Long-term treatment with TGFbeta1 impairs mechanotransduction in bovine aortic endothelial cells.

BACKGROUND AND PURPOSE: Vascular endothelial cells play a role in the physiological response to mechanical stress. Transforming growth factor beta1 (TGFbeta1) induces morphological changes in endothelial cells, and this may alter their mechanosensitive responses. The aim of this study was to examine the effects of TGFbeta1 on hypotonic stress (HTS)-induced responses in bovine aortic endothelial cells (BAECs). EXPERIMENTAL APPROACH: Cultured BAECs were treated with 3 ng ml(-1) TGFbeta1 for 24 h (24h-TGFbeta1) or 7 days (7d-TGFbeta1). Cytosolic actin fibres were stained with rhodamine-phalloidin. Intracellular Ca2+ concentration was measured using fura2. Tyrosine phosphorylation and RhoA expression were assessed by Western blotting. Expression of RhoA mRNA was assessed by real-time PCR. KEY RESULTS: BAECs developed pseudopod-like processes within 24 h and showed a fibroblast-like appearance after 7 days. HTS induced Ca2+ transients via endogenous ATP release in both control and 24h-TGFbeta1 BAECs but not in 7d-TGFbeta1 BAECs. We have previously shown that HTS-induced ATP release is mediated by sequential activation of RhoA and tyrosine kinases. The basal amount of membrane-bound RhoA was significantly lower in 7d-TGFbeta1 than in 24h-TGFbeta1 or control BAECs. HTS increased the membrane-bound RhoA to the same fractional level in 24h-TGFbeta1 and control BAECs, but its net maximal amount was significantly lower in 7d-TGFbeta1. HTS-induced downstream signals of RhoA activation, i.e. the tyrosine phosphorylation of FAK and paxillin, were markedly suppressed in 7d-TGFbeta1 BAECs. CONCLUSIONS AND IMPLICATIONS: These results indicate that long-term treatment with TGFbeta1 does not impair mechanoreception in BAECs but impairs mechanotransduction by affecting RhoA membrane translocation.

Sustained contraction and loss of NO production in TGFbeta1-treated endothelial cells.

BACKGROUND AND PURPOSE: Transforming growth factor beta1 (TGFbeta1) is generated in atherosclerotic and injured vessel walls. We examined whether the endothelial-to-mesenchymal transdifferentiation induced by TGFbeta1 affects endothelial functions. EXPERIMENTAL APPROACH: Bovine aortic endothelial cells (BAECs) were treated with 3 ng ml(-1) TGFbeta1 for 7 days. Contraction of TGFbeta1-treated BAECs was assessed by collagen gel contraction assay. Protein expression and phosphorylation were assessed by Western blotting. Intracellular Ca2+ concentration and NO production were measured using fura2 and DAF-2, respectively. KEY RESULTS: TGFbeta1-treated BAECs showed dense actin fibers and expressed smooth muscle marker proteins; they also changed into smooth muscle-like, spindle-shaped cells in collagen gel cultures. ATP (10 microM) induced a gradual contraction of collagen gels containing TGFbeta1-treated BAECs but not of gels containing control BAECs. ATP-induced contraction of TGFbeta1-treated BAECs was not reversed by the removal of ATP but was partially suppressed by a high concentration of sodium nitroprusside (1 microM). TGFbeta1-treated BAECs showed sustained phosphorylation of myosin light chain in response to ATP and low levels of basal MYPT1 expression. ATP-induced Ca2+ transients as well as eNOS protein expression were not affected by TGFbeta1 in BAECs. However, ATP-induced NO production was significantly reduced in TGFbeta1-treated BAECs. Anti-TGFbeta1 antibody abolished all of these TGFbeta1-induced changes in BAECs. CONCLUSIONS AND IMPLICATIONS: Mesenchymal transdifferentiation induced by TGFbeta1 leads to sustained contraction and reduced NO production in endothelial cells. Such effects, therefore, would not be beneficial for vascular integrity.

Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration.

Cell migration is a fundamental biological process involving membrane polarization and cytoskeletal dynamics, both of which are regulated by Rho family GTPases. Among these molecules, Rac is crucial for generating the actin-rich lamellipodial protrusion, a principal part of the driving force for movement. The CDM family proteins, Caenorhabditis elegans CED-5, human DOCK180 and Drosophila melanogaster Myoblast City (MBC), are implicated to mediate membrane extension by functioning upstream of Rac. Although genetic analysis has shown that CED-5 and Myoblast City are crucial for migration of particular types of cells, physiological relevance of the CDM family proteins in mammals remains unknown. Here we show that DOCK2, a haematopoietic cell-specific CDM family protein, is indispensable for lymphocyte chemotaxis. DOCK2-deficient mice (DOCK2-/-) exhibited migration defects of T and B lymphocytes, but not of monocytes, in response to chemokines, resulting in several abnormalities including T lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. In DOCK2-/- lymphocytes, chemokine-induced Rac activation and actin polymerization were almost totally abolished. Thus, in lymphocyte migration DOCK2 functions as a central regulator that mediates cytoskeletal reorganization through Rac activation.

Alterations of Ca2+ mobilizing properties in migrating endothelial cells.

Endothelial migration is one of the major events of pathological neovascularization. We compared the characteristics of Ca2+ mobilization in nonconfluent, confluent, and migrating endothelial cells. Migration of endothelial cells was induced by wounding the confluent cell monolayer. The basal intracellular Ca2+ concentration was lower in migrating cells and higher in confluent cells than in nonconfluent cells. Thapsigargin (TG)-induced Ca2+ leak and TG-evoked Ca2+ entry were accelerated in migrating cells, whereas the latter was suppressed in confluent cells. The ATP-induced Ca2+ transient was also much larger in migrating cells than in confluent cells. These alterations were also observed in a cell as an intracellular polarization, i.e., the leading edge showed an acceleration of TG-evoked Ca2+ entry and an augmentation of the ATP-induced Ca2+ transient. Endothelial migration was significantly suppressed by TG or cyclopiazonic acid. These observations suggest that the alterations of Ca2+ store site-related Ca2+ mobilizations, i.e., Ca2+ sequestration, release, and TG-evoked Ca2+ entry, may be involved in the cellular mechanisms of endothelial migration.

Involvement of Rho-kinase and tyrosine kinase in hypotonic stress-induced ATP release in bovine aortic endothelial cells.

Hypotonic stress induces ATP release followed by Ca2+ oscillations in bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress-induced ATP release. Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-mediated Ca2+ oscillations induced by hypotonic stress. ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-treating the cells with Y-27632, a selective inhibitor of Rho-kinase, also suppressed the hypotonic stress-induced ATP release and Ca2+ oscillations, indicating that Rho-mediated activation of Rho-kinase may be involved in the hypotonic ATP release. Hypotonic stress also induced a transient rearrangement of the actin cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress-induced ATP release nor the Ca2+ oscillations. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathways are involved in hypotonic stress-induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs.

The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

Impairment of endothelial nitric oxide production by acute glucose overload.

We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM D-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677-685, 1998) that glucose overload impairs Ca(2+) mobilization due to an accumulation of superoxide anions (O(2)(-)) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to Ca(2+) entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an O(2)(-) donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular Ca(2+) concentration increase induced by the Ca(2+) ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and high D-glucose-treated cells in Ca(2+)-free solution. These results indicate that glucose overload impairs NO production by the O(2)(-)-mediated attenuation of Ca(2+) entry.

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

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

Hypotonic stress-induced NO production in endothelium depends on endogenous ATP.

The mechanism by which mechanical stress induces nitric oxide (NO) synthesis in endothelium is still controversial. Hypotonic stress (HTS, -20%) induced ATP release, which evoked Ca(2+) transients in bovine aortic endothelial cells (BAEC). HTS also induced NO synthesis, assessed by DAF-2 fluorescence, which was suppressed by inhibiting endogenous ATP-induced Ca(2+) transients with suramin or neomycin. Exogenously applied ATP mimicked these responses. Pretreatment with wortmannin did not affect DAF-2 fluorescence, suggesting that Akt phosphorylation was not involved in HTS-induced NO synthesis. These results indicate that endogenous ATP plays a central role in HTS-induced NO synthesis in BAEC.

Hypotonic stress-induced dual Ca(2+) responses in bovine aortic endothelial cells.

We have investigated the effects of hypotonic stress on intracellular calcium concentration ([Ca(2+)](i)) in bovine aortic endothelial cells. Reducing extracellular osmolarity by 5% to 40% elicited a steep Ca(2+) transient both in normal Krebs and Ca(2+)-free solutions. The hypotonic stress-induced Ca(2+) transient was inhibited by phospholipase C inhibitors (neomycin and U-73122), a P(2)-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyrase), suggesting that the hypotonic stress-induced Ca(2+) transient is mediated by ATP. A luciferin-luciferase assay confirmed that 40% hypotonic stress released 91.0 amol/cell of ATP in 10 min. When the hypotonic stress-induced fast Ca(2+) transient was inhibited by neomycin, suramin, or apyrase, a gradual [Ca(2+)](i) increase was observed instead. This hypotonic stress-induced gradual [Ca(2+)](i) increase was inhibited by a phospholipase A(2) inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [Ca(2+)](i) increase with an ED(50) of 13.3 microM. These observations indicate that hypotonic stress induces a dual Ca(2+) response in bovine aortic endothelial cells, i.e., an ATP-mediated fast Ca(2+) transient and an arachidonic acid-mediated gradual Ca(2+) increase, the former being the predominant response in normal conditions.

Inhibition of Ca(2+) signalling by p130, a phospholipase-C-related catalytically inactive protein: critical role of the p130 pleckstrin homology domain.

p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P(3). Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca(2+) signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca(2+) concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P(3); the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.

Effect of eotaxin and platelet-activating factor on airway inflammation and hyperresponsiveness in guinea pigs in vivo.

Although eotaxin causes selective infiltration of eosinophils into the lung, its role in airway hyperresponsiveness remains unclear. We studied the effects of local administration of eotaxin on airway inflammation and hyperresponsiveness in guinea pigs in vivo. Airway responsiveness to inhaled histamine and differential cell counts in bronchoalveolar lavage fluid (BALF) were evaluated 12 h, 24 h, 3 d, and 7 d after intratracheal instillation of eotaxin. Significant eosinophilia in BALF was observed between 6 h and 7 d after eotaxin administration. Histologically, eosinophil accumulation was observed in the airways but not in the alveoli. In contrast, eotaxin did not affect airway responsiveness between 12 h and 7 d after its administration. We then studied the effects on airway responsiveness of subthreshold doses of interleukin 5, leukotriene D(4) (LTD(4)), and platelet-activating factor (PAF) combined with eotaxin. Neither interleukin 5 nor LTD(4) affected airway responsiveness. After eotaxin treatment, PAF significantly enhanced airway responsiveness without further increases in eosinophil counts. Eotaxin plus PAF significantly increased in eosinophil peroxidase activity in BALF compared with control and with eotaxin alone. These data indicate that eotaxin alone causes eosinophil accumulation in the airways but not hyperresponsiveness, and that additional factors such as PAF are needed to activate eosinophils for the development of airway hyperresponsiveness.

Superoxide anion impairs Ca(2+) mobilization in cultured human nasal epithelial cells.

We examined the effects of superoxide anion (O(-2)) on the intracellular Ca(2+) concentration in cultured human nasal epithelial cells. The cells were exposed to O(-2) by pretreatment with xanthine (X) and xanthine oxidase (XO); control cells were treated with X alone. When Ca(2+)-containing Krebs solution was reperfused in the thapsigargin-treated, store-depleted cells, reapplication-induced intracellular Ca(2+) concentration elevation was significantly smaller in X/XO-treated cells than in the control cells, suggesting that O(-2) impairs Ca(2+) release-activated Ca(2+) entry (CRAC). Bath application of ATP induced a steep Ca(2+) transient in both control and X/XO-treated cells. However, the concentration-response curve of the ATP-induced Ca(2+) transient was shifted to a higher concentration in X/XO-treated cells. The impairments of CRAC and ATP-induced Ca(2+) transient induced by X/XO were reversed by superoxide dismutase. Furthermore, all these X/XO-induced effects were also observed in cells pretreated with pyrogallol, also an O(-2) donor. These results indicate that O(-2) impairs at least two mechanisms involved in Ca(2+) mobilization in human nasal epithelial cells, i.e., CRAC and ATP-induced Ca(2+) release.

Effects of superoxide anion on intracellular Ca2+ concentration in rabbit pulmonary arterial smooth muscle cells.

AIM: To study the effect of superoxide anion on the Ca2+ homeostasis in smooth muscle cells isolated from the rabbit pulmonary artery. METHODS: Intracellular Ca2+ concentration ([Ca2+]i) was investigated using cell suspension of freshly isolated smooth muscle cells from rabbit pulmonary artery (PASMC). Fura-2 fluorescent ratio obtained at 340 nm and 380 nm wave lengths was measured as an indicator of [Ca2+]i. RESULTS: ATP 30 mumol.L-1 induced a transient increase in the ratio (Ca2+ transient). Thapsigargin, an inhibitor of sarcoplasmic Ca2+ ATPase, induced a phasic increase in the ratio due to Ca2+ leak from intracellular store sites, but not the sustained increase, thereby suggesting the absence of Ca2+ release-activated Ca2+ entry (CRAC) mechanism in PASMC. When PASMC were exposed to superoxide anion by the pretreatment with xanthine and xanthine oxidase (X/XO) for 30 min, sustained component of ATP-induced Ca2+ transient was elevated. The ratios at 5 and 10 min after ATP application (delta ratio5 min and delta ratio10 min) were increased from 0.091 +/- 0.022 to 0.149 +/- 0.048 (P < 0.05) and from 0.021 +/- 0.020 to 0.117 +/- 0.047 (P < 0.01), respectively. But, thapsigargin-induced [Ca2+]i transient was not affected by X/XO. CONCLUSION: Superoxide anion makes ATP-induced Ca2+ transient sluggish, and does not affect Ca2+ leak pathway in PASMC.

Increase in calcium in smooth muscle cells of the rabbit bladder induced by acetylcholine and ATP.

Cultured smooth muscle cells from rabbit urinary bladder were loaded with fura-2. Changes in intracellular Ca concentration [Ca2+]i produced by acetylcholine (ACh) or adenosine triphosphate (ATP) were estimated by measuring the fluorescence ratio F340/F380. Western blot analysis and immunohistochemical techniques showed that the cultured cells retained alpha-smooth muscle actin. ATP produced a rapid but transient increase in [Ca2+]i and ACh produced a delayed, prolonged increase. Application of ACh after ATP in Ca-free solution failed to elevate [Ca2+]i suggesting that both ACh and ATP release Ca2+ from the same intracellular stores. Following application of ACh but not ATP in Ca-free Krebs solution, reintroduction of Ca2+ produced elevation of [Ca2+]i, indicating that ACh causes prolonged opening of channels in the membrane. The sustained increase induced by ACh was abolished by nicardipine (blocker of Ca2+ voltage dependent channel ICa(V)) or quinine (blocker of non-selective cation channels). Although the elevations to ACh or ATP were abolished by neomycin (an inhibitor of phospholipase C) the different time courses suggest that the mechanisms of release of Ca2+ from intracellular stores or the pathway for refilling the stores is different.

Acute glucose overload abolishes Ca2+ oscillation in cultured endothelial cells from bovine aorta: a possible role of superoxide anion.

Effects of acute glucose overload on [Ca2+]i were investigated in cultured endothelial cells from bovine aorta. Application of 0.1 micromol/L ATP elicited an oscillatory increase in [Ca2+]i (Ca2+ oscillation) in Krebs solution containing 11.5 mmol/L glucose. The frequency of Ca2+ oscillation induced by ATP increased in a concentration-dependent manner, ranging between 0.03 and 1 micromol/L. When cells were preincubated with 23 mmol/L glucose-containing Krebs solution (high glucose solution) for 3 hours, 0.1 micromol/L ATP failed to induce Ca2+ oscillation but evoked only a phasic followed by sustained increase in [Ca2+]i. Application of a higher concentration of ATP (10 micromol/L) evoked a transient increase in [Ca2+]i both in control and high glucose-treated cells. However, the falling phase of [Ca2+]i was prolonged in high glucose-treated cells. Thapsigargin (1 micromol/L), an inhibitor of endoplasmic Ca2+-ATPase, induced a transient followed by a sustained increase in [Ca2+]i in control cells. Preincubation with high glucose solution increased the rate of rise of the thapsigargin-induced increase in [Ca2+]i and abolished the sustained increase, suggesting that glucose overload accelerates Ca2+ leak from intracellular store sites and impairs Ca2+ release-activated Ca2+ entry. We found that all of the glucose overload-induced changes in Ca2+ mobilization could be mimicked by xanthine with xanthine oxidase and abolished by superoxide dismutase. These results indicate that acute glucose overload accumulates superoxide anion in bovine aortic endothelial cells, thereby diminishing ATP-induced Ca2+ oscillation through the impairment of Ca2+ homeostasis.

Effects of acute glucose overload on histamine H2 receptor-mediated Ca2+ mobilization in bovine cerebral endothelial cells.

[Ca2+]i and whole-cell membrane current were measured in microvascular endothelial cells from bovine brain. The effects of histamine on [Ca2+]i were examined, and the acute effect of changing extracellular glucose concentration on Ca2+ homeostasis was investigated. Application of 10 micromol/l histamine evoked an initially transient and then sustained increase in [Ca2+]i in normal Krebs solution, but only the transient component in Ca2+-free solution, thereby indicating that histamine mobilizes Ca2+ both from intracellular store sites and extracellular space. The effects of histamine on [Ca2+]i were inhibited by the H2 antagonists, ranitidine and cimetidine, but not by the H1 antagonist, pyrilamine. Incubation of the cells for 2 h in solutions containing low (1.1 and 2.3 mmol/l) or high (23 mmol/l) concentrations of glucose did not influence the resting level of [Ca2+]i. Treatment with low concentrations of glucose did not impair histamine-induced Ca2+ mobilization. On the other hand, when histamine was applied to the cells pretreated with 23 mmol/l glucose, it failed to mobilize Ca2+ from both intracellular store sites and extracellular space. The effect of histamine was mimicked by dibutyryl cyclic AMP, but glucose overload failed to inhibit this, suggesting that glucose overload inhibits H2 receptor-mediated cyclic AMP production. Glucose overload-induced impairment of histamine action was reversed by pretreatment with staurosporine and calphostin C and mimicked by phorbol-12,13-dibutyrate, thereby suggesting the involvement of protein kinase C in the high glucose-induced inhibition of Ca2+ mobilization. Whole-cell membrane current measurement showed that there was no difference in the membrane currents between control and high glucose-treated cells. These results indicate that in bovine brain microvascular endothelial cells, histamine induces Ca2+ release from intracellular store sites and subsequent entry from the extracellular space through the activation of H2 receptors. Glucose overload acutely inhibits histamine-induced Ca2+ mobilization by the activation of protein kinase C.

Hydrogen peroxide induced responses of cat tracheal smooth muscle cells.

1. The effects of hydrogen peroxide (H2O2) (10(-6)-10(-3) M) on membrane potential, membrane currents, intracellular calcium concentration, resting muscle tone and contractions elicited by electrical field stimulation (EFS) and carbachol were examined in cat tracheal strips and isolated smooth muscle cells. 2. H2O2 (10(-4) and 10(-5) M) enhanced the amplitude of contractions and excitatory junction potentials (e.j.p.) evoked by EFS without changing muscle tone and resting membrane potential of the tracheal smooth muscle, and enhanced the contraction induced by carbachol (10(-3) M). At an increased concentration (10(-3) M), H2O2 elevated resting muscle tone and marginally hyperpolarized the membrane in the majority of the cells. 3. In 51 out of 56 cells examined, H2O2 (10(-6)-10(-3) M) elicited an outward current at a holding potential of -40 mV and enhanced the frequency of the spontaneous transient outward current (STOC). In 20 cells the outward current was preceded by a small inward current. In the other cells, H2O2 elicited only an inward current or did not affect the background current. 4. In Ca2+ free solution the action of H2O2 on the resting muscle tone, STOCs, background current and on the current induced by ramp depolarization was significantly reduced. 5. H2O2 (10(-4) M) increased the intracellular ionized calcium concentration both in the absence and presence of external Ca2+. However, the effect developed faster and was of a higher amplitude in the presence of external Ca2+. 6. These results suggest that H2O2 increases intracellular Ca2+, with a subsequent augmentation of stimulation-evoked contractions, and enhances Ca2+ and voltage-sensitive potassium conductance.

The electrical properties and responses to nerve stimulation of the proximal urethra of the male rabbit.

OBJECTIVE: To identify the nature of neurotransmitters acting on the smooth muscle cells of the proximal urethra and the electrical activity underlying the mechanical responses to nerve stimulation. MATERIALS AND METHODS: The electrical activity of longitudinal strips of proximal urethra obtained from male rabbits was recorded with microelectrode and double sucrose-gap techniques. Intramural nerves were stimulated with 200 microseconds pulses. Drugs which selectively affected neurotransmission were added to the perfusing fluid. RESULTS: The mean resting potential of smooth muscle cells was -39 mV and they had infrequent spontaneous action potentials. The frequency was increased by noradrenaline without depolarization; ATP produced depolarization with increased action-potential frequency. Small (< or = 16 mV) spontaneous depolarizations and hyperpolarizations could be recorded from < 50% of cells; large (< or = 40 mV) hyperpolarizations also occurred. Electrical field stimulation evoked excitatory and inhibitory junction potentials (EJPs and IJPs). Most EJPs reached a maximum within 500 ms, decayed with a time-constant of 200-300 ms and were blocked by alpha, beta-methylene ATP. IJPs had a latency of > 1 s and showed much variation in amplitude and duration. IJPs could still be recorded in the presence of N-nitro-L-arginine methyl ester (L-NAME). In the double sucrose-gap, depolarization and associated contraction were reduced by alpha, beta-methylene ATP. Further reduction occurred with phentolamine and the remaining response was blocked by atropine. A large hyperpolarization could occur in response to a single stimulus but this decreased progressively with repeated stimulation. The hyperpolarization was partially blocked by L-NAME. CONCLUSIONS: The proximal urethra receives excitatory innervation involving three neurotransmitters, i.e. ATP, acetylcholine and noradrenaline, and inhibition is associated with hyperpolarization which results at least in part from action of non-nitrergic nerves.