Angiotensin II causes calcium entry into bovine adrenal chromaffin cells via pathway(s) activated by depletion of intracellular calcium stores

The characteristics and properties of the increase in cytosolic [Ca²⁺] that occurs in bovine adrenal medullary chromaffin cells on exposure to angiotensin II have been investigated. In fura-2 loaded cells exposure to a maximally effective concentration of angiotensin II (100 nM) caused a rapid, but transient increase in cytosolic [Ca²⁺] followed by a lower plateau that was sustained as long as external Ca²⁺ was present. In the absence of external Ca²⁺ only the initial brief transient was observed. In cells previously treated with thapsigargin in Ca²⁺-free medium to deplete the internal Ca²⁺ stores, angiotensin II caused no increase in cytosolic [Ca²⁺] when external Ca²⁺ was absent. Reintroduction of external Ca²⁺ to thapsigargin-treated, store-depleted cells caused a sustained increase in cytosolic [Ca²⁺] that was not further increased upon exposure to angiotensin II. Analysis of the data suggests that in bovine chromaffin cells angiotensin II causes Ca²⁺ entry via a pathway(s) activated as a consequence of internal store mobilization, and entry through this pathway(s) forms the majority of the sustained Ca²⁺ influx evoked by angiotensin II.     

Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea

Contractions of guinea pig trachea in the absence and presence of indomethacin to LTD₄ > LTC₄ > K⁺ > histamine > acetylcholine were reduced following a 45 minute exposure of the tissues to calcium-free Krebs' solution (Ca²⁺-free Krebs' solution), were further reduced by a transient exposure to EGTA (1.25 mM) in Ca²⁺-free Krebs' solution and were virtually abolished when tested in the presence of EGTA (0.125 mM) in Ca²⁺-free Krebs' solution. In normal Krebs' solution (2.5 mM Ca²⁺) the Ca²⁺ entry blockers nifedipine (N) ? D-600 > verapamil (V) > diltiazem (D) almost completely abolished the contractions to K⁺ but blocked only a component of the maximum response to the other agonists. After exposure to Ca²⁺-free Krebs' solution for 45 minutes, any residual contractions to LTC₄ & LTD₄, were reversed by low concentrations of N (0.3 μM) or D-600 (2.1 μM). Leukotrienes appear to mobilize a superficial and a bound store of Ca²⁺ which gains entry through at least two types of Ca²⁺ channels (or mechanisms), one of which is blocked by N and D600. K⁺-induced contractions appear to be dependent on superficial and tightly bound Ca²⁺ but entry is solely through channels which are blocked by the Ca²⁺ entry blockers studied. Contraction to histamine and acetylcholine persisted following exposure of the tissues to Ca²⁺ free Krebs' solution but contractile activity was virtually abolished in Ca²⁺ free Krebs' solution containing EGTA. Residual contractions to histamine and part of the residual contractions to acetylcholine in Ca²⁺-free Krebs' solution were blocked by low dose N (0.3μM) or D600 (2.1 μM). These findings suggest a major role for extracellular Ca²⁺ during spasmogen-induced contraction in this tissue.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

Up-regulation of Ca2+ influx mediated by store-operated channels in HL60 cells induced to differentiate by 1α,25-Dihydroxyvitamin D3

The physiologically active form of vitamin D, 1α,25-dihydroxyvitamin D₃ (1,25D₃), induces promyelocytic HL60 cells to differentiate towards monocyte-like cells. During this differentiation increased cytosolic calcium (Ca_i²⁺) and expression of surface receptors for chemotactic factors “prime” the cell for the activation of monocyte functions and the triggering of the respiratory burst pathway. We examined whether the Ca²⁺ influx mediated by store-operated channels (SOC) contributed to the increased Ca_i²⁺ following exposure of HL60 cells to 10⁻⁷ M 1,25D₃. Cells treated with 1,25D₃ for 72 hr demonstrated a rapid transient rise in Ca_i²⁺ followed by a second, phasic, increase in Ca_i²⁺ in response to the purinergic agonist ATP. This second Ca_i²⁺ transient was blocked by Ni²⁺, SKF 96365, or withdrawal of extracellular Ca²⁺⁺. In cells suspended in Ca²⁺-free medium, peak changes (Δ) in [Ca²⁺]_i elicited by ATP-induced Ca²⁺ mobilization occurred with similar EC₅₀ values in differentiated and vehicle (EtOH)-treated cells; however, peak [Ca²⁺]_i was reduced by 55% in 1,25D₃-treated cells. Decreased Ca²⁺ mobilization was associated with a 25–35% reduction in intracellular Ca²⁺ stores (determined with ionomycin). 1,25D₃-treated cells exposed to ATP or thapsigargin (Tg) in Ca²⁺-free medium for 3 min with subsequent addition of 1 mM Ca²⁺ exhibited a respective 80% or 120% stimulation in peak [Ca²⁺]_i compared to EtOH-treated cells. Enhanced Ca²⁺ influx mediated by SOC was also seen in these cells as an increase in the rate of Mn²⁺ entry after exposure to ATP or Tg. At 96 hr after addition of 1,25D₃, when differentiated phenotype was established, basal Ca²⁺_i and Ca²⁺ entry mediated by SOC returned to control values, but Ca²⁺ store size remained reduced. Up-regulation of Ca²⁺ influx via the SOC pathway during 1,25D₃-induced differentiation may contribute to the functional properties of the maturing monocyte, or to the resetting of molecular programs responsible for the changing phenotype. J. Cell. Physiol. 172:284–295, 1997. © 1997 Wiley-Liss, Inc.     

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Activation of a capacitative Ca entry pathway by store depletion in cultured hippocampal neurones

Intracellular Ca²⁺ ([Ca²⁺]_i) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca²⁺, the cholinergic agonist carbachol (200 μM) and the sarco-endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (0.4 μM) both transiently elevated [Ca²⁺]_i. A subsequent addition of Ca²⁺ into the bathing medium caused a second [Ca²⁺]_i change which was blocked by lanthanum (50 μM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca²⁺ entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca²⁺ entry in excitable cells.     

Activation of the Ca2+ “receptor” on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: Evidence for receptor activation and inactivation,intracellular Ca2+ redistribution,and divalent cation modulation

Earlier studies have demonstrated that a high (mM) extracellular Ca²⁺ concentration triggers intracellular [Ca²⁺] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni²⁺, elicited rapid and concentration-dependent elevations of cytosolic [Ca²⁺]. The peak change in cytosolic [Ca²⁺] increased monotonically with the application of [Ni²⁺] in the 50–5,000 μM range in solutions containing 1.25 mM-[Ca²⁺] and 0.8 mM-[Mg²⁺]. The resulting concentration-response function suggested Ni²⁺-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni²⁺ applications in the range 5–1,500 μM-[Ni²⁺] inhibited subsequent responses to a maximally effective [Ni²⁺] of 5,000 μM. Ni²⁺-induced cytosolic [Ca²⁺] responses were not dependent on extracellular [Ca²⁺]. Thus, when 5,000 μM-[Ni²⁺] was applied to osteoclasts in Ca²⁺-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (≤5 nM-[Ca²⁺] and 0.8 mM-[Mg²⁺]), cytosolic [Ca²⁺] responses resembled those obtained in the presence of 1.25 mM-[Ca²⁺]. Prior depletion of intracellular Ca²⁺ stores by ionomycin prevented Ni²⁺-induced cytosolic [Ca²⁺] responses, suggesting a major role for intracellular Ca²⁺ redistribution in the response to Ni²⁺. The effects of Ni²⁺ were also modulated by the extracellular concentration of the divalent cations, Ca²⁺ and Mg²⁺. When these cations were not added to the culture medium (0 μM-[Ca²⁺] and [Mg²⁺]), even low [Ni²⁺] ranging between 5 pM and 50 μM elicited progressively larger cytosolic [Ca²⁺] transients. However, the response magnitude decreased at higher, 250–5,000 μM-[Ni²⁺], resulting in a “hooked” concentration-response curve. Furthermore, increasing extracellular [Mg²⁺] or [Ca²⁺] (0–1 mM) diminished the response to 50 μM-[Ni²⁺], a concentration on the rising phase of the “hook.” Similar increases (0–10 mM) in extracellular [Mg²⁺] or [Ca²⁺] increased the response to 5,000 μM-[Ni²⁺], a concentration on the falling phase of the “hook”. These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni²⁺ as well as the divalent cations, Ca²⁺ and Mg²⁺. Receptor occupancy apparently activates intracellular Ca²⁺ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca²⁺ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Activation of Phosphatidylinositol-linked Novel D<Subscript>1</Subscript> Dopamine Receptor Contributes to the Calcium Mobilization in Cultured Rat Prefrontal Cortical Astrocytes

Recent evidences indicate the existence of an atypical D₁ dopamine receptor other than traditional D₁ dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase C_β (PLC_β). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide (PI)-linked D₁ dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca²⁺]_i) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959 caused a transient dose-dependent increase in [Ca²⁺]_i. Application of D₁ receptor, but not D₂, α₁ adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca²⁺]_i rise, indicating that activation of the D₁ dopamine receptor was essential for this response. Increase in [Ca²⁺]_i was a two-step process characterized by an initial increase in [Ca²⁺]_i mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated calcium channels, and capacitative Ca²⁺ entry. Furthermore, SKF83959-stimulated increase in [Ca²⁺]_i was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D₁ receptor by SKF83959 mediates a dose-dependent mobilization of [Ca²⁺]_i via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 μM) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) from 72 ± 5 to 3042 ± 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca²⁺]_i, the effect of LPC was examined in the Ca²⁺-free solution containing EGTA. Under the Ca²⁺ -free conditions, LPC did not increase [Ca²⁺]_i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca²⁺-present condition. Addition of ryanodine (10 μM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca²⁺-free and Ca²⁺ -present conditions. Preincubation of the myocytes with d-propranolol (50 μM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca²⁺ -free and Ca²⁺ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca²⁺]_i, and the Ca²⁺ -independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

Effects of bradykinin on Ca2+ mobilization and prostaglandin E2 release in human periodontal ligament cells.

In fura-2-loaded human periodontal ligament (HPDL) cells, bradykinin induced a rapidly transient increase and subsequently sustained increase in cytosolic Ca²⁺ ([Ca²⁺]_i). When external Ca²⁺ was chelated by EGTA, the transient peak of [Ca²⁺]_i was reduced and the sustained level was abolished, implying the Ca²⁺ mobilization consists of intracellular Ca²⁺ release and Ca²⁺ influx. Thapsigargin, a specific Ca²⁺-ATPase inhibitor for inositol 1,4,5-trisphosphate (1,4,5-1P₃)-sensitive Ca²⁺ pool, induced an increase in [Ca²⁺]_i in the absence of external Ca²⁺. After depletion of the intracellular Ca²⁺ pool by thapsigargin, the increase in [Ca²⁺]_i induced by bradykinin was obviously reduced. Bradykinin also stimulated formation of inositol polyphosphates including 1,4,5-IP₃. These results suggest that bradykinin stimulates intracellular Ca²⁺ release from the 1,4,5-1P₃-sensitive Ca²⁺ pool. Bradykinin stimulated prostaglandin E₂ (PGE₂) release in the presence of external Ca²⁺, but not in the absence of external Ca²⁺. Ca²⁺ ionophore A23187 and thapsigargin evoked the release of PGE₂ in the presence of external Ca²⁺ despite no activation of bradykinin receptors. These results indicate that bradykinin induces Ca²⁺ mobilization via activation of phospholipase C and PGE₂ release caused by the Ca²⁺ influx in HPDL cells.     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field

The effect of exposure of single rat pituitary cells to 50 Hz sine wave magnetic fields of various strengths on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was studied by using dual-emission microfluorimetry, using indo-1 as probe. A 30 min exposure of the cells to vertical 50 μT peak magnetic field triggered a long-lasting increase in [Ca²⁺]_i from a basal value of about 185 ± 4 nM to 326 ± 41 nM (S.E.; n = 150). The vertical and horizontal components of the static magnetic field were 57 and 15 μT, respectively. The 50 Hz ambient magnetic field was always below 0.1 μT rms. The effect was observed both at 25 ± 2 °C and at 37 ± 2 °C. Responsive cells, for which [Ca²⁺]_i rose to values above 309 nM, were identified as lactotrophs and represented 29% of the total pituitaries. [Ca²⁺]_i increase, for the most part, was due to Ca²⁺ influx through voltage-dependent dihydropiridine-sensitive calcium channels inhibited by PN 200-110. However, neither Ca²⁺ channel blockers nor removal of Ca²⁺ from the external medium during exposure completely prevented the field-induced [Ca²⁺]_i increase. Additional experiments using an MTT colorimetric assay showed that alteration of Ca²⁺ homeostasis of lactotrophs was associated with impairment of some mitochondrial processes. © Wiley-Liss, Inc.     

Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca²⁺]_i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca²⁺]_i in a dose-dependent manner. This elevation of [Ca²⁺]_i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca²⁺ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca²⁺]_i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca²⁺-free condition, orexin A failed to induce any changes of Ca²⁺ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca²⁺]_i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca²⁺ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca²⁺]_i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca²⁺]_i in neurons from deep layers of PFC, which depends on extracellular Ca²⁺ influx via L-type Ca²⁺ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.     

Oleic acid-induced Ca2+ mobilization in human platelets: is oleic acid an intracellular messenger?

The purpose of this study was to explore the effect of oleic acid (OA) on intracellular Ca²⁺ mobilization in human platelets. When applied extracellularly, OA produced a concentration dependent rise in cytosolic [Ca²⁺] [Ca²⁺]_cyt) when extracellular [Ca²⁺] ([Ca²⁺]_ext was zero (presence of EGTA), suggesting that OA caused an intracellular release of Ca²⁺. Intracellular Ca²⁺ release was directly proportional to entry of OA into platelets and OA entry was indirectly proportional to [Ca²⁺]_ext. In permeabilized platelets, OA caused the release of ⁴⁵Ca²⁺ from ATP dependent intracellular stores. Finally, our results show that thrombin stimulated the release of [³H]OA from platelet phospholipids. The saturated fatty acids stearic and palmitic acid did not stimulate an increase in [Ca²⁺]_cyt under these conditions, but the unsaturated fatty acid, linolenic acid produced effects similar to those of OA, suggesting specificity among fatty acids for effects on [Ca²⁺]_cyt. Taken together, our experiments suggest that OA which has been incorporated into platelet phospholipids was released intothe cytosol by thrombinstimulation. Our experiments also show that OA stimulates Ca²⁺ release from intracellular stores. These results support the hypothesis that OA may serve as an intracellular messenger in human platelets.     

The effect of chlorpromazine on intracellular Ca<Superscript>2+</Superscript> concentration in macrophages

Using Fura-2AM microfluorimetry, it was shown for the first time that neuroleptic chlorpromazine causes intracellular Ca²⁺ concentration increase in macrophages due to Ca²⁺ mobilization from intracellular Ca²⁺ stores and subsequent Ca²⁺ entry from the external medium. Chlorpromazine-induced Ca²⁺ entry is inhibited by La³⁺ and 2-aminoethoxydiphenyl borate and is associated with Ca²⁺ store depletion.