Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

Release of Ca(2+) from intracellular stores and entry of extracellular Ca(2+) are involved in sea squirt sperm activation.

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

Three distinct Ca(2+) influx pathways couple acetylcholine receptor activation to catecholamine secretion from PC12 cells

Amperometry and microfluorimetry were employed to investigate the Ca(2+)-dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine-evoked exocytosis was entirely dependent on extracellular Ca(2+) but was only partly blocked by Cd(2+), a nonselective blocker of voltage-gated Ca(2+) channels. Secretion and rises of [Ca(2+)](i) observed in response to nicotine could be almost completely blocked by methyllycaconitine and alpha-bungarotoxin, indicating that such release was mediated by receptors composed of alpha7 nicotinic acetylcholine receptor subunits. Secretion and [Ca(2+)](i) rises could also be fully blocked by co-application of Cd(2+) and Zn(2+). Release evoked by muscarine was also fully dependent on extracellular Ca(2+). Muscarinic receptor activation stimulated release of Ca(2+) from a caffeine-sensitive intracellular store, and release from this store induced capacitative Ca(2+) entry that could be blocked by La(3+) and Zn(2+). This Ca(2+) entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca(2+)](i) and exocytosis via Ca(2+) influx through voltage-gated Ca(2+) channels, alpha7 subunit-containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca(2+) entry.     

Recombinant expression of the plasma membrane Na(+)/Ca(2+) exchanger affects local and global Ca(2+) homeostasis in Chinese hamster ovary cells

The cardiac type Na(+)/Ca(2+) exchanger (NCX1) has been transiently expressed in Chinese hamster ovary cells, which do not contain an endogenous exchanger, together with aequorin chimeras that are targeted to different intracellular compartments to investigate intracellular Ca(2+) homeostasis. The expression of NCX decreased the endoplasmic reticulum Ca(2+) concentration, [Ca(2+)](er), in resting cells, showing that the exchanger was operative under these conditions. It induced a greater reduction in the height of the mitochondrial and cytosolic Ca(2+) transients in agonist-stimulated cells than would have been expected from the [Ca(2+)](er) decrease. It also had a major effect on the sub-plasma membrane Ca(2+) concentration, [Ca(2+)](pm): after a transient [Ca(2+)](pm) rise induced by the activation of capacitative Ca(2+) influx, [Ca(2+)](pm) settled to a value about 3-fold higher than in controls. The sustained [Ca(2+)](pm) increase after the transient was due to the operation of the exchanger, either directly by operating in the Ca(2+) entry mode, or indirectly by removing the Ca(2+) inhibition on the capacitative Ca(2+) influx channels.     

Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 microm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 microm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 microm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 microm Gd3+, as well as 30-100 microm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 microm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2)]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 microm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+) entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.     

Tyrosine phosphorylation is involved in Ca(2+)entry in human gingival fibroblasts

Bradykinin (1 microM) and histamine (100 microM) evoked an initial transient increase and a subsequent sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fura-2-loaded human gingival fibroblasts, which may be attributed to Ca(2+) release from intracellular stores and Ca(2+) entry from extracellular sites, respectively. In fibroblasts pretreated with tyrosine kinase inhibitors such as herbimycin A (1 microM) and tyrphostin 47 (20 microM), the sustained level of [Ca(2+)](i) induced by bradykinin and histamine increased, but not the initial peak level. In the absence of external Ca(2+), bradykinin and histamine induced only the transient increase in [Ca(2+)](i), but a subsequent addition of Ca(2+) to the medium resulted in a sustained increase in [Ca(2+)](i) caused by Ca(2+)entry. Thapsigargin, an inhibitor of Ca(2+)-ATPase in inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores, mimicked the effect of bradykinin and histamine. In the fibroblasts pretreated with tyrosine kinase inhibitors, the bradykinin-, histamine- and thapsigargin-induced Ca(2+) entry was clearly enhanced, but not the transient [Ca(2+)](i) increase. Tyrosine phosphatase inhibitor benzylphosphonic acid (200 microM) had no effect on Ca(2+)entry or transient [Ca(2+)](i) increase. These results suggest that tyrosine phosphorylation is involved in Ca(2+) entry in human gingival fibroblasts.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

Novel effects of propranolol. Release of internal Ca(2+) followed by activation of capacitative Ca(2+) entry in Madin Darby canine kidney cells

The effect of propranolol on Ca(2+) signalling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Propranolol increased cytosolic free Ca(2+) levels ([Ca(2+)](i)) in a concentration-dependent manner between 0.1 and 1 mM. The response was partly inhibited by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with 0.2 mM propranolol partly inhibited the [Ca(2+)](i) rise induced by 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with thapsigargin abolished propranolol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 0.2 mM propranolol in Ca(2+)-free medium. Propranolol (0.2 mM) inhibited 25% of thapsigargin-induced capacitative Ca(2+) entry. Suppression of 1,4,5-trisphosphate (IP(3)) formation by 2 microM U73122, a phospholipase C inhibitor, did not alter 0.2 mM propranolol-induced internal Ca(2+) release. Propranolol (1 mM) also increased [Ca(2+)](i) in human neutrophils. Collectively, we have found that 0.2 mM propranolol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive Ca(2+) stores in an IP(3)-independent manner, followed by Ca(2+) influx from external space. Independently, propranolol was able to inhibit thapsigargin-induced capacitative Ca(2+) entry.     

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.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.     

Novel effects of gossypol, a chemical contraceptive in man: mobilization of internal Ca(2+) and activation of external Ca(2+) entry in intact cells

The effect of gossypol on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Gossypol evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 2 and 20 microM. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with gossypol nearly abolished the [Ca(2+)](i) increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 5 microM gossypol in Ca(2+)-free medium. This Ca(2+) entry was decreased by 25 microM econazole, 50 microM SKF96365 and 40 microM aristolochic acid (a phospholipase A(2) inhibitor). Pretreatment with aristolochic acid inhibited 5 microM gossypol-induced internal Ca(2+) release by 55%, but suppression of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 microM) also increased [Ca(2+)](i) in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from external space.     

EGF enhances Ca(2+) mobilization and capacitative Ca(2+) entry in mouse mammary epithelial cells

Effects of epidermal growth factor (EGF) on the intracellular Ca(2+) ([Ca(2+)](i)) responses to nucleotides, Ca(2+) release from thapsigargin-sensitive stores and capacitative Ca(2+) entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti-PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S-phase of cell cycle. EGF treatment apparently increased the number of PCNA-stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca(2+)](i) response. EGF treatment for 1-2 days in culture, however, enhanced [Ca(2+)](i) responses including [Ca(2+)](i) increase by ATP, UTP and other nucelotides, Ca(2+) release from thapsigargin-sensitive stores, as well as capacitative Ca(2+) entry. Genistein, a tyrosine kinase inhibitor, prevented EGF-induced cell proliferation and the [Ca(2+) ](i) responses in a dose-dependent manner. These results indicate that EGF treatment enhances Ca(2+) mobilization and capacitative Ca(2+) entry, well correlated with cellular proliferation in mammary epithelial cells.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.