Biomedical subjects

H Ohshika

Publications and source records attributed to H Ohshika.

At least 55 records · Page 3Linked to original sources

PubMed1995

Tubulin stimulates adenylyl cyclase activity in rat striatal membranes via transfer of guanine nucleotide to Gs protein.

Previous studies of rat cerebral cortex and rat C6 glioma cells have demonstrated that dimeric tubulin is capable of activating the G proteins Gs and Gil via transfer of guanine nucleotide from tubulin to Gs alpha and Gil alpha. To provide further information regarding cytoskeletal modulation of adenylyl cyclase, the present study examined effects of tubulin on the activation of the enzyme in rat striatal membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analog 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of adenylyl cyclase by approximately 130%. Furthermore, tubulin-GppNHp activated SKF 38393-sensitive adenylyl cyclase and potentiated forskolin-stimulated activity of the enzyme. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analog [32p]p3 (4-azidoanilido)-p1-5'-GTP ([32P]AAGTP), was incubated with striatal membranes, AAGTP was transferred from tubulin to Gs alpha as well as Gi alpha with the extents of nucleotide transfers being 7.6 +/- 0.8% and 17.8 +/- 1.4% of AAGTP originally bound to tubulin, respectively. These results indicate that, in rat striatum, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gs alpha, supporting the hypothesis that tubulin contributes to the regulation of neuronal signal transduction.

Adenylyl Cyclases↗

PubMed1995

Impairment of adenylyl cyclase signal transduction in mecobalamin-deficient rats.

This study examined alterations in the beta-adrenoceptor-G5-adenylyl cyclase system in cerebral cortex membranes from vitamin B12-deficient rats fed a diet lacking vitamin B12 (mecobalamin) for 15 weeks. Basal, 5(7)-guanylylimidodiphosphate (GppNHp)-, isoproterenol-, and forskolin-stimulated adenylyl cyclase activities were significantly reduced in mecobalamin-deficient rats compared with those in control rats. However, no significant differences were observed in the amount and function of G5- estimated by immunoblotting and guanine nucleotide photoaffinity labeling, respectively, or in the densities and the dissociation constants of beta-adrenoceptors, estimated by [125I] pindolol binding, between control and the deficient rats. These results indicate that vitamin B12 deficiency results in the impairment of the coupling among the beta-adrenoceptor, G5- and the catalytic subunit of adenylyl cyclase, and in dysfunction of the catalytic subunit of the enzyme, suggesting that vitamin B12 participates in the regulation of neuronal adenylyl cyclase signal transduction.

Adenylyl Cyclase Inhibitors↗

PubMed1995

[The significance of platelet alpha 2-adrenoceptor and sympathetic nerve activity in the hypertensive mechanism under essential hypertension--with special reference to pressor response to norepinephrine].

Although many studies have examined the metabolism of catecholamines and cardiovascular responsiveness to norepinephrine in essential and various experimental hypertension, the role of sympathetic nervous system in the pathogenesis of hypertension has not been elucidated. In this study, therefore,the role of sympathetic nerve activity related to platelet alpha 2-adrenoceptor was investigated to clarify the mechanism in which sympathetic nervous system augments the blood pressure elevation in patients with essential hypertension (EHT). Tritiated yohimbine binding was used to estimate platelet membrane alpha 2-adrenoceptor characteristics in 27 hospitalized patients with mild to moderate EHT and 27 normotensive subjects (NT) receiving a regular diet containing 120mEq/day of sodium and 75mEq/day of potassium. In this study, mean arterial pressure (MAP) and plasma norepinephrine concentration (pNE) was significantly higher in EHT than those in NT. Total binding sites (Bmax) and dissociation constant (Kd) for 3H-yoshimbine in EHT was also significantly higher than those in NT. There was a significant positive correlation between Bmax and age in NT, but not in EHT. A significant positive correlation was observed between the pressor response to infused norepinephrine (NE-R:increments in MAP induced by i.v. infused 0.2 micrograms/kg/min of NE) and Bmax both in NT and EHT. On the other hand, no significant correlation was found between NE-R and Kd in NT and EHT. In addition, Bmax was correlated inversely with PNE in both NT and EHT. These findings suggest that down-regulation mechanism exists in platelet alpha 2-adrenoceptor number responded to PNE levels. Moreover, the increased density of alpha-adrenoceptor might have something to do with the augmented NE-R in EHT, indicating an important pathophysiological role of this receptor in the hypertensive mechanisms in EHT.

Adult↗

PubMed1995

Reversible inhibition of gap junctional intercellular communication, synchronous contraction, and synchronism of intracellular Ca2+ fluctuation in cultured neonatal rat cardiac myocytes by heptanol.

We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca2+ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca2+ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca2+ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca2+ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca2+ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca2+ fluctuations, which facilitate synchronized contraction of cardiac myocytes.

Alcohols↗

PubMed1995

Beta-adrenergic receptor-mediated calcium mobilization in the human Jurkat T cell line.

The regulation of Ca2+ mobilization by beta-adrenergic receptor in the human Jurkat T cell line was investigated. Jurkat cells had a single class of beta-adrenergic receptor binding sites. Isoproterenol (ISP) caused the increase in cytosolic free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. In the absence of extracellular Ca2+, the pretreatment with OKT3, an anti-CD3 antibody, did not affect a transient increase in [Ca2+]i induced by the subsequent addition of ISP, and vice versa. On the other hand, the pretreatment with thapsigargin abolished the response of [Ca2+]i to the subsequent additions of ISP and OKT3. In permeabilized Jurkat cells, the addition of cAMP released Ca2+ from the intracellular Ca2+ pool. Neither nimodipine nor H8, a protein kinase A inhibitor, affected the increase in [Ca2+]i induced by ISP. The results suggest that cAMP accumulated by the activation of beta-adrenergic receptor may directly release Ca2+ from the inositol trisphosphate-insensitive intracellular Ca2+ pool in Jurkat T cells.

Calcium↗

PubMed1995

Participation of tubulin in the stimulatory regulation of adenylyl cyclase in rat cerebral cortex membranes.

This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by approximately 156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for beta-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gs alpha as well as Gi alpha. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to Gs alpha, as well as affecting the Gi-mediated inhibitory pathway.

Adenylyl Cyclases↗

PubMed1995

Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

Adenylyl Cyclases↗

PubMed1995

[Analysis of GTP-binding protein function with a photoaffinity GTP analog].

The mechanism whereby hormones or neurotransmitters activate G proteins and their intracellular effectors can be studied in reconstituted systems using purified components. However, the regulation of receptor-G protein signaling appears to be substantially more complex in the cell and several additional components participate in this event. To study the relationship among G proteins receptors, and effectors in complex systems, such as membranes of permeable cells, it is necessary to employ methods that selectively allow the examination of G protein activation. One such method is photoaffinity labeling using a hydrolysis-resistant, photoaffinity GTP analog, P3 (4-azidoanilido)-P1-guanosine 5'-triphosphate (AAGTP). Here we describe the synthesis and purification of [32P]AAGTP as well as a procedure suitable studying the G protein function in membrane preparations. Photoaffinity labeling in rat cerebral cortex membranes showed that at least four G proteins (GsH, GsL, Gi, and Go) were labeled by [32P]AAGTP. [32P]AAGTP labeling on Gs and Gi was altered in concert with the activation states of those G proteins. An agonist-specific increase in [32P]AAGTP labeling of the G protein alpha-subunit in a membrane preparation has also been demonstrated. Thus, the photoaffinity labeling method with [32P]AAGTP makes it possible to investigate the behavior of individual G proteins in complex systems such as membrane preparations.

Affinity Labels↗

PubMed1995

Effects of 2-deoxyglucose, a metabolic inhibitor, on spontaneous contraction and adrenoceptor responsiveness in cultured rat ventricular myocytes.

Although it is well known that myocardial ischemia induces the depletion of myocardial ATP and sustained myocardial dysfunction, the mechanisms causing impaired myocardial function have not been elucidated completely. To clarify the relationship between ATP depletion and myocardial contractility, we investigated the influence of myocardial ATP depletion on spontaneous beating in cultured rat ventricular myocytes. Furthermore, because catecholamines have been used to improve myocardial contraction in the ischemic heart, we attempted to determine whether the ATP depletion per se alters the contractile responses to alpha 1- and beta-adrenoceptor stimulation. After 24 hr of culture in the presence of a metabolic inhibitor, 2-deoxyglucose (2DG, 5mM), myocardial contractility decreased to 19% of the vehicle level, and returned to normal after the removal of 2DG. The beating rate did not show any alterations in the vehicle, in the presence of 2DG (2DG [+/+]) or after the removal of 2DG (2DG [+/-]). Norepinephrine (NE) caused significant decreases in beating rate and increases in contractility in all groups. Isoproterenol (ISP) caused significant increases in beating rate and contractility in all groups. In the 2DG (+/+) group, the contractility was significantly lower as compared to other groups during NE or ISP stimulation. However, the percent change of contractility was similar to those of other groups after NE or ISP stimulation in the 2DG (+/+) group. These results suggest that decreased myocardial ATP causes the decreased contractility and does not affect the alpha 1- or beta-adrenoceptor-mediated responses.

Adenosine Triphosphate↗

PubMed1995

Gq/11 communicates with thromboxane A2 receptors in human astrocytoma cells, rabbit astrocytes and human platelets.

Western blot analysis was performed to clarify the presence of trimeric G protein subfamily in membranes derived from human astrocytoma cells (1321N1), cultured rabbit astrocytes and human platelets, using G protein antisera GS alpha, Gi alpha, Gq/11 alpha, and G beta were found to exist in membranes derived from human astrocytoma cells and rabbit astrocytes as well as human platelets. However, only small amount of G(o) alpha was detected in any membranes. Gq/11 alpha was expressed much more in human platelets than in human astrocytoma cells or rabbit astrocytes. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of thromboxane A2 (TXA2), activated GTPase in membranes derived from human astrocytoma cells, rabbit astrocytes and human platelets with different potencies. STA2-induced GTPase activation in human platelet membranes was partly inhibited by treatment with QL antibody at 0 degrees C for 90 min. Furthermore, STA2-induced GTPase activation in membranes derived from human astrocytoma cells and rabbit astrocytes were potently inhibited by treatment with QL antibody. The results obtained indicate that TXA2 receptors in human astrocytoma cells and rabbit astrocytes communicate with Gq/11 as well as in human platelets.

Animals↗

PubMed1995

Calcium antagonistic actions of tetrandrine depend on cell types.

We examined the effects of tetrandrine (TET) on Ca2+ mobilization in various types of cells using inositol trisphosphate-generating drugs and compared it with those using the microsomal Ca(2+)-ATPase inhibitor thapsigargin (TG) which is a tool for analyzing Ca2+ store-regulated Ca2+ entry (capacitative Ca2+ entry). In rat pheochromocytoma PC12 cells, 100 microM TET abolished high K+ (30 mM)-induced sustained increase in [Ca2+]i and partially inhibited bradykinin (1 microM)-induced or TG (100 nM)-induced Ca2+ entry. In NIH/3T3 fibroblasts, 100 microM TET abolished Ca2+ entry induced by bombesin (1 microM) or TG (100 nM). In rat glioma C6 cells, the addition of 100 microM TET reduced the sustained elevation of [Ca2+]i induced by endothelin 1 (10 nM) or TG (100 nM) declining to the resting level. In rat parotid acinar cells, 100 microM TET abolished a sustained increase in [Ca2+]i induced by carbachol (100 microM) or TG (100 nM). In human leukemia T-cell line Jurkat, 100 microM TET did not inhibit Ca2+ entry evoked by the anti-CD3 antibody OKT3 (10 micrograms/ml) or TG (100 nM). The present results suggest that the action of TET on Ca2+ entry is dependent on cell types.

Alkaloids↗

PubMed1994

Impaired expression of Gs alpha protein mRNA in rat ventricular myocardium with aging.

We have recently reported that the responsiveness of adrenoceptors is decreased with aging in rat ventricular myocardium. Thus, the current study determined aging-dependent changes in: (a) characteristics of myocardial G proteins as determined by Western blot analysis; (b) steady-state levels of G protein mRNA as determined by Northern blot analysis; and (c) the intropic response to isoproterenol, a beta-adrenoceptor agonist. Cardiac preparations were isolated from male Wistar rats of 6 (adult) and 24 (old) months old. Compared with adults, aging decreased the combined level of the three Gs alpha subunits (45, 47 and 52 kDa) by a total of 23% in ventricular membrane preparations. In contrast, levels of Gi alpha (40/41 kDa), Gq alpha (42 kDa), Go alpha (39 kDa) and G common beta (35/36 kDa) immunoreactivity were not affected by aging in the same membrane preparations. In ventricular myocardium, steady-state levels of Gs alpha mRNA (1.9 kb) decreased by 20-28% between 6 and 24 months of age with no change in Gi alpha mRNA (2.4 kb). An aging-associated decline in beta-adrenergic stimulation was observed in the maximum positive inotropic effect elicited by isoproterenol in the presence of prazosin in left papillary muscles, with no change in ED50 values. These results suggest that age-related changes in cardiac excitation and contraction coupling following beta-adrenoceptor stimulation are mediated, at least in part, by Gs alpha protein dysfunction.

Aging↗

PubMed1994

The expression, phosphorylation, and localization of connexin 43 and gap-junctional intercellular communication during the establishment of a synchronized contraction of cultured neonatal rat cardiac myocytes.

We analyzed the expression, phosphorylation, and localization of the major cardiac gap-junction protein connexin 43 (Cx43) during the establishment of a synchronized contraction in confluent monolayers of primary cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctions by the microinjection-dye transfer method. Monitoring of the beating rate and synchronization by Fotonic Sensor showed that at Day 1 of culture cardiac myocytes contracted spontaneously but irregularly, that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. At Day 7, the confluent cells exhibited synchronous contraction with a relatively constant rate (125 +/- 20 beats/min). Cardiac myocytes expressed a large amount of Cx43 mRNA even at Day 1 and maintained the expression until at least Day 7. Immunofluorescence of Cx43 showed that the localization of Cx43-positive spots was mostly restricted to cell-cell contacts between myocytes and that few Cx43-positive spots were present between myocytes and fibroblasts or between fibroblasts. The amount of Cx43 protein, the proportion of phosphorylated forms to the nonphosphorylated one, and the number and total area of Cx43-positive spots increased with culture time. Gap-junctional intercellular communication measured by dye transfer assay was also increased with culture time and correlated well with the number and total area of Cx43-positive spots. Our systematic study suggests that a concerted action of the expression, phosphorylation, and localization of Cx43 and gap-junctional intercellular communication plays a major role in the reestablishment of synchronous beating of cultured neonatal rat cardiac myocytes.

Animals↗

PubMed1994

Antidepressants directly influence in situ binding of guanine nucleotide in synaptic membrane.

The present study examined the in vitro effects of antidepressants on functional photoaffinity labeling of GTP binding protein. Saturation binding studies were performed by incubating membranes with increasing concentrations of [32P]-AAGTP, followed by UV irradiation and SDS-PAGE. The specifically bound isotherms for each of the G proteins studied showed characteristics of a one site model. Scatchard analysis revealed increases in the Bmax and Kd of AAGTP binding for each of the G proteins (especially stimulatory G proteins) with the addition of antidepressants such as amitriptyline, clomipramine, desipramine and mianserin but not with monoamine oxidase inhibitor, antipsychotics and axiolytics. These results suggested that drugs having antidepressive properties may directly affect G protein, especially Gs protein.

Affinity Labels↗

PubMed1994

Role of alpha 1-adrenoceptor subtypes which mediate positive chronotropy in neonatal rat cardiac myocytes.

We investigated the involvement of alpha 1-adrenoceptor subtypes in the positive chronotropic response to norepinephrine (NE) in neonatal rat cardiac myocytes at day 3 of culture. The cardiac myocytes at day 3 of culture exhibited a dose-dependent positive chronotropic response to NE in the presence of propranolol, a beta-adrenoceptor antagonist. The positive chronotropic responses to NE were completely antagonized by the alpha 1-adrenoceptor antagonist prazosin. The NE-induced positive chronotropic response was inhibited 68% by the alpha 1B-adrenoceptor antagonist, chloroethylclonidine (CEC), but partially (41%) so by the alpha 1A-adrenoceptor antagonist, WB4101. In the membrane fraction derived from cardiac myocytes at day 3 of culture, pretreatment with CEC decreased the Bmax of the alpha 1-adrenoceptor to 22% of the control value. The NE-induced positive chronotropic response was inhibited 62 and 77% by the voltage-gated Ca2+ channel blocker such as nifedipine and verapamil, respectively. These findings indicate (1) that cultured neonatal rat cardiac myocytes possess both alpha 1-adrenoceptor subtypes, i.e., alpha 1A and alpha 1B, (2) that the predominant alpha 1-adrenoceptor subtypes mediating NE-induced positive chronotropy in neonatal rat cardiac myocytes at day 3 of culture are alpha 1B-subtypes, and (3) that NE-induced positive chronotropy may be caused via voltage-gated Ca2+ channel activation.

Animals↗

PubMed1994

Hypoxia inverts the negative chronotropic response to norepinephrine in normoxia in cultured neonatal rat cardiac myocytes: role of the alpha 1 adrenergic signal transduction system.

In the present study, we investigated the effect of hypoxia on the chronotropic response to norepinephrine (NE) of cultured neonatal rat ventricular myocytes. We measured beating of myocytes with the Fotonic sensor, using a newly developed method for a noncontact displacement measurement. The beating rate counted with the sensor had a high correlation coefficient with that counted visually under a microscope (r = 0.997, P < 0.01). NE concentrations of 10(-8) - 10(-4) M caused negative chronotropy dose dependently in the presence of 5 x 10(-7) M propranolol. NE-induced chronotropy was completely antagonized by 10(-6) M prazosin. Three hours hypoxia decreased the spontaneous beating rate 40% (P < 0.01). Negative chronotropy induced by 10(-4) M NE in normoxia was inverted to positive and was antagonized by prazosin. Hypoxia increased the basal level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to 190% (P < 0.01), while NE-stimulated Ins(1,4,5)P3 production was significantly suppressed. Immunoblotting analysis of G protein subunits demonstrated no quantitative changes in Gi alpha, Gq alpha, Go alpha and G beta common subunits in hypoxia. In a saturation binding assay with [3H]prazosin, Kd values were increased to 152% by hypoxia (P < 0.05) without significant change in Bmax. Basal activity of low Km-GTPase was increased to 122% by hypoxia (P < 0.05). These results suggest that the hypoxia-induced increase in low-Km GTPase activity, which could stimulate phospholipase C by an activated alpha GTP subunit of G protein and consequently induce receptor-independent increase in Ins(1,4,5)P3, may be responsible for the inversion of the NE-induced negative chronotropic response in normoxia.

Animals↗

PubMed1994

Alteration of tubulin-Gi protein interaction in rat cerebral cortex with aging.

The ability of the tubulin dimer to interact with and to modulate the Gi function inhibiting adenylyl cyclase was examined in cerebral cortex membranes from 2-month-old and 24-month-old rats. The hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp)-dependent inhibition of adenylyl cyclase was significantly decreased in cerebral cortex membranes from 24-month-old rats. Tubulin, prepared from rat brains by polymerization with GppNHp, caused inhibition of adenylyl cyclase (approximately 28%) in 2-month-old rats. Tubulin-GppNHp-dependent inhibition of adenylyl cyclase in 24-month-old rats was significantly attenuated (approximately 15%). In 2-month-old rats, when tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to Gi alpha. Transfer of AAGTP from tubulin to Gi alpha was reduced in 24-month-old rats. Furthermore, photoaffinity labeling of [32P]AAGTP to Gi alpha in cortex membranes was significantly decreased in 24-month-old rats. No differences were observed in the amounts of Gs alpha, Gi alpha, or G beta subunits and tubulin, estimated by immunoblotting, in cortex membranes from 2-month-old and 24-month-old rats. These results suggest that the ability of tubulin to interact with Gi and thereby modulate the inhibitory regulation of adenylyl cyclase is reduced in the cerebral cortex of 24-month-old rats.

Adenylyl Cyclase Inhibitors↗

PubMed1994

Multiple calcium signaling pathways in Mardin-Darby canine kidney cells.

Effects of various receptor agonists on cytoplasmic Ca2+ concentration ([Ca2+]i) were examined in fura-2-loaded Mardin-Darby canine kidney (MDCK) monolayer cells. Carbachol (100 microM) increased [Ca2+]i which slightly declined to a sustained increase in [Ca2+]i. On the other hand, [Ca2+]i elevated by 100 nM bradykinin (BK) declined to a resting level of [Ca2+]i even in the presence of BK. After washout of BK, the subsequent addition of a higher concentration of BK (1 microM) caused a smaller increase in [Ca2+]i than that induced by 100 nM BK. Isoproterenol (100 microM) did not increase [Ca2+]i by itself but caused an transient increase in [Ca2+]i in the presence of 1 mM isobutyl-methylxanthine (IBMX). Prostaglandin E1 (1 microM) resulted in a slight increase in [Ca2+]i which was potentiatedin the presence of 1 mM IBMX. The microsomal Ca(2+)-ATPase inhibitor thapsigargin (100 nM) caused a sustained increase in [Ca2+]i. These results suggest that MDCK cells have multiple Ca2+ signaling pathways which may regulate epithelial cell functions.

1-Methyl-3-isobutylxanthine↗