrations between 10 and 200 microM NPC-14686 increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by 90%. In Ca(2+)-free medium, pretreatment with 100 microM NPC-14686 nearly abolished the [Ca(2+)](i) increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and abolished the [Ca(2+)](i) increase induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 microM) induced a slight [Ca(2+)](i) increase after pretreatment with 2 microM CCCP and 1 microM thapsigargin. Addition of 3 mM Ca(2+) elicited a [Ca(2+)](i) increase in cells pretreated with 100 microM NPC-14686 in Ca(2+)-free medium. Inhibition of inositol-1,4,5-trisphosphate (IP(3)) production by suppressing phospholipase C with 2 microM U73122 did not alter NPC-14686-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with 10 or 200 microM NPC-14686 for 1-30 min decreased cell viability by 10-20% concentration dependently. Collectively, the results demonstrate that, in MDCK tubular cells, NPC-14686 induced Ca(2+) release followed by Ca(2+) entry, with the latter playing a major role. NPC-14686 appears to release intracellular Ca(2+) in an IP(3)-uncoupled manner. NPC-14686 may be of mild cytotoxicity.     

German cockroach extract activates protease-activated receptor 2 in human airway epithelial cells

BACKGROUND: The German cockroach has been reported to act as an allergen that might be associated with a protease reaction in asthma. However, the molecular identities of the antigens in German cockroach extract (GCE) with protease activity and the protease-activated receptors (PARs) that are activated by GCE in human airway epithelial cells have not been characterized. OBJECTIVE: We investigated the direct effect of GCE on Ca(2+) signaling in human airway epithelial cells and the type of PARs activated by GCE. METHODS: The Ca(2+)-sensitive dye Fura2 was used to determine intracellular Ca(2+) concentration ([Ca(2+)](i)) by means of spectrofluorometry. RESULTS: GCE induced a baseline type of [Ca(2+)](i) oscillations in a dose-dependent manner. The oscillations persisted for long periods of time in the absence of Ca(2+) entry across the plasma membrane, suggesting that the observed [Ca(2+)](i) increases were due to Ca(2+) release from intracellular stores. Accordingly, after depleting endoplasmic reticulum Ca(2+) with thapsigargin, an endoplasmic reticulum Ca(2+) ATPase inhibitor, the GCE-mediated [Ca(2+)](i) signals were abolished. Whereas desensitization of PAR-1, PAR-3, and PAR-4 had no effect on GCE-mediated Ca(2+) mobilization, no GCE-mediated [Ca(2+)](i) increase was observed after desensitization of PAR-2. CONCLUSIONS: These results indicate that GCE has a direct effect on human airway epithelial cells, in particular generating [Ca(2+)](i) oscillations through Ca(2+) release from thapsigargin-sensitive Ca(2+) stores through activation of PAR-2.     

Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ release

Selective activation of neuronal functions by Ca(2+) is determined by the kinetic profile of the intracellular calcium ([Ca(2+)](i)) signal in addition to its amplitude. Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca(2+) current and the resulting rise and decay of [Ca(2+)](i) in differentiated pheochromocytoma (PC12) cells. We show that equal amounts of Ca(2+) entering through N-type and L-type voltage-gated Ca(2+) channels result in significantly different [Ca(2+)](i) temporal profiles. When the contribution of N-type channels was reduced by omega-conotoxin MVIIA treatment, a faster [Ca(2+)](i) decay was observed. Conversely, when the contribution of L-type channels was reduced by nifedipine treatment, [Ca(2+)](i) decay was slower. Potentiating L-type current with BayK8644, or inactivating N-type channels by shifting the holding potential to -40 mV, both resulted in a more rapid decay of [Ca(2+)](i). Channel-specific differences in [Ca(2+)](i) decay rates were abolished by depleting intracellular Ca(2+) stores with thapsigargin or by blocking ryanodine receptors with ryanodine, suggesting the involvement of Ca(2+)-induced Ca(2+) release (CICR). Further support for involvement of CICR is provided by the demonstration that caffeine slowed [Ca(2+)](i) decay while ryanodine at high concentrations increased the rate of [Ca(2+)](i) decay. We conclude that Ca(2+) entering through N-type channels is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR provides a mechanism whereby the kinetics of intracellular Ca(2+) leaves a fingerprint of the route of entry, potentially encoding the selective activation of a subset of Ca(2+)-sensitive processes within the neuron.     

GABAA-receptor-mediated increase in intracellular Ca2+ concentration in the regenerating retina of adult newt

We used optical recording with the Ca(2+)-sensitive dye, fura-2, in living slice preparations from the newt retina at different stages of regeneration. gamma-Aminobutyric acid (GABA) induced pronounced [Ca(2+)](i) rise in progenitor cells and differentiating ganglion cells in the 'intermediate' stage of retinal regeneration. This [Ca(2+)](i) rise became less pronounced at the beginning of synapse formation in the late regenerating retina. At the late period of the late regenerating retina with the IPL thickness comparable to that of the control retina, GABA-induced [Ca(2+)](i) rise became undetectable or sometimes a small decrease in [Ca(2+)](i) was observed in regenerated ganglion cells. In contrast, N-methyl-d-aspartate (NMDA)-induced [Ca(2+)](i) rise appeared in premature ganglion cells and became prominent gradually as the regeneration proceeded. The [Ca(2+)](i) rise to GABA was mediated by GABA(A) receptors. This was shown by inhibition of GABA-induced Ca(2+) response with the preincubation of the GABA(A) receptor antagonist, bicuculline. The [Ca(2+)](i) rise due to GABA was suppressed in the absence of extracellular Ca(2+) or in the presence of the L-type voltage-gated Ca(2+) channel blocker, verapamil, suggesting that Ca(2+) may be entered through L-type Ca(2+) channels. Transient appearance of [Ca(2+)](i) rise to GABA during regeneration and origin of GABA-induced [Ca(2+)](i) rise were similar to those in the developing retina [J. Neurobiol. 24 (1993) 1600]. These similarities may suggest that common mechanisms may control neurogenesis and/or synaptogenesis during development and regeneration.     

Ischemia but not anoxia evokes vesicular and Ca(2+)-independent glutamate release in the dorsal vagal complex in vitro

Whole cell recordings of fura-2 dialyzed vagal neurons of brain stem slices were used to monitor interstitial glutamate accumulation within the dorsal vagal complex. Anoxia produced a sustained outward current (60 pA) and a moderate [Ca(2+)](i) rise (40 nM). These responses were neither mimicked by [1S,3R]-1-aminocyclo-pentane-1, 3-dicarboxylic acid nor affected by Ca(2+)-free solution, 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide in glucose-free saline (in vitro ischemia) as well as ouabain or iodoacetate elicited an initial anoxia-like [Ca(2+)](i) increase that turned after several minutes into a prominent Ca(2+) transient (0.9 microM) and inward current (-1.8 nA). APV plus CNQX (plus methoxyverapamil) inhibited this inward current as well as accompanying spontaneous synaptic activity, and reduced the secondary [Ca(2+)](i) rise to values similar to those during anoxia. Each of the latter drugs delayed onset of both ischemic current and prominent [Ca(2+)](i) rise by several minutes and attenuated their magnitudes by up to 40%. Ca(2+)-free solution induced a twofold delay of the ischemic inward current and suppressed the prominent Ca(2+) increase but not the initial moderate [Ca(2+)](i) rise. Cyclopiazonic acid or arachidonic acid in Ca(2+)-free saline delayed further the ischemic current, whereas neither inhibitors of glutamate uptake (dihydrokainate, D,L-threo-beta-hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the Cl(-) channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid had any effect. In summary, the response to metabolic arrest is due to activation of ionotropic glutamate receptors causing Ca(2+) entry via N-methyl-D-aspartate receptors and voltage-activated Ca(2+) channels. An early Ca(2+)-dependent exocytotic phase of ischemic glutamate release is followed by nonvesicular release, not mediated by reversed glutamate uptake or Cl(-) channels. The results also show that glycolysis prevents glutamate release during anoxia.     

Beta-chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia

Activation of beta-chemokine receptors, co-receptors for human immunodeficiency virus type-1 (HIV-1), stimulates movement and secretion in microglia, possibly through a Ca(2+)-dependent mechanism. We studied chemokine activation of Ca(2+) signaling processes in microglia. Human fetal microglia were grown in primary culture and chemokine-induced increases in intracellular calcium concentration ([Ca(2+)](i)) were measured in single cells using indo-1-based microfluorimetry. Application of 50 ng/ml regulated on activation, normal T expressed and secreted (RANTES; 120 s) evoked responses in 26% of the microglia (187/719 cells). [Ca(2+)](i) increased from a basal level of 66+/-6 nM to peak at 268+/-23 nM (n=187). Chemokine-evoked responses rapidly desensitized as indicated by the rapid return to basal [Ca(2+)](i) levels in the maintained presence of RANTES. The removal of extracellular Ca(2+) or stimulation in the presence of Ni(2+) (2mM) or La(3+) (100 microM) blocked the RANTES-elicited [Ca(2+)](i) increase. The L-type calcium channel antagonist nimodipine (10 microM) inhibited the RANTES-mediated increase in [Ca(2+)](i) by 80+/-16%. Thus, the RANTES-evoked calcium transient appears to result from Ca(2+) influx with little if any release from intracellular stores. Application of gp120(clade) (E) and gp120(CM235) (50 ng/ml) neither mimicked nor antagonized the RANTES-evoked response. Application of 50 ng/ml eotaxin (120 s) evoked an increase in [Ca(2+)](i) in 13% of the human microglia in culture (61/469 cells). The HIV-1 regulatory protein Tat (50 ng/ml) increased the [Ca(2+)](i) in a subset of eotaxin-responsive cells (16/30). The L-type calcium channel antagonist nimodipine (3 microM) inhibited eotaxin- and Tat-mediated increases in [Ca(2+)](i) by 88+/-6% and 93+/-6%, respectively. Thus, activation of CCR3 appears to evoke Ca(2+) influx through L-type Ca(2+) channels.These results indicate that beta-chemokines, RANTES and eotaxin, activate a nimodipine sensitive Ca(2+) influx pathway in human fetal microglia. HIV-1 Tat protein mimicked chemokine-mediated Ca(2+) signaling and may modulate the migratory and secretory responses of microglia.     

Fibronectin induces pseudopod formation and cell migration by mobilizing internal Ca(2+) in blastoderm cells from medaka embryos

During vertebrate embryogenesis, blastoderm cells at the gastrula stage migrate to new locations for subsequent development. The cellular mechanism of migration was studied in medaka (Oryzias latipes) embryos at the early gastrula stage. When fibronectin was applied iontophoretically or by the puff method, cell surface protrusion known as pseudopods and a local [Ca(2+)](i) rise at the site of application were observed in approximately half of the isolated blastoderm cells. When the pseudopod adhered to the substrate, the cell body moved toward the direction of the pseudopod as [Ca(2+)](i) declined and the pseudopod was withdrawn. Local puff application of ionomycin, a Ca(2+) ionophore, in the presence of external Ca(2+) induced protrusions of the plasma membrane similar to pseudopods, suggesting that the [Ca(2+)](i) rise itself is causing pseudopod formation. On the other hand, fibronectin induced pseudopods even in the absence of external Ca(2+), suggesting the mobilization of Ca(2+) from internal stores. In accordance with this interpretation, fibronectin failed to induce [Ca(2+)](i) rises after pretreatment with thapsigargin, a blocker of Ca(2+)-ATPase in the endoplasmic reticulum. Furthermore, chelating internal Ca(2+) with BAPTA prevented fibronectin from inducing pseudopods. U-73122, a blocker of phospholipase C, completely suppressed both the [Ca(2+)](i) rise and morphological changes accompanied with fibronectin application, suggesting involvement of the inositol phosphate pathway. On the other hand, caffeine evoked a [Ca(2+)](i) rise in a great majority of the fibronectin-responsive cells and the percentage of fibronectin-responsive cells was greatly reduced by a blocking dose of ryanodine. These results suggest that fibronectin activates phospholipase C and the initial [Ca(2+)](i) rise through IP(3) receptors further activates ryanodine receptors, achieving the local [Ca(2+)](i) rise. The decay time course of [Ca(2+)](i) after fibronectin application was prolonged in the absence of external Na(+). DCB, an inhibitor of Na(+)/Ca(2+) exchangers, also prolonged the time course of the [Ca(2+)](i) decay, suggesting the contribution of Na(+)/Ca(2+) exchangers. Cytochalasin D, an inhibitor of actin polymerization by binding to the barbed end of F-actin, induced swelling in fibronectin-responsive cells and prevented fibronectin from inducing pseudopod formation without suppressing the [Ca(2+)](i) rise. These results support the hypothesis that fibronectin facilitates cell migration via pseudopod formation during gastrulation.     

Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells

BACKGROUND: The present study examined whether the sex steroids, estradiol and progesterone, could alter cytoplasmic calcium concentrations ([Ca(2+)](cyt)) in human granulosa-lutein cells. METHODS: Human granulosa cells were obtained at the time of oocyte retrieval for IVF and cultured for 3-7 days. Cells were loaded with Fura-2 AM and changes in [Ca(2+)](cyt) of single cells were studied using a dynamic digital Ca(2+) imaging system. RESULTS: Both estradiol and progesterone stimulated elevations of [Ca(2+)](cyt) in Ca(2+)-containing medium within seconds of exposure of the granulosa-lutein cells to the steroid, but only estradiol caused an increase in [Ca(2+)](cyt) in Ca(2+)-free medium. Both ICI-182780 and RU 486 stimulated [Ca(2+)](cyt) increases and inhibited the effects of estradiol and progesterone, respectively. Tamoxifen also induced transient increases in [Ca(2+)](cyt) concentrations but inhibited the effects of both estradiol and progesterone. The inhibitory effects of tamoxifen, ICI-182780 and RU 4486 on [Ca(2+)](cyt) responses to estradiol and progesterone could be reversed with higher concentrations of estradiol and progesterone, respectively. The [Ca(2+)](cyt) effects induced with tamoxifen could not be eliminated by prior treatment with RU 486 or ICI-182780. CONCLUSION: These results provide strong evidence that both estradiol and progesterone as well as the steroid antagonists, tamoxifen, RU 486 and ICI-182780, can act on human granulosa-lutein cells through a non-genomic mechanism.     

Osmotic membrane stretch increases cytosolic Ca(2+) and inhibits bone resorption activity in rat osteoclasts

Although the importance of mechanical stress on bone metabolism is well known, the intracellular mechanisms involved are not well understood. To evaluate the role of mechanical stress on osteoclastic function, we investigated the effects of membrane stretch induced by osmotic cell swelling on cytosolic Ca(2+) and bone resorption activity in freshly isolated rat osteoclasts. The intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microspectrofluorimetry. Exposure to hypotonic solution (211-151 mOsm) caused cell swelling and reversibly increased [Ca(2+)](i) in the osteoclasts. This [Ca(2+)](i) increase was abolished by the omission of extracellular Ca(2+), but was not affected by the depletion of intracellular Ca(2+) stores. Gd(3+) and La(3+) inhibited the swelling-induced [Ca(2+)](i) increase, while nifedipine and Bay K 8644 did not. Neither protein kinase A inhibitors (Rp-cAMP, H-89) nor protein kinase C inhibitors (staurosporine, chelerythrine) affected the [Ca(2+)](i) increase. Membrane depolarization was not essential for the [Ca(2+)](i) increase either. To assess the effects of membrane stretch on the bone resorption activity of osteoclasts, we investigated actin ring formation, the intracellular structure responsible for bone resorption in osteoclasts. Hypotonic stimulation acutely disrupted actin ring formation in an extracellular Ca(2+)-dependent manner, and this disruption was prevented by Gd(3+). Moreover, Ca(2+) ionophore (ionomycin) also induced disruption of the actin rings. These results indicate that mechanical stress inhibits osteoclastic bone resorption activity, possibly via the elevation of [Ca(2+)](i) through stretch-activated, non-selective cation channels.     

Polycyclic aromatic hydrocarbon diol epoxides increase cytosolic Ca(2+) of airway epithelial cells

Polycyclic aromatic hydrocarbons (PAHs) increase cytosolic Ca(2+) concentration ([Ca(2+)](i)) in lymphocytes and mammary epithelial cells, but little is known regarding their effects on [Ca(2+)](i) in airway epithelium. We hypothesized that benzo[a]pyrene (BP) and/or anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), a carcinogenic BP metabolite, increases [Ca(2+)](i) in untransformed human small airway epithelial (SAE) cells and that their effects on [Ca(2+)](i) are directly proportional to carcinogenicity. SAE [Ca(2+)](i) was determined by a ratiometric digital Ca(2+) imaging system. BPDE increased SAE [Ca(2+)](i) within 20 s in media with high (1 mM) and low (10 nM) Ca(2+) at a threshold concentration of 0.2 nM. Elevation of [Ca(2+)](i) persisted longer with high Ca(2+). Neither BP nor solvent altered [Ca(2+)](i). Thapsigargin and inositol 1,4,5- phosphate receptor (InsP(3)R) antagonists inhibited this BPDE action with low Ca(2+). We conclude that BPDE but not BP increases [Ca(2+)](i) partly by mobilizing Ca(2+) from cytosolic stores through an InsP(3)R. The most potent carcinogenic PAH diol epoxide increased in SAE [Ca(2+)](i) at the lowest threshold concentration, suggesting that carcinogenicity is directly proportional to the action of PAHs on SAE [Ca(2+)](i). Short-term exposure to BPDE 36 to 48 h before the study rendered SAE cells less sensitive to BPDE, suggesting that BPDE may also induce persistent changes in Ca(2+) signaling pathways.     

Na(+) and Ca(2+) homeostasis pathways, cell death and protection after oxygen-glucose-deprivation in organotypic hippocampal slice cultures

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.     

Hypocretin/orexin peptide signaling in the ascending arousal system: elevation of intracellular calcium in the mouse dorsal raphe and laterodorsal tegmentum

Dysfunction of the hypocretin/orexin (Hcrt/Orx) peptide system is closely linked to the sleep disorder narcolepsy, suggesting that it is also central to the normal regulation of sleep and wakefulness. Indeed, Hcrt/Orx peptides produce long-lasting excitation of arousal-related neurons, including those in the laterodorsal tegmentum (LDT) and the dorsal raphe (DR), although the mechanisms underlying these actions are not understood. Since Hcrt/Orx mobilizes intracellular calcium ([Ca(2+)](i)) in cells transfected with orexin receptors and since receptor-mediated Ca(2+) transients are ubiquitous signaling mechanisms, we investigated whether Hcrt/Orx regulates [Ca(2+)](i) in the LDT and DR. Changes in [Ca(2+)](i) were monitored by fluorescence changes of fura-2 AM loaded cells in young mouse brain slices. We found Hcrt/Orx (Orexin-A, 30-1,000 nM) evoked long-lasting increases in [Ca(2+)](i) with differing temporal profiles ranging from spiking to smooth plateaus. A fragment of Hcrt/Orx (16-33) failed to evoke changes in [Ca(2+)](i) and changes were not blocked by TTX or ionotropic glutamate receptor antagonists, suggesting they resulted from specific activation of postsynaptic orexin receptors. Unlike orexin receptor-transfected cells, Hcrt/Orx-responses were not attenuated by depletion of Ca(2+) stores with cyclopiazonic acid (CPA; 3-30 microM), thapsigargin (3 microM), or ryanodine (20 microM), although store-depletion by either CPA or ryanodine blocked Ca(2+) mobilization by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 30 microM). In contrast, Hcrt/Orx responses were strongly attenuated by lowering extracellular Ca(2+) ( approximately 20 microM) but were not inhibited by concentrations of KB-R7943 (10 microM) selective for blockade of sodium/calcium exchange. Nifedipine (10 microM), inhibited Hcrt/Orx responses but was more effective at abolishing spiking than plateau responses. Bay K 8644 (5-10 microM), an L-type calcium channel agonist, potentiated responses. Finally, responses were attenuated by inhibitors of protein kinase C (PKC) but not by inhibitors of adenylyl cyclase. Collectively, our findings indicate that Hcrt/Orx signaling in the reticular activating system involves elevation of [Ca(2+)](i) by a PKC-involved influx of Ca(2+) across the plasma membrane, in part, via L-type calcium channels. Thus the physiological release of Hcrt/Orx may help regulate Ca(2+)-dependent processes such as gene expression and NO production in the LDT and DR in relation with behavioral state. Accordingly, the loss of Hcrt/Orx signaling in narcolepsy would be expected to disrupt calcium-dependent processes in these and other target structures.     

Intracellular Mg(2+) hyperpolarizes rabbit coronary artery smooth muscle cells by differential modulation of Ca(2+)(i)-dependent ion channels

We investigated the role of intracellular Mg(2+) ([Mg(2+)](i)) in the regulation of membrane potential ( V(m)) in rabbit coronary artery smooth muscle cells. V(m), membrane currents and intracellular Ca(2+) ([Ca(2+)](i)) were measured using standard patch-clamp and microfluorometry techniques. When [Ca(2+)](i) was increased by caffeine, V(m) depolarized at low [Mg(2+)](i) (0.1 mM), but hyperpolarized at high [Mg(2+)](i) (> or =1.2 mM). Effects of [Mg(2+)](i) on caffeine-induced currents were investigated. [Mg(2+)](i) selectively facilitated the activation of Ca(2+)-activated K(+) currents ( I(K,Ca)), while Ca(2+)-activated Cl(-) currents ( I(Cl,Ca)) were unaffected. Simultaneous recording of [Ca(2+)](i) and I(K,Ca) at different [Mg(2+)](i) showed that [Mg(2+)](i) increased the Ca(2+) sensitivity of I(K,Ca). [Ca(2+)](i) also inhibited voltage-dependent K(+) (K(V)) currents, although this effect was significant only at low [Mg(2+)](i). These results imply that the relative contributions of I(K,Ca), I(Cl,Ca) and K(V) currents to V(m) during an increase in [Ca(2+)](i) are affected by [Mg(2+)](i): at low [Mg(2+)](i), activation of I(Cl,Ca) and inhibition of K(V) currents depolarized V(m); at high [Mg(2+)](i) the activation of I(K,Ca) predominated, resulting in hyperpolarization of V(m). In conclusion, [Mg(2+)](i) hyperpolarizes V(m) by selective facilitation of I(K,Ca) and may thus possibly contributes to the relaxation of the coronary artery.