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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 Ca2+ concentration ([Ca2+]i) in fluo-4-loaded endothelial cells of isolated mouse aortic strips in situ. The increase in [Ca2+]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 [Ca2+]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 Ca2+, but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase. The spatiotemporal properties of the [Ca2+]i response were completely different from those of a Ca2+ wave induced by ATP, a Ca2+-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 Ca2+ influx, termed ‘Ca2+ spot’, in endothelial cells via activation of elementary Ca2+ 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.

The caspase activation cascade and mitochondrial changes are major biochemical reactions in the apoptotic cell death machinery. We attempted to clarify the temporal relationship between caspase activation, cytochrome c release, mitochondrial depolarization, and morphological changes that take place during tumor necrosis factor (TNF)-α-induced cell death in HeLa cells. These reactions were analyzed at the single-cell level with 0.5 – 1 min resolution by using green fluorescent protein (GFP)-variant-derived probes and chemical probes. Cytochrome c release, caspase activation, and cellular shrinkage were always observed in this order within 10 min in all dying cells. This sequence of events was thus considered a critical pathway of cell death. Mitochondrial depolarization was also observed in all dying cells observed, but frequently occurred after caspase activation and cellular shrinkage. Mitochondrial depolarization is therefore likely to be a reaction that does not induce caspase activation and subsequent cellular shrinkage. Mitochondrial changes are important for apoptotic cell death; moreover, cytochrome c release, and not depolarization, is a key reaction related to cell death. In addition, we also found that the apoptotic pathway proceeds only when cells are exposed to TNF-α. These findings suggest that the entire cell death process proceeds rapidly during TNF-α exposure. Keywords:: tumor necrosis factor (TNF)-α, cytochrome c, mitochondrial depolarization, caspase, real-time imaging

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive approach used for stimulating the brain, and has proven effective in the treatment of depression, however the mechanism of its antidepressant action is unknown. Recently, we have reported the induction of kf-1 in rat frontal cortex and hippocampus after chronic antidepressant treatment and repeated electroconvulsive treatment (ECT). In this study, we demonstrated the induction of kf-1 after rTMS in the rat frontal cortex and hippocampus, but not in hypothalamus. Our data suggest that kf-1 may be a common functional molecule that is increased after antidepressant treatment, ECT and rTMS. In conclusion, it is proposed that induction of kf-1 may be associated with the treatment induced adaptive neural plasticity in the brain, which is a long-term target for their antidepressant action.

Although the therapeutic action of antidepressants most likely involves the regulation of serotonergic and noradrenergic signal transduction, no consensus has been reached concerning their precise molecular or cellular mechanisms of action. In the present study, we demonstrated that chronic treatment with a tricyclic antidepressant (imipramine) and a selective serotonin reuptake inhibitor (sertraline) reduced the expression of Ndrg2 mRNA and protein in the rat frontal cortex. Ndrg2 is a member of the N-Myc downstream-regulated genes. Interestingly, repeated ECT also significantly decreased Ndrg2 expression in this region of the brain. These data suggest that Ndrg2 may be a common functional molecule that is decreased after antidepressant treatment and ECT. Although, the functional role of Ndrg2 in the central nervous system remains unclear, our findings suggest that Ndrg2 may be associated with treatment-induced adaptive neural plasticity in the brain, a chronic target of antidepressant action. In conclusion, we have identified Ndrg2 as a candidate target molecule of antidepressants and ECT.

Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-α-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-α-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination. Keywords:: fluorescence resonance energy transfer, green fluorescent protein, tumor necrosis factor-α, cell death, caspase

Diacylglycerol kinase (DG kinase) is a key enzyme in vascular contraction; however, alterations of the regulatory mechanisms in vascular dysfunction are poorly understood. In this study, the effect of a novel DG kinase inhibitor, stemphone, on vascular contraction was investigated. The conventional DG kinase inhibitor, 6-[2-(4-[(4-fluorophenyl)phenyl-methylene]-1-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-α] pyrimidine-5-one (R59022) (0.1–30 μM), inhibited thromboxane A2 analogue 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619)-induced sustained contractions in mouse aorta and porcine coronary artery in a dose-dependent manner. Treatment with stemphone did not affect contractions in these tissues. However, stemphone significantly inhibited (>0.3 μM) U46619-induced spontaneous phasic contraction in mouse portal vein. This inhibitory effect was not detected following R59022 treatment in portal vein. Therefore, stemphone demonstrated selectivity in terms of portal vein contraction. Under high glucose (22.2 mM) conditions, U46619-induced contraction was enhanced in these three types of vascular tissue. Inhibitory effects of R59022 were attenuated under these conditions; however, effects of stemphone were observed. These results indicated that stemphone could inhibit portal vein contraction under high glucose conditions, for example, diabetes. These data suggested the possibility that DG kinase may be a target of hyperportal pressure. Total mass of DG was enhanced under high glucose conditions. DG was derived from incorporated glucose via de novo synthesis in the absence of phospholipase C pathway mediation. This enhanced DG under high glucose conditions activated a calcium-independent protein kinase C (PKC). This PKC was associated with calcium-independent DG kinase activation. Treatment with stemphone also inhibited calcium-independent DG kinase. These signal transduction pathways were distinguishable from a DG–PKC pathway under normal glucose conditions. The present investigation suggested that stemphone selectively inhibited overcontraction of portal vein induced by high glucose levels. This phenomenon was attributable to inhibition of calcium-independent DG kinase activation that occurred under high glucose conditions mediated by both DG synthesized from glucose and calcium-independent PKC activation. British Journal of Pharmacology (2004) 143, 166–178. doi:10.1038/sj.bjp.0705910

Although blockade by antidepressants of monoamine uptake into nerve endings is one of the cornerstones of the monoamine hypothesis of depression, there is a clear discrepancy between the rapid effects of antidepressants in increasing synaptic concentrations of monoamine and the lack of immediate clinical efficiency of antidepressant treatment. Novel biological approaches beyond the 'monoamine hypothesis' are definitely expected to cause paradigm shifts in depression research. Functional genomics are powerful tools that can be used to identify genes affected by antidepressants or by other effective therapeutic manipulations. Using RNA fingerprinting technique, we have previously identified several cDNA fragments as antidepressant related genes and from these, original cDNA microarrays were developed. Some of these candidate genes may encode common functional molecules induced by chronic antidepressant treatment. Defining the roles of these genes in drug-induced neural plasticity is likely to transform the course of research on the biological basis of depression. Such detailed knowledge will have profound effects on the diagnosis, prevention, and treatment of depression.

We investigated portal vein (PV) contractility in diabetes using a mouse model (ob/ob mouse) of spontaneous noninsulin-dependent diabetic mellitus. Spontaneous phasic contraction in control mice (C57Bl) was increased in the presence of the thromboxane A(2) analog 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F(2)alpha (U46619) in a time- and concentration-dependent manner. This response was enhanced under high glucose conditions (22.2 mM). Diacylglycerol (DG) was synthesized from glucose and was not affected by phospholipase C (PLC) inhibition under resting conditions in normal glucose. Inhibition of DG-induced PKC activation with 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo-(2,3-alpha)pyrrolo(3,4-c)-carbazole (Gö6976), a calcium-dependent protein kinase C (PKC) inhibitor, was only observed under normal glucose conditions. High glucose levels enhanced PLC-independent DG formation followed by an induction of total phosphatidylinositol turnover via calcium-independent PKC activation in C57Bl mice. In ob/ob mice, the high glucose-induced enhancement of PV contraction in response to U46619 was suppressed. These findings suggest that these differences are associated with long-term exposure of tissue to a hyperglycemic state. Under high glucose conditions, DG derived from glucose fell below 50% in C57Bl mice. Moreover, the DG-related calcium-independent PKC was desensitized in ob/ob mice. These results suggest that suppression of the glucose-induced enhancement of PV contraction involves both a decrease in glucose-derived DG formation and reduction of the glucose sensitivity of DG-related PKC.

The present investigation was conducted in order to determine whether lysophosphatidic acid (LPA) induces itch-scratch responses (ISRs) in mice. Intradermal administration of LPA induces ISRs; furthermore, the time course for LPA-induced ISRs was similar to that for histamine-induced responses. Comparative study of the pruritogenic activity revealed that histamine possessed a potent effect characterized by a dose-response relationship; however, prostaglandin D2 failed to induce this response. Pretreatment with ketotifen, a histamine H1 receptor antagonist, and capsaicin inhibited LPA-induced ISRs. Additionally, LPA-induced ISRs were abolished by Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). These findings suggest that LPA-induced ISRs are attributable to histamine- and substance-P-mediated pathways. Moreover, the Rho/ROCK-mediated pathway may be involved.

The mechanotransduction mechanisms play an important role in regulation of specific cellular response or maintenance of cellular homeostasis in a wide variety of cell types. Increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is an important signal in the first step of mechanotransduction. Mechanosensitive (MS) cation channels are thought to be a putative pathway of Ca(2+) entry; however, the molecular mechanisms remain unclear. We have previously demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid present in human plasma, sensitizes the response of [Ca(2+)](i) to mechanical stress in cultured smooth muscle cells, cultured lung epithelial cells, and cultured lens epithelial cells. Using real-time confocal microscopy, local increases in [Ca(2+)](i) in several regions within the cell subjected to mechanical stress were clearly visualized in cultured bovine lens epithelial cells and cultured vascular endothelial cells in the presence of LPA. We called the phenomenon "Ca(2+) spots". Pharmacological studies revealed that the Ca(2+) spot is an elementary Ca(2+)-influx event through MS channels. In this review, possible physiological and pathophysiological roles of LPA as a mechanosensitizer are discussed.

Lysophosphatidic acid (LPA) has been shown to be a chemoattractant in in vitro studies. The present study was carried out to determine whether LPA enhances infiltration of inflammatory cells in in vivo studies with guinea pigs. LPA (1 – 10 μg/ml), when by guinea pigs for 5 min, substantially increased the numbers of eosinophils and neutrophils in the bronchoalveolar lavege fluid (BALF), which was recovered at over 4 h after the inhalation of LPA. Infiltration in BALF was significantly inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). LPA also increased superoxide production of eosinophils and neutrophils. In contrast, Y-27632 inhibited superoxide production. These findings suggest that LPA may contribute to infiltration and activation of inflammatory cells in bronchial asthma; furthermore, the Rho/ROCK-mediated pathway may be involved.

The investigation of mechanotransduction in the cardiovascular system is essentially important for elucidating the cellular and molecular mechanisms involved in not only the maintenance of hemodynamic homeostasis but also etiology of cardiovascular diseases including arteriosclerosis. The present review summarizes the latest research performed by six academic groups, and presented at the 75th Annual Meeting of the Japanese Pharmacological Society. Technology of cellular biomechanics is also required for research and clinical application of a vascular hybrid tissue responding to pulsatile stress. 1) Vascular tissue engineering: Design of pulsatile stress-responsive scaffold and in vivo vascular wall reconstruction (T. Matsuda); 2) Cellular mechanisms of mechanosensitive calcium transients in vascular endothelium (M. Oike et al.); 3) Cross-talk of stimulation with fluid flow and lysophosphatidic acid in vascular endothelial cells (K. Momose et al.); 4) Mechanotransduction of vascular smooth muscles: Rate-dependent stretch-induced protein phosphorylations and contractile activation (K. Obara et al.); 5) Lipid mediators in vascular myogenic tone (I. Laher et al.); and 6) Caldiomyocyte regulates its mechanical output in response to mechanical load (S. Sugiura et al.).

Dysfunction of vascular contraction in diabetes has been reported; however, the mechanisms are poorly understood. In this study, calcium sensitization involving increases in contraction in streptozotocin-induced diabetic rat aorta was detected. We hypothesize that an alteration in the intracellular signalling system plays a role in the dysfunction of vascular contractility in diabetes. Therefore, diacylglycerol (DG) kinase as a key enzyme of phosphatidylinositol (PI) turnover was investigated. Treatment with norepinephrine (NE) caused time- and dose-dependent activation of DG kinase in control rats. This activation required simultaneous increases in intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activation. In diabetic rats, hyper-reactivity of DG kinase involving inactivation in the resting state and over-activation in NE stimulation was observed. During hyper-reactivity, [Ca2+]i dependency of DG kinase was enhanced. Treatment with 50 mM KCl induced significant escalation in activity; moreover, basal activation of PKC was detected only in diabetes. These results suggested that PKC had been activated in the resting state. In contrast, these conditions were insufficient for DG kinase activation due to the absence of [Ca2+]i elevation. During NE-stimulation, PKC activation was maintained and [Ca2+]i increased. Therefore, DG kinase was activated and an elevation in calcium dependency enhanced this activation. The present study suggested that DG kinase hyper-reactivity in diabetes involved both an increase in [Ca2+]i and basal activation of PKC. This phenomenon may be associated with increased vascular contraction in diabetes mediated by acceleration of PI-turnover. Keywords: Vascular smooth muscle, insulin-dependent diabetes mellitus, norepinephrine, phosphatidylinositol-turnover, calcium, contraction, protein kinase C Introduction Insulin-dependent diabetes mellitus (IDDM) results as a consequence of decreased insulin secretion and/or desensitization of insulin receptors, leading to complicating dysfunctions in many types of tissue (Ozturk et al., 1996). Diabetic patients appear to be particularly prone to disorders of the neurological, cardiovascular, gastrointestinal and reproductive systems (Raccah, 1998). It has been suggested that these complications are due to abnormalities in the nervous system. Recently, correlations between changes in intracellular signalling pathways and complications in many cell types have also been demonstrated (Ozturk et al., 1996; Schmidt et al., 1999). Clarification of this relationship may provide new information regarding the development of a treatment for diabetes. Phosphatidylinositol turnover (PI-turnover) occurs in numerous cell types; moreover, this process is involved in the intracellular signalling pathway. Changes in PI-turnover activity are believed to influence cellular functions. In PI-turnover, diacylglycerol (DG) plays an important role in cellular function as an endogenous activator of protein kinase C (PKC). Diacylglycerol kinase (DG kinase) phosphorylates DG, generating phosphatidic acid (PA) (Flores et al., 1996). Consequently, endogenous DG levels can be altered by DG kinase activity. We previously found that the carbachol (CCh)-induced muscarinic receptor mediated DG kinase activation in guinea-pig taenia coli (Nobe et al., 1994). Furthermore, we examined the mechanisms of regulation of DG kinase activation and discovered that both an increase in intracellular calcium concentration ([Ca2+]i) and PKC activation were required (Nobe et al., 1995; 1997). These observations suggested that regulation of DG kinase by PKC was an important feedback mechanism in PI-turnover. On the basis of these data, alterations in DG kinase activity in some tissues isolated from streptozotocin (STZ)-induced diabetic rats were detected (Nobe et al., 1998). In that investigation, DG kinase activity in diabetic aortic smooth muscle indicated marked changes. However, the mechanism(s) has not been clarified. The objective of this study was to clarify the mechanism(s) of DG kinase activity change in diabetic rat aorta in order to understand the relation between alterations in the intracellular signalling pathway and dysfunctions in vascular smooth muscle contractility. The present findings suggest that both increases in DG kinase calcium dependency and basal activation of PKC are involved in the hyper-reactivity of DG kinase, which may lead to enhancement of aortic contractility in diabetes.

Inhalation of lysophosphatidic acid (LPA, 1-100 microg/ml) for 2 min enhanced the airway response induced by intravenous injection of ACh in guinea pigs. At 30 min after inhalation of LPA, the airway response to ACh was two fold higher than that before inhalation. This enhancement of airway response to ACh was partially inhibited by capsaicin desensitization or bilateral vagotomy. These results suggested that the enhancement of airway response to ACh induced by LPA may be due to the activation of capsaicin-sensitive fibers. It can be also contribute to bronchial asthma or other types of pulmonary disease such as cough variant asthma and atopic cough.

Abstract —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 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 μmol/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.

We have previously identified 204 partial cDNA fragments (ADRG1–204) as antidepressant related genes/expressed sequence tags. Then, we developed our original cDNA microarrays, on which the 194 clones out of ADRG1–204 were spotted. With this ADRG microarray, we found that the expression of a spot, ADRG55, which representing cysteine string protein (CSP), was significantly increased in rat brain after chronic treatment with a selective serotonin reuptake inhibitor, sertraline. In the present study, reverse transcription-polymerase chain reaction analysis confirmed the induction of CSP at mRNA levels in rat frontal cortex after chronic treatment with two different classes of antidepressants, imipramine or sertraline. Western blot analysis also revealed that CSP-immunoreactivity was increased after antidepressant treatment. In conclusion, our data suggest that CSP is one of the common functional molecules induced after chronic antidepressant treatment.

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 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.

Previously, we have identified 200 cDNA fragments as antidepressant related genes/ESTs. In this study, using these cDNAs, we developed our original cDNA microarray for rapid secondary screening of candidate genes as the novel therapeutic targets. With this microarray, we found that the expression of a novel gene, ADRG34, was significantly increased in rat hippocampus which had been chronically treated with a selective serotonin reuptake inhibitor antidepressant, sertraline. RT-PCR analysis also demonstrated the induction of ADRG34 at mRNA levels in rat hippocampus and the frontal cortex. This cDNA encoded 685 amino acid residues containing a RING-H2 finger motif at the carboxy-terminal. Sequence analysis of ADRG34 with the EMBL/GenBank database showed significant homology to mouse and human kf-1 gene. Our data suggest that ADRG34, a possible rat homologue of kf-1, may be one of the common functional molecules induced after chronic antidepressant treatment.

The effects of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response of the cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) to hypotonic stress were studied in cultured bovine lens epithelial cells, to test whether LPA affects cellular swelling-mediated increase in [Ca 2+ ] i , which may relate to formation of sugar cataracts. Exposure of the cells to a 30% hypotonic stress caused only a slight increase in [Ca 2+ ] i . Pretreatment with LPA (10 μM) significantly augmented the hypotonic stress-induced [Ca 2+ ] i response, whereas addition of LPA to the cells did not affect [Ca 2+ ] i . The hypotonic stress-induced increase in [Ca 2+ ] i in the presence of LPA was inhibited by Gd 3+ , a blocker of mechanosensitive cation channels, but not by nicardipine, a L-type Ca 2+ 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 Ca 2+ influx through Gd 3+ -sensitive stretch-activated ion channels, and not via Ca 2+ release from intracellular stores. On the other hand, LPA did not affect the [Ca 2+ ] i response to ATP, a Ca 2+ mobilizing agonist. Therefore, LPA sensitizes the hypotonic stress-induced [Ca 2+ ] i response in lens epithelial cells, suggesting that LPA potentiates the development of cataracts induced by cellular swelling such as sugar cataract.

4,5-Diaminofluorescein (DAF-2) is a newly developed indicator of nitric oxide (NO). Two amino groups of DAF-2 are oxidized by NO. We investigated the effects of reducers on the NO-induced oxidation of DAF-2. NOC-5 (0.1-10 microM), a NO-donor, concentration-dependently elicited fluorescence with 10 microM DAF-2. The rate of the fluorescence reaction was dependent on the width of the excitation band path. The presence of catecholamines (1 microM), but not tyrosine or phenylephrine, attenuated the fluorescence induced by NOC-5. Ascorbate and other reducers like dithiothreitol, 2-mercaptoethanol, or glutathione (all 1 mM) abolished the fluorescence. These results suggest that reducers attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on cytoplasmic free calcium ion concentration ([Ca2+]i) were investigated in primary cultured rat hepatocytes. A high concentration (4.0 microM) of TBT increased resting levels of [Ca2+]i and then induced cell blebs resulting in cell death within 2 h. The increase in [Ca2+]i, but not the cell death, depended on the presence of extracellular Ca2+, suggesting that the increase in [Ca2+]i is not critical for the cytotoxicity of TBT. A low concentration (0.1 microM) of TBT did not have any toxic effect (decrease in ATP content, decrease in viability, and shape change) on cultured hepatocytes and did not change [Ca2+]i. However, the calcium responses induced by phenylephrine, [Arg8]-vasopressin, and ATP were suppressed in the cells pretreated with 0.1 microM TBT for 30 min. The suppression was not observed in the cells pretreated with 0.1 microM TBT for only 1 min. Pretreatment with 0.1 microM TBT for 30 min had no effect on the inositol 1,4,5-triphosphate content or its increase in response to hormonal stimulation. These results suggest that TBT suppresses hormone-induced calcium responses at nontoxic low concentrations.

The purpose of this study was to examine whether tetrahydrobiopterin (BH 4 ), one of the cofactors of nitric oxide (NO) synthase, attenuates endothelial cell death induced by 3-morpholinosydnonimine-N-ethylcarbamide (SIN-I), which is known to produce both superoxide and NO. Endothelial cell death was assessed by the release of intracellular lactate dehydrogenase (LDH). Addition of SIN-1 (500, 1.000 μM) to endothelial cells induced cell death from 6 h after its addition. The SIN-I-induced endothelial cell death was strongly reduced by treatment with carboxy-PTIO, a NO scavenger, or superoxide dismutase (SOD). Iron chelators and hydroxyl radical scavengers also reduced the SIN-I-induced endothelial cell death. Interestingly. the SIN-1-induced endothelial cell death was also reduced by treatment with catalase. Thus NO. superoxide. hydroxyl radical. and hydrogen peroxide are likely to be implicated in SIN-1-induced endothelial cell death. Moreover, pretreatment with sepiapterin, a precursor of BH 4 synthesis, reduced the SIN-I-induced endothelial cell death and increased the intracellular BH 4 content. Both the protective effect of sepiapterin and the increase in intracellular BH 4 content were prevented by co-pretreatment with N-acetylserotonin (NAS). an inhibitor of BH 4 synthesis. The protective effect of sepiapterin also was observed when uptake of trypan blue was used as another marker of cell death. These findings suggest that BH 4 has a protective effect against endothelial cell death caused by the presence of NO and superoxide. The protective effect of BH 4 may at least partly involve scavenging of superoxide or hydrogen peroxide or both. because we and other groups previously found that BH 4 has a scavenging activity for reactive oxygen species.

The purpose of this study was to characterize the protective effect of tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, against NO-induced endothelial cell injury. The addition of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, to endothelial cells induced the release of lactate dehydrogenase (LDH), a marker for cell injury. The SNAP-induced endothelial cell injury was markedly reduced by pretreatment with sepiapterin, a precursor of BH4 synthesis. On the other hand, exogenous BH4 had little effect on the SNAP-induced endothelial cell injury. We recently found that NO-induced endothelial cell injury involves a part of H202 production, since the injury was blocked by the treatment with catalase. Although BH4 released reactive oxygen species (ROS) in cell-free conditions, the increase in intracellular BH4 by pretreatment with sepiapterin strongly reduced H202-induced intracellular oxidative stress. These findings suggest that the increase in intracellular BH4 content but not extracellular BH4, strongly attenuates NO-induced endothelial cell injury by at least one of the mechanisms by which BH4 reduces H202-induced oxidative stress. Intracellular BH4 seems mainly to play a role as an antioxidant or as a ROS-scavenger.

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, attenuates NO-induced endothelial cell death. S-Nitroso-N-acetyl-DL-penicillamine (SNAP) was used as a NO donor. Endothelial cell death was assessed by the leakage of intracellular lactate dehydrogenase (LDH). Addition of SNAP to endothelial cells time- and concentration-dependently induced endothelial cell death. The SNAP-induced endothelial cell death was strongly reduced by the treatment with carboxy-PTIO, a NO scavenger, or catalase, but not with superoxide dismutase (SOD). Moreover, pretreatment with sepiapterin, a precursor of BH4, increased intracellular BH4 content, and strongly reduced the SNAP-induced endothelial cell death. Both the increase in BH4 content and the protective effects of sepiapterin were prevented by co-pretreatment with N-acetylserotonin (NAS), an inhibitor of BH4 synthesis. These findings suggest that the cytotoxicity of NO released from SNAP involves H2O2 production, and increase in intracellular BH4 content attenuates NO-induced endothelial cell death. Scavenging of H2O2 by BH4 may be at least one of the mechanisms by which BH4 reduces NO-induced endothelial cell death.

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.

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 purpose of this study was to characterize the protective effect of N G -nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on oxidative stress-induced endothelial cell injury. Intracellular oxidative stress was induced by 1-chloro-2,4-dinitrobenzene, a glutathione (GSH) depleting agent, and the leakage of intracellular lactate dehydrogenase was measured as a marker of cell injury. Addition of l-chloro-2,4-dinitrobenzene (100-500 μM) induced leakage of lactate dehydrogenase from endothelial cells, and the leakage of lactate dehydrogenase was strongly attenuated by L-NAME, but not by N G -methyl-L-arginine, also an inhibitor of nitric oxide synthase. However, cell injury induced by the Ca 2+ ionophore ionomycin was not affected by L-NAME or N G -methyl-L-arginine. Moreover, neither L-NAME nor N G -methyl-L-arginine affected GSH depleting agent-induced or H 2 O 2 -induced cell injury in a rat foetal lung fibroblast cell line which lacks nitric oxide synthase. These results suggest that the protective effect of L-NAME is likely to be related to nitric oxide synthase, while the inhibition of nitric oxide production may not be involved in the protective effect of L-NAME, since N G -methyl-L-arginine did not affect endothelial cell injury.

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.

We previously reported that lysophosphatidic acid (LPA) regulates Ca²⁺ influx of fluid flow in stimulated endothelial cells and that LPA and shear stress showed increment and suppressive effects on phenylephrine-induced vasoconstriction and acetylcholine-induced vasodilatation, respectively. However, a vasoconstrictive effect of LPA alone in the presence of shear stress was not found. The present study examined the effect of LPA alone in the presence of shear stress on Ca²⁺ responses in endothelial and smooth muscle cells and contraction in mouse aortic strip using real-time 2-photon laser scanning microscopy and a custom-made parallel-plate flow chamber. Application of micromolar LPA and high shear stress elicited movement of endothelial cells after Ca²⁺ responses. The endothelial cells moved along the major axis of smooth muscle cells, a direction that was identical to that found during vasoconstriction evoked by the application of phenylephrine. The frequency of Ca²⁺ oscillations in smooth muscle cells was highest according to endothelial movement. Vasoconstriction evoked by LPA and shear stress was significantly reduced by the application of a thromboxane A₂ receptor antagonist, a cyclooxygenase inhibitor, and a thromboxane synthase inhibitor. These results suggest that micromolar LPA and high shear stress elicit vasoconstriction that is caused by Ca²⁺-dependent contraction in medial smooth muscle cells. Thromboxane A₂ may be involved in that response.

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.

The mechanism of increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) by removal of extracellular Na + , which phenomena were reported previously (Japan.J. Pharmacol. 61 83–91 1993), was investigated in cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent Ca 2+ indicator, fura-2, by digital ratio imaging microscopy. Isotonic substitution of choline chloride for NaCl induced a transient increase in [Ca 2+ ] i . The pretreatment of thapsigargin (0.5 μM), but not nicardipine (10 μM), suppressed the transient increase completely. In solutions containing micromolar concentrations of free Ca 2+ (nominally Ca 2+ -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 [Ca 2+ ] i , indicating that removal of extracellular Na + releases Ca 2+ from intracellular stores including inositol 1,4,5-trisphosphate (IP 3 )-releasable pools. The Na + -free-induced transient increase required the presence of more than micromolar concentrations of extracellular free Ca 2+ and releasable Ca 2+ within the stores, but ryanodine did not affect the transient increase. These results suggest that undetectable influx of Ca 2+ by the reverse-mode action of the Na + /Ca 2+ exchanger can release Ca 2+ from the thapsigargin-sensitive intracellular stores including IP3-releasable pools.

In order to directly detect nitric oxide (NO) liberated from isolated tissue, a practical and convenient method using a nitric oxide-sensitive electrode is described. To avoid the nonselective signal caused by ionic substances, the electrode was covered with three layers but remains permeable for gaseous substances. In a solution bubbled with 20% oxygen (pO2, approximately 150 mm Hg), administration of S-nitroso-N-acetyl-d,l-penicillamine (SNAP) at concentrations greater than 10−7 mol/L elicited an electrode response. Based on a comparison with the chemical determination of NO released from SNAP, the electrode may be able to detect nitric oxide around nmol/L. At least 30 nmol NO per liter in anoxic conditions was reported to be detected by this electrode (Matsui, 1995). In a specially designed small chamber, the electrode was attached on the surface of endothelial side of the isolated aorta of the guinea pig. When carbachol was added to the chamber, the electrode responded when the solution was bubbled with 20% but not with 40% or 95% of oxygen, suggesting a much faster decomposition of nitric oxide in the presence of higher concentrations of oxygen. The electrode response to carbachol was abolished in the presence of NG-monomethyl-L-arginine or nitro arginine. These results suggest that the electrode method described in this manuscript is suitable for detecting nitric oxide liberated from isolated tissues when comparatively low oxygen levels are present in the physiological salt solution.

Sodium nitroprusside is widely used in pharmacological studies as a potent vasodilator or a nitric oxide donor. The mechanisms of cellular death induced by sodium nitroprusside were investigated in murine neuroblastoma N1E-115 cells. Sodium nitroprusside reduced the cellular viability, and the DNA extracted from treated cells showed a ladder-like intranucleosomal fragmentation pattern, which is an indication of apoptosis. The DNA fragmentations were also visualized by in situ nick translation. The cellular death was attenuated by cycloheximide, indicating that ongoing protein synthesis was essential for the initiation of the degenerative response. However, other nitric oxide donors did not decrease the cellular viability. The nitric oxide scavenger, hemoglobin, had no effect on sodium nitroprusside-induced cellular death. Furthermore, sodium cyanide, which is formed by the metabolism of sodium nitroprusside, did not cause cellular death. On the other hand, hydrogen peroxide, another product of sodium nitroprusside metabolism, reduced the cellular viability and induced DNA fragmentation. In addition, the cell damage induced by sodium nitroprusside was enhanced by a medium without fetal bovine serum. In conclusion, we proposed that hydrogen peroxide is the important toxic species for induction of apoptosis in N1E-115 cells exposed to sodium nitroprusside.

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 μM) 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.

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.

[ 3 H]9-Methyl-7-bromoeudistomin D ([ 3 H]MBED), the most powerful Ca 2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [ 3 H]MBED binding. [ 3 H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5′-(β,γ-methylene)triphosphate. The B max value was 170 times more than that of [ 3 H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [ 3 H]MBED binding protein was different from [ 3 H]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.

The regulatory mechanisms of diacylglycerol (DG) kinase activity were studied in guinea pig taenia coli. In an octylglycoside mixed micellar assay system, DG kinase activities were distributed in both membrane and cytosolic fractions. Treatment of the tissue with carbachol (CCh) increased the activity in the membrane fraction and decreased the cytosolic fraction without affecting total DG kinase activity. The Km value of DG kinase in the membrane fraction was unchanged by treatment with CCh, although Vmax was increased. These findings suggest that DG kinase may be translocated from the cytosol to the membrane by CCh-stimulation. Increase in DG content by treatment of tissue with a cell-permeable species of DG, dioctanoyl-sn-glycerol, did not induce DG kinase translocation. Each treatment with protein kinase C (PKC) inhibitor and PKC-desensitization blocked CCh-induced DG kinase translocation; and phorbol ester induced the translocation only in intracellular calcium-accumulated tissues. Considering these results, CCh-induced DG kinase activation appears to involve DG kinase translocation from the cytosol to the membrane in association with both PKC and intracellular calcium concentration rather than cellular DG content.

Changes in diacylglycerol kinase (DG kinase) activity in carbachol (CCh)-stimulated guinea pig taenia coli were investigated. In a mixed micellar assay system, added 1,2-dioctanoyl- sn -glycerol (diC8) and endogenous DG were competitively bound to common DG kinase isozymes from guinea pig taenia coli and phosphorylated, suggesting that diC8 is useful as a probe of agonist effects on DG kinase activity. In phosphorus-32 ([ 32 P]Pi)- and diC8-prelabeled guinea pig taenia coli, diC8 was phosphorylated by DG kinase to [ 32 P]dioctanoyl-phosphatidic acid ([ 32 P]diC8-PA). CCh increased the accumulation of both [ 32 P]diC8-PA and endogenous [ 32 P]phosphatidic acid ([ 32 P]PA) in a time- and dose-dependent manner (0.1–100 μM CCh). CCh-induced increases in [ 32 P]diC8-PA and [ 32 P]PA were inhibited by 1 μM atropine and 3 μM DG kinase inhibitor (R59022). These findings indicated the activation of DG kinase by muscarinic receptor stimulation in guinea pig taenia coli. Therefore, DG kinase activation may play an important role in CCh-induced PA formation. CCh-induced [ 32 P]diC8-PA and [ 32 P]PA accumulation was dependent on intracellular calcium concentrations. However, a KCl-induced increase in intracellular calcium, without receptor stimulation, was ineffective. Moreover, treatment with phorbol ester also increased accumulation of both PA species in KCl-treated tissues. These findings suggest that muscarinic receptor mediated activation of DG kinase may require both an increase in intracellular calcium and PKC activation in guinea pig taenia coli.

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.

The soluble fraction from the ileal longitudinal muscle of guinea pigs was examined for the presence of an endogenous modulator of muscarinic receptors. In the presence of the soluble fraction, the binding of [ 3 H]quinuclidinyl benzilate to the membranes from the tissue was inhibited in a concentration-dependent manner. The inhibitory activity in the soluble fraction was heat stable, but was inactivated by trypsin treatment. Several protease inhibitors had no effect on the inhibitory activity. These results suggest the exsistence of an endogenous protein that inhibits the binding of the muscarinic ligand to the receptor. Ultrafiltration demonstrated that the protein factor had a molecular mass of more than 10,000 Da. Saturation binding and dissociation kinetic experiments indicate neither a competitive nor allosteric mode of inhibitory action and suggest that an irreversible block or internalization of muscarinic receptors is induced by the endogenous protein.

In studies on the intrinsic contraction mechanisms of smooth muscle, various factors such as cell-to-cell interaction, penetration of chemicals to the trigger regions, or extracellular spaces in the tissue may influence the results. The use of single smooth muscle cells in such investigations may help to eliminate these factors. Single smooth muscle cells can be isolated from guinea pig taenia coli by digesting the minced tissue with a combination of collagenase and soybean trypsin inhibitor. The cells are spindle shaped, 100-200 microns in length and 10 microns in diameter. Three kinds of techniques for determination of contractile responses of single smooth muscle cells have been established: 1) fixation method, 2) perfusion method, and 3) capillary adhesion method. Using these techniques, the cells appeared to be contracted in a dose-dependent manner (graded response), while another mode of the contractile response, the "all-or-none response", has also been proposed. Scanning and transmission electron micrographs of the single cells revealed several evaginations of various sizes and shapes on the surface and electron dense bodies at the neck of the evaginations. ED50 obtained with the single cells, prepared from taenia coli of guinea pigs with collagenase in the presence of trypsin inhibitor was similar to that obtained with the intact tissue. Digestion of the tissue with highly purified collagenase and papain yields a large number of single cells with higher sensitivity to agonists. Characteristics of cells isolated from ileal longitudinal muscle of guinea pig are similar to those of cells isolated from taenia coli. The magnitude of contraction and sensitivity to acetylcholine were different in each cell. Incubation of isolated tissues with various concentrations of agonists followed by measurement of cell length in the tissue showed that the heterogeneous responses of the cells were intrinsic characteristics of smooth muscle cells and not due to damage incurred during the isolation procedure. Since isolated smooth muscle cells possess contractility, these might preserve the characteristics of smooth muscle, so they can be used for smooth muscle research. It must be considered, however, that each cell in the smooth muscle tissue has different contractility.

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 Ca2+-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+]0 to generate the [Ca2+]i oscillations was observed. An inorganic Ca2+-antagonist, LaCl3, immediately suppressed the [Ca2+]i oscillations, but the treatment with verapamil or nicardipine, Ca2+-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. These results suggest the following: (1) The generation of the spontaneous [Ca2+]i oscillations is highly dependent on [Ca2+]o but not due to Ca2+ influx through voltage-dependent Ca2+ channels; (2) Thapsigargin-sensitive Ca2+ pumping pools, which may be inositol 1,4,5-trisphosphate (IP3)-releasable Ca2+ pools, play an important role in generating the spontaneous [Ca2+]i oscillations, and the uptake of Ca2+ into the pools is highly dependent on Ca2+ influx across the plasma membrane.

The biosynthetic mechanism for determining the side-chain length of ubiquinone in rat heart mitochondria was investigated. The biosynthesis of nonaprenyl ubiquinone (UQ-9) and decaprenyl ubiquinone (UQ-10) in the mitochondria from rat hearts previously perfused with mevalonolactone was accelerated depending on the concentration of mevalonolactone. Furthermore the synthesis ratio between UQ-10 and UQ-9 (UQ-10/UQ-9) increased in accordance with the increasing concentration of mevalonolactone used. In addition, an enhancement of the synthesis ratio (UQ-10/UQ-9) was observed when the rats were treated with isoproterenol to increase the activity of 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme which forms mevalonate. Moreover, the addition of isopentenyl pyrophosphate, which is a metabolite of mevalonate, elevated the synthetic ratios UQ-10/UQ-9 in intact mitochondria and decaprenyl pyrophosphate/solanesyl pyrophosphate in the partially purified polyprenyl pyrophosphate synthetase from rat heart. These results suggest that the HMG-CoA reductase could be involved as a determining factor of the side-chain length of ubiquinone in rat heart.