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Using real-time two-photon laser scanning microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive lipid mediator, causes shear stress-dependent oscillatory local increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fluo-4-loaded endothelial cells of isolated mouse aortic strips in situ. The increase in [Ca(2+)](i) occurred independently in the individual endothelial cells in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.3-10 μm) and the shear stress (10-80 dyn cm(-2)). The response was inhibited by removing extracellular Ca(2+), but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. These results were almost the same as those in the previous investigation using cultured bovine aortic endothelial cells, and suggest that LPA enhanced the shear stress-induced oscillatory Ca(2+) influx, termed 'Ca(2+) spot', in endothelial cells via activation of elementary Ca(2+) influx. In conclusion, the present study demonstrates, for the first time, that LPA functions as an endogenous sensitizer for mechanotransduction in endothelial cells in shear conditions in aortic strips in situ as well as in cultured cells. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Recent developments in optoelectronics permit real-time Ca(2+) imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca(2+) imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-microm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 microm). When serial sectioning of 21 sequential images at 0.3-microm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca(2+)-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca(2+)](i) of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca(2+) imaging in blood vessels in situ.

The mechanotransduction mechanism is believed to play an important role in maintenance of cellular homeostasis in a wide variety of cell types. In particular, the mechanotransduction system in vascular endothelial cells may be an essential mechanism for local hemodynamic control. Elevations in intracellular free Ca2+ concentration ([Ca2]i) are an important signal in the initial step of mechanotransduction and mechanosensitive (MS) cation channels are thought to be a putative pathway; however, the molecular mechanisms remain unclear. We found that lysophosphatidic acid (LPA), a bioactive phospholipid, sensitizes the response of [Ca2+]i to mechanical stress in several cell types. Employing real-time confocal microscopy, local increases in [Ca2+]i in several regions within the cell during application of mechanical stress were clearly visualized in bovine lens epithelial and endothelial cells in the presence of LPA. The phenomenon was termed "Ca2+ spots". Pharmacological studies revealed that Ca2+ spots arise due to influx through MS channels. In this report, our data indicating the possible significance of LPA as an endogenous factor involved in regulation of mechanotransduction is reviewed. Furthermore, our findings suggest that the Ca2+ spot is a novel phenomenon occurring as an elementary Ca2+-influx event through MS channels directly coupled with the initial step in mechanotransduction.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).

Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow-induced [Ca(2+)](i) response in fluo 4-loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca(2+)](i) was localized to a circular area with a diameter of <4 microm and spread concentrically, resulting in a mean global increase in [Ca(2+)](i). The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.1 to 10 micromol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca(2+) or by the application of Gd(3+), an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca(2+)-ATPASE: It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca(2+)](i) response arises from Ca(2+) influx through Gd(3+)-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. Therefore, we called the phenomenon Ca(2+) spots. We conclude that LPA positively regulates fluid flow-induced local and oscillatory [Ca(2+)](i) increase, ie, the Ca(2+) spots, in endothelial cells via the activation of elementary Ca(2+) influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Local increases in the intracellular Ca2+ concentration ([Ca2+]i) in several regions within the bovine lens epithelial cell during application of mechanical stress were clearly visualized in the presence of lysophosphatidic acid (LPA), a bioactive lysophospholipid, using real-time confocal microscopy. We called the phenomenon 'Ca2+ spots'. Ca2+ spots started in a circular area with a radius of about 1.5 m. These Ca2+ spots spread concentrically, resulting in a mean global increase in [Ca2+]i. The local increase often occurred in a stepwise manner or repetitively at the same region. The spatiotemporal properties of the Ca2+ spots were completely different from those of the Ca2+ wave induced by ATP, a Ca2+-mobilizing agonist. Ca2+ spots were inhibited by decreasing the extracellular Ca2+ concentration or by the presence of Gd3+, an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, suggesting that Ca2+ spots arise from Ca2+ influx through Gd3+-sensitive MS channels. On the assumption that, in lens epithelial cells, the open probability of the MS channel is 0.4, the membrane potential is 56 mV and the channel conductance is 50 pS, the estimated maximum flux of Ca2+ in a Ca2+ spot (0.4 x 10-17 to 4.7 x 10-17 mol x s(-1)) was comparable to currents of one or a few MS channels. On real-time three-dimensional confocal imaging analysis, which permitted simultaneous imaging of basal and apical planes of cells at 37.6 ms intervals, Ca2+ spots on the apical plane were more clearly visualized than those on the basal plane. From these results, we propose that the Ca2+ spot is an elementary Ca2+-influx event through MS channels directly coupled with the first step in mechanoreception In addition, our results strongly suggest that LPA functions as an endogenous factor affecting mechanotransduction systems.

Changes in intracellular calcium concentration ([Ca2+]i) and focal adhesion sites of cultured bovine aortic endothelial cells (BAECs) were simultaneously visualized in real time. Local [Ca2+]i transients were observed at the rear edges of spontaneously migrating BAECs. Furthermore, the majority of starting regions of [Ca2+]i transients retracted continuously. Frequency of [Ca2+]i transients increased with the application of fluid flow. The majority of starting regions of flow-induced [Ca2+]i transients retracted following the occurrence of [Ca2+]i transients. In addition, retracted areas were distributed in the upstream regions of the cell. Application of GdCl3, a mechanosensitive cation channel blocker, resulted in a clear reduction of [Ca2+]i transients and rear retractions in cases of spontaneous and flow-induced BAEC migration. Flow-induced directional rear retractions were also inhibited. Consequently, we conclude that local [Ca2+]i transients play an important role in the migration of BAECs with respect to rear retraction. Furthermore, flow-induced [Ca2+]i transients regulate directional rear retraction under flow conditions.

The effects of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response of the cytosolic free Ca2+ concentration ([Ca2+]i) to hypotonic stress were studied in cultured bovine lens epithelial cells, to test whether LPA affects cellular swelling-mediated increase in [Ca2+]i, which may relate to formation of sugar cataracts. Exposure of the cells to a 30% hypotonic stress caused only a slight increase in [Ca2+]i. Pretreatment with LPA (10 microM) significantly augmented the hypotonic stress-induced [Ca2+]i response, whereas addition of LPA to the cells did not affect [Ca2+]i. The hypotonic stress-induced increase in [Ca2+]i in the presence of LPA was inhibited by Gd3+, a blocker of mechanosensitive cation channels, but not by nicardipine, a L-type Ca2+ channel blocker, or thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump. These results show that LPA sensitizes the response to hypotonic stress via increase in Ca2+ influx through Gd3+-sensitive stretch-activated ion channels, and not via Ca2+ release from intracellular stores. On the other hand, LPA did not affect the [Ca2+]i response to ATP, a Ca2+ mobilizing agonist. Therefore, LPA sensitizes the hypotonic stress-induced [Ca2+]i response in lens epithelial cells, suggesting that LPA potentiates the development of cataracts induced by cellular swelling such as sugar cataract.

Nicotine dose-dependently induced cytotoxicity in human glioma (KG-1-C) and glioblastoma (GBS-1, T98G) cell lines, but could not induce internucleosomal DNA cleavage, in contrast to apoptosing human myelogenous leukemic cell lines. Human glioma/glioblastoma cell lines thus might have a chromatin structure resistant to endonuclease digestion. Nicotine induced a rapid increase in the intracellular calcium concentration. Confocal experiments with Fluo-3 fluorescence revealed that nicotine elevated the free Ca2+ concentration in both nuclear and cytoplasmic regions of the cells, and the elevation of Ca2+ in the nuclear region was more pronounced than that of the cytoplasmic region. The present study suggests that nuclear accumulation of Ca2+ is an important initial step for cell death induction by nicotine.

We investigated changes in intracellular pH (pHi) in relation to intracellular Ca2+ concentration ([Ca2+]i) in primary cultured hippocampal neurons treated with glutamate. [Ca2+]i and pHi were imaged with fluorescent dyes and confocal microscopy. Exposure to 1 mM glutamate for 10 min increased [Ca2+]i and evoked acidosis. These changes persisted for at least 60 min, even after removal of glutamate. The increase in [Ca2+]i and the acidosis were not observed in Ca2+-free solution and were attenuated in the presence of MK-801, an NMDA receptor antagonist. We also found that the increase in [Ca2+]i and acidosis could be induced by addition of Ca2+ to the extracellular solution in the cells pretreated with glutamate in Ca2+-free solution, even if glutamate did not exist in the extracellular solution. On the other hand, ionomycin and Br-A23187, calcium ionophores, increased [Ca2+]i to almost the same level as glutamate and increased pHi. Extracellular Ca2+ was also indispensable for the increase in [Ca2+]i and the alkalosis. These results suggest the followings: 1) intracellular acidosis by glutamate is dependent on the presence of extracellular Ca2+; 2) the acidosis does not result from only the increase in [Ca2+]i; and 3) glutamate induces the irreversible disorder of regulatory mechanisms of [Ca2+]i not only by Ca2+-dependent process, but also by Ca2+-independent process.

We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca2+]i in the presence of LPA and this increase was concentration-dependent (1-10 microM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca2+]i. The mechanical stress-induced increase in [Ca2+]i in the presence of LPA was inhibited by removing extracellular Ca2+ or by addition of Gd3+, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca2+ influx through cation-selective mechanosensitive channels, but does not sensitise Ca2+ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca2+ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca2+ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca2+]i induced by mechanical stress.

Diacylglycerol kinase (DG kinase) is activated by various stimuli in many types of cells. We reported earlier that carbachol (CCh) induced DG kinase translocation from the cytosolic fraction to the membrane fraction in guinea pig taenia coli (Biochem. Pharmacol., 50: 591-599, 1995). In this study, the regulation mechanisms of DG kinase translocation are reported, based on the following findings: 1) CCh sustained an increase in DG kinase in the membrane fraction and a decrease in the cytosolic fraction; 2) blocking calcium influx by removing extracellular calcium did not affect the CCh-induced sustained DG kinase translocation; 3) exposing purified protein kinase C (PKC) to DG kinase increased DG kinase affinity to octylglycoside micelles only with the enzyme extracted from the cytosolic fraction; and 4) CCh-induced DG kinase translocation was reversed by removing CCh, and the serine/threonine phosphatase inhibitor, okadaic acid, blocked the reversal of the translocation. These results suggest that CCh-induced DG kinase translocation is promoted by both a transient increase in intracellular calcium, which may be released from the intracellular store, and by DG kinase phosphorylation by PKC.

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

We previously reported that lysophosphatidic acid (LPA) sensitized mechanical stress-induced intracellular free Ca2+ concentration response (Biochem. Biophys. Res. Commun. 208, 19-25, 1995). In the present study, the signal transduction pathway of the sensitizing effect of LPA was investigated in cultured longitudinal muscle cells from guinea pig ileum. Suramin, a putative LPA receptor antagonist, did not affect the response in the presence of 30 nM LPA, suggesting that the response is induced via activation of suramin-insensitive LPA receptor. Neither pertussis toxin nor wortmannin inhibited the LPA-sensitized response, indicating that G(i/o)- and phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathways are not involved in the sensitizing effect. C3 ADP ribosyltransferase had no effect on the response, whereas formation of actin-stress fiber in the presence of LPA was completely inhibited, suggesting rho-related cytoskeletal change is not involved in the response. In contrast, a phospholipase C (PLC) inhibitor, U73122, completely inhibited the response, but broad spectrum kinase inhibitors, staurosporine and H7, had no effect on the response. In addition, tyrosine kinase inhibitor, genistein, but not tyrphostin partially inhibited the response. These results suggest that LPA sensitizes the mechanical stress-induced response via activation of PLC, but not protein kinase C. Additionally, tyrphostin-insensitive tyrosine kinase, which is related to other pathway than G(i/o)- and rho-mediated pathways, may be involved in the response.

The cytotoxic activity of sodium 5,6-benzylidene-L-ascorbate (SBA) against human KG-1-C glioma and T98G glioblastoma cell lines was augmented by pretreatment of the cells with L-buthionine-[S, R]-sulfoximine (BSO), which reduced the intracellular glutathione concentrations. SBA produced shrunken cells and large DNA fragments, without the induction of nuclear and internucleosomal DNA fragmentation. The rapid elevation of intracellular free Ca2+ concentration observed after SBA treatment was further augmented by BSO pretreatment. A confocal experiment with Fluo-3 fluorescence revealed that SBA markedly elevated the free Ca2+ concentration in the nuclear region, but did not significantly affect that in the cytoplasmic region. The present study suggests that the nuclear accumulation of Ca2+ is an important initial step for cell death induction by SBA.

We conformed that lysophosphatidic acid (LPA), which is known to be released from activated platelets, sensitizes response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stimulation in cultured epithelial cells (REPF-LC-AI cells) from human lung carcinoma. [Ca2+]i was transiently increased by spritzing of bath solution onto cells as mechanical stimulation in the presence of LPA with concentration-dependent manner (10-100 nM). The transient increase induced by the mechanical stimulation in the presence of LPA was inhibited by 10 microM Ga3+ or removing extracellular Ca2+, but not by 10 microM nicaridipine, suggesting that LPA sensitizes mechanical stimulation-induced Ca2+ influx through stretch-activated ion channels. Phosphatidic acid (1 microM), but not lysophosphatidycholine (10 microM), histamine (100 nM), bradykinin (10 nM), nor ionomycin (100 nM), caused the same effect as that of LPA. This effect was observed in confluent cells, but not in subconfluent cells. These results show that LPA sensitizes mechanoreceptor-linked response in human lung epithelial cells, suggesting a possibility that LPA affects lung function, in particular, during pathological state.

The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms.

We have previously reported that lysophosphatidic acid (LPA) sensitizes mechanical stress-induced cytosolic free Ca2+ concentration ([Ca2+]i) response related to Ca2+ entry through Gd(3+)-sensitive ion channels (Biochem. Biophys. Res. Commun. 208 19-25 1995). Here we examined the contribution of Ca2 release from intracellular stores to the mechanical stress-induced [Ca2+]i response sensitized by LPA in cultured longitudinal muscle cells from guinea pig ileum. Although the percentage of responsive cells to the mechanical stress in the presence of 30 nM LPA declined by decreasing extracellular Ca2+ concentration to less than 20 microM, the amplitude of the mechanical stress-induced [Ca2+]i transient did not depend on extracellular Ca2+ concentrations (10 microM-1.8 mM). The [Ca2+]i transient was completely abolished by treatment with thapsigargin. In addition, the amplitude of the [Ca2+]i transient gradually decreased after ryanodine and caffeine treatment. These results indicate that the mechanical stress-induced [Ca2+]i transient in the presence of LPA is mainly due to Ca2+ release from ryanodine-sensitive intracellular stores and may be triggered by Ca2+ influx through Gd(3+)-sensitive ion channels.

The mechanism of increase in intracellular Ca2+ concentration ([Ca2+]i) by removal of extracellular Na+, which phenomena were reported previously (Japan. J. Pharmacol. 63 83-91 1993), was investigated in cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent Ca2+ indicator, fura-2, by digital ratio imaging microscopy. Isotonic substitution of choline chloride for NaCl induced a transient increase in [Ca2+]i. The pretreatment of thapsigargin (0.5 microM), but not nicardipine (10 microM), suppressed the transient increase completely. In solutions containing micromolar concentrations of free Ca2+ (nominally Ca2+-free solution), the Na+-free induced transient increase was observed, but neither the second cell exposure to the Na+-free solution nor the following application of histamine increased [Ca2+]i, indicating that removal of extracellular Na+ releases Ca2+ from intracellular stores including inositol 1,4,5-trisphosphate (IP3)-releasable pools. The Na+-free induced transient increase required the presence of more than micromolar concentrations of extracellular free Ca2+ and releasable Ca2+ within the stores, but ryanodine did not affect the transient increase. These results suggest that undetectable influx of Ca2+ by the reverse-mode action of the Na+/Ca2+ exchanger can release Ca2+ from the thapsigargin-sensitive intracellular stores including IP3-releasable pools.

Cytosolic Ca2+ transients induced by hepatocyte growth factor (HGF) were imaged in primary cultured rat hepatocytes using newly developed rapid scanning confocal microscopes and indo-1. HGF (40 ng/ml) increased cytosolic free Ca2+ concentration ([Ca2+]i) in about 60% of hepatocytes, in 45% of which the increases were oscillatory. In each of the oscillatory hepatocytes, the repetitive increases in [Ca2+]i originated from a specific same region adjacent to the cell membrane and propagated across the cell like waves. Phenylephrine (10 microM) also induced Ca2+ waves. The locus where HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves were originated was the same, and there was a correlation in the peak height between HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves in each cell, although the mechanisms of inositol 1,4,5-trisphosphate (ins(1,4,5)P3) formation induced by HGF should be different from those by phenylephrine. On the other hand, there was no correlation between sensitivity of each cell to HGF and that to phenylephrine which were measured as latent periods prior to Ca2+ rises after an addition of the agonists. These results suggested the following: the spatial patterns of Ca2+ waves were decided by a common mechanism, probably not the propagation of ins(1,4,5)P3 but the distribution of ins(1,4,5)P3-sensitive Ca2+ pools; sensitivities of each cell to the agonists did not mainly depend on the common mechanism.

[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5'-(beta,gamma-methylene)triphosphate. The Bmax value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine-binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine-binding protein in the central nervous system.

We found that lysophosphatidic acid (LPA) sensitizes response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stimulation in cultured longitudinal muscle cells from guinea pig ileum. [Ca2+]i was transiently increased by spritzing of bath solution onto cells as mechanical stimulation in the presence of LPA, but not in absence of LPA. The effect was reversible and concentration-dependent (1-30 nM). Ga3+ but not nicardipine inhibited the [Ca2+]i transient in the presence of LPA. Phosphatidic acid also induced the sensitization, but the effective concentration was more than 10 times higher than in LPA. Histamine and carbachol did not have any sensitizing effect to mechanical stimulation. These results show that LPA sensitizes mechanoreceptor-linked response, suggesting that LPA may play an important role in mechanotransduction mechanisms as an endogenous regulatory factor.

Single smooth muscle cells were isolated from guinea pig trachea and cultured for 48 hours. The cells were then loaded with a fluorescent-free calcium indicator, fluo-3. Concentrations of cytoplasmic-free calcium ([Ca2+]i) were determined by using a confocal laser scanning fluorescence imaging system. Cells exposed to histamine at concentrations less than 1.0 microM did not indicate any changes of the fluorescence intensities, but stayed at the basal level of [Ca2+]i. Cells exposed to histamine at a concentration in the range of 1.5 to 10 microM indicated marked increases in [Ca2+]i. A further increase in [Ca2+]i by application of higher concentrations of histamine was not observed. The threshold concentration of histamine to increase [Ca2+]i and peak [Ca2+]i varied from cell to cell. In addition, half-maximal response time was shortened with increasing concentrations of histamine from 1.5 to 100 microM. These results indicate that [Ca2+]i responses to histamine is an all-or-none type in each cell. The heterogeneity in the required threshold concentration of histamine to increase in [Ca2+]i and the concentration dependency in half-maximal response time of histamine-induced [Ca2+]i increase may be related to the graded responses of histamine-induced contractions in preparations of the tracheal tissue.

3',3",5',5"-Tetraiodophenolsulfonephthalein (I4PSP) stimulated both Ca2+ transient levels and aggregation in response to thrombin in platelets. Ca2+ uptake into skeletal sarcoplasmic reticulum (SR) was inhibited by I4PSP (IC50, 1.8 microM) whereas that of red blood cell ghosts was not affected by it. Furthermore, I4PSP inhibited the SR Ca(2+)-ATPase activity (IC50, 1.1 microM). Kinetic analysis of the inhibitory effects of I4PSP reveals that I4PSP shows an inhibition of noncompetitive and competitive type with respect to low and high concentrations of ATP, respectively. The mode of inhibition by I4PSP is an uncompetitive type with respect to Ca2+. I4PSP decreased the decomposition rate of the phosphorylated enzyme intermediate (EP). The change in the tryptophan fluorescence of SR Ca(2+)-ATPase induced by ATP was reduced by I4PSP indicating inhibition of the EP transition from Ca2E1P to E2P. The concentration-inhibitory response curves for I4PSP in Ca(2+)-ATPase activities and fluorescence changes were closely correlated. These results suggest that I4PSP slows down the structure transition from Ca2E1P to E2P, resulting in inhibition of the Ca(2+)-ATPase activity. On the basis of these results, it is suggested that the stimulation of Ca2+ transient levels in platelets and their aggregation in response to thrombin are due to the inhibition of Ca2+ pump in intracellular Ca2+ stores by I4PSP.

The relationship between spontaneous Ca2+ oscillations and cell proliferation was investigated in cultured longitudinal muscle cells from guinea pig ileum. BAPTA, caffeine, thapsigargine and La3+ suppressed the spontaneous Ca2+ oscillations and the DNA synthesis at 5.0 mM, 10.0 mM, 15.62 nM and 0.5 mM, respectively, whereas verapamil, nicardipine and ryanodine did not suppress either. However, BAPTA and La3+ did not inhibit the DNA synthesis at 1.25 mM and 0.13 mM, but did suppress the Ca2+ oscillations even at 1.0 mM and 0.1 mM, respectively. These results show that the spontaneous Ca2+ oscillations are not prerequisite for the cell proliferation.

We previously described that carbachol (CCh) increased intracellular phosphatidic acid (PA) in guinea pig taenia coli and the increase was inhibited by diacylglycerol (DG) kinase inhibitor, R59022. The present study was conducted to clarify the role of DG kinase in CCh-induced increase in cytosolic free calcium and force development. Application of CCh to taenia coli induced transient increase in calcium level and contraction followed by sustained and lower calcium level and smaller contraction, and R59022 inhibited the latter responses specifically. The sustained responses were also evoked by 50 mM KCl, but these responses were slightly inhibited by R59022. Inhibition of calcium influx reduced responses to CCh but not KCl. Therefore, we consider that the sustained contraction induced by CCh is attributed to activation of DG kinase which generates PA and accelerates calcium influx.

The cytoplasmic free calcium ion concentration ([Ca2+]i) of cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent [Ca2+]i indicator, fura-2, was measured by digital ratio imaging microscopy. Spontaneous [Ca2+]i oscillations were observed in 25% to 80% of the cells, which differed with the batches of the cultured cells after 5 to 8 days in culture. The frequency and amplitude of the [Ca2+]i oscillations in each individual cell were usually regular, but heterogeneity between neighboring cells was observed. The spontaneous [Ca2+]i oscillations were also observed even after incubation of the cells under a serum-free condition for 72 hr. Exchange of extracellular solution to Ca(2+)-free solution containing EGTA or BAPTA immediately stopped the [Ca2+]i oscillations. The ratio of the oscillating cells was dependent on the extracellular calcium ion concentration ([Ca2+]o); and heterogeneity in the range of the [Ca2+]o to generate the [Ca2+]i oscillations was observed. An inorganic Ca(2+)-antagonist, LaCl3, immediately suppressed the [Ca2+]i oscillations, but the treatment with verapamil or nicardipine, Ca(2+)-channel blockers, did not have any effect on the [Ca2+]i oscillations. An inhibitor of the intracellular Ca2+ pump, thapsigargin, induced a transient increase in [Ca2+]i and then inhibited the spontaneous [Ca2+]i oscillations. Neomycin, a compound known to inhibit phosphoinositide turnover, inhibited the [Ca2+]i oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of calcitonin (CT) on hepatic glycogenolysis was investigated after a single subcutaneous administration of the hormone to intact rats. Administration of CT (porcine CT; 80 MRC mU/100 g BW) produced a significant decrease in glycogen content of the liver, and corresponding increases in phosphorylase a activity and calcium content of the particulate glycogen fraction in the liver. These alterations were observed with the dose of CT at physiological level. The removal of calcium by 1 mM EGTA treatment of the hepatic particulate glycogen caused a clear reduction in the increase in phosphorylase a activity produced by CT administration. Meanwhile, the enzyme activity in 1 mM EGTA-treated particulate glycogen of the liver in both control and CT (80 MRC mU/100 g BW)-treated rats was significantly enhanced by the addition of calcium ion (10 microM). Furthermore, a single intraperitoneal administration of calcium chloride (2.0 mg Ca/100 g BW) to intact rats produced a remarkable increase in phosphorylase a activity and calcium content of the hepatic glycogen particulate fraction. These results suggest that the promotion of hepatic glycogenolysis by CT administration may result from the increase in phosphorylase a activity mediated by cellular calcium.

The ultrastructural changes of single smooth muscle cells of guinea pig taenia coli during contraction evoked by Ca2+ (Ca) and carbachol (CCh) were investigated by transmission and scanning electron microscopy. Cells, prepared in the presence of a high concentration (140 mM) of KCl, without addition of Ca, exhibited smooth surfaced contour. The contraction of these cells were dose dependent upon Ca. With the increase in Ca concentration, the contraction was accompanied with formation of processes (0.15 micron thick and 1 micron long), membrane evaginations of various sizes and shapes and cores densely packed with filaments at the bottoms of all evaginations. Cells, prepared in the presence of 0.18 mM Ca and at a physiological concentration (2.7 mM) of KCl, had two populations: One had shallow invaginations of the cell membrane and the other had spinous processes. Cells of both types were responsive to CCh and the spinous processes disappeared during contraction but evagination did not appear. Differences in ultrastructural changes during contractions between single smooth muscle cells and intact tissues may be due to the fact that single cells are mechanically free from adjacent cells and connective tissues. It is also possible that membrane properties change during isolation, particularly when cells are isolated in Ca free medium.

The effect of calcitonin (CT) on alanine aminotransferase activity and calcium content in the hepatic mitochondria of intact rats was investigated. A single subcutaneous administration of CT (synthetic [Asu1,7] eel calcitonin; 80 MRC mU/100 g body weight) produced a significant increase in alanine aminotransferase activity and a corresponding elevation of calcium content. Those increases were also observed with the lowest dose of CT (20 MRC mU/100 g). The mitochondrial calcium depletion by 10 mM EGTA treatment produced a remarkable reduction in alanine aminotransferase activity increased by CT, although EGTA treatment did not alter basal enzyme activity. Addition of calcium ion (1-10(3) microM) into the EGTA-treated mitochondria, however, produced a significant increase in alanine aminotransferase activity. The enzyme activity increased by CT was not inhibited by the presence of calmodulin inhibitor, W-7 (1-10(3) microM). These results suggest that the mitochondrial calcium increased by CT administration directly activates alanine aminotransferase in the hepatic mitochondria of rats.

The effect of calcitonin (CT) on calcium content and glucose-6-phosphatase activity in the hepatic microsomes of rats was investigated. A single sc administration of CT (80 MRC mU/100 g body weight) produced a significant increase in microsomal calcium content and a corresponding elevation of microsomal glucose-6-phosphatase activity. An appreciable effect of CT was observed at a dose of 20 MRC mU/100 g body weight. On the other hand, the removal of calcium by 10 mM EGTA washing of the microsomes caused a marked reduction of glucose-6-phosphatase activity raised by CT administration. The addition of calcium ion at the amount of 10(-2)-10(3) nmoles Ca2+ per mg protein in the microsomes produced a significant increase in glucose-6-phosphatase activity. The present results suggest that microsomal calcium increased by CT administration activates glucose-6-phosphatase in the hepatic microsomes of rats.

45Ca-loaded single smooth muscle cells were prepared from taenia coli of guinea pig. The cells were perfused continuously with physiological salt solution on membrane filter with pore size of 12 microns and radioactivity in the effluent was determined. The 45Ca-efflux curves consisted of two straight lines in semilogarithmic figure representing loosely and tightly bound calcium compartments with half time of 1.87 +/- 0.27 and 6.78 +/- 0.43 min, respectively. The loosely bound calcium was supposed to be localized on plasma membrane. Size of the tightly bound 45Ca was dependent on period for calcium-depletion of the preparation prior to 45Ca-loading. The efflux was stimulated by acetylcholine and the stimulated efflux returned to the spontaneous efflux level instantly after removal of acetylcholine. This stimulation was abolished by atropine. Repetitive stimulation failed to increase 45Ca-efflux while the rate of the spontaneous 45Ca-efflux did not change significantly. These results suggested that, in taenia coli of guinea pig, at least three calcium compartments existed, loosely bound calcium, tightly bound calcium and more tightly bound calcium which can be released by acetylcholine, in addition to calcium in extracellular fluid. Advantages and disadvantages of using single smooth muscle cells for investigation of calcium movement and some technical improvement for determination of 45Ca-efflux from single cells were described.

The effect of calcitonin (CT) on fructose 1,6-diphosphatase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to rats. Administration of CT (synthetic [Asu1,7] eel CT; 80 MRC mU/100 g body weight) produced significant increase in fructose 1,6-diphosphatase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The binding of calcium by 10 microM EGTA in the hepatic cytosol caused a clear reduction of the increase in fructose 1,6-diphosphatase activity produced by CT administration. The enzyme activity of CT-treated rats was markedly reduced by W-7 (100 microM), calmodulin inhibitor, while that of control rats was not significantly altered by the drug. Meanwhile, fructose 1,6-diphosphatase activity in the hepatic cytosol obtained from normal rats was significantly enhanced by addition of calcium ion (0.1-5.0 microM). This increase was also clearly inhibited by W-7. These results suggest that CT increases fructose 1,6-diphosphatase activity in the hepatic cytosol of rats, and that this hormonal regulation may depend on calmodulin, mediated through calcium increased in the cytosol.