Minoru Tsuboi

Possible mechanism of regulating adenylate cyclase activity in adipocyte membranes from exercise-trained male rats

(-)-Isoproterenol-stimulated adenylate cyclase activities were significantly greater in membranes from exercise-trained male rats than in sedentary male rats. GTP-inhibition of forskolin (10 microM)-stimulated cyclase activities were observed in sedentary membranes, whereas the inhibitory actions of GTP were significantly reduced in membranes from trained rat adipocytes. Treatment of membranes with islet-activating protein, a pertusis toxin, completely abolished the differences in GTP-inhibition of forskolin-stimulated cyclase activities between the two groups. The amounts of the inhibitory regulatory protein (41kDa/40kDa polypeptides) were about 40% less in membranes from trained rats than in sedentary membranes, whereas that of the stimulatory regulatory protein (a 45kDa polypeptide) was equivalent. It is concluded that the enhanced cyclase activities of adipocyte membranes from trained male rats appear to result from, in part, an attenuation of the inhibitory pathway due to a specific decrease in the amount of the inhibitory regulatory proteins.

Enzymic determination of serum oxalate

A new colorimetric, enzymic method for determination of serum oxalate is described using oxalate oxidase from barley seedlings. This method, based on a specific reaction of oxalate with oxidase is rapid, simple and precise. The percentage recoveries of oxalate added to serum ranged from 91.5 to 103. The oxalate content in the serum obtained from 50 healthy non-fasting donors varied from 0.06 mg/dl to 0.41 mg/dl. The proposed method showed a good correlation with a chemical method.

Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 microM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [32P]phosphoinositide breakdown, the first peak of carbachol (1 microM)-stimulated DAG accumulation was found to be related to the breakdown of [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2). The second peak was found to be related to [32P]PIP2 breakdown. Carbachol stimulated the release of [3H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [3H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [3H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-microM)-induced release of [3H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown [32P]PIP and of [32P]PIP2 and that the second peak is predominantly associated with [32P]PIP2 breakdown and phosphatidylcholine hydrolysis.

Protein kinase C-dependent diacylglycerol formation is mediated via Ca2+ / calmodulin in parotid cells

The kinetics of carbachol-induced sn-1,2-diacylglycerol (DAG) formation and the underlying mechanism(s) involved in parotid acinar cells were investigated. Supramaximal concentrations of carbachol for amylase secretion (10 microM) caused a transient rise in DAG levels at 10 s. In contrast, this rapid rise was not elicited by 1 microM carbachol, which is the maximally effective concentration for amylase secretion. Carbachol (10 microM) also increased DAG levels linearly up to 20 min, which were sustained for up to a further 10 min. DAG formation stimulated by 1 microM carbachol was biphasic; the first peak was observed after 5 min and the second after 20 min. DAG formation induced by 0.01-0.1 microM carbachol was concentration-dependent and monophasic, peaking at 5 min. The second peak evoked by carbachol was partly inhibited by Ca2+ deprivation from the extracellular space, whereas the first peak was not. Similar results were obtained in experiments using Ca2+ antagonists such as verapamil and LaCl3. The protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and staurosporine, and a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), significantly inhibited the second DAG peak produced by 1 microM carbachol, but did not alter the first peak. The degree of inhibition of the second peak by these antagonists was comparable. Furthermore, the inhibitory effect of staurosporine and W-7 was concentration-dependent. The A23187-induced accumulation of DAG also was abolished by both staurosporine and W-7. These data indicate that a protein kinase C-dependent mechanism(s) is involved in mediating the second DAG accumulation peak induced by 1 microM carbachol and is mainly regulated by the Ca(2+)-calmodulin complex.

Effect of mammalian lignans on f MLP‐induced oxidative bursts in human polymorphonuclear leucocytes

Abstract— We examined the effects of mammalian lignans, enterolactone, prestegane B and 2,3‐dibenzylbutane‐1,4‐diol (DBB) on superoxide production and luminol‐dependent chemiluminescence (LCL) response in human polymorphonuclear leucocytes (PMNs). The three lignans had no direct effect on the responses of human PMNs. DBB and prestegane B enhanced the superoxide production and LCL response induced by formylmethionyl‐leucyl‐phenyl‐alanine (f MLP), but enterolactone inhibited f MLP‐induced effects. The effects of DBB were stronger than those of prestegane B and the effects of DBB were inhibited by bromophenacyl bromide, mepacrine, N‐(6‐aminophenyl)‐5‐chloro‐t‐naphthalene sulphonamide and trifluoroperazine, but not by gossypol, nordihydroguaretic acid, indomethacin, staurosporine, 1‐(5‐isoquinolinesulphonyl)‐2‐methylpiperazine dihydrochloride or (R,S)‐2‐methoxy‐3‐(octadecyl‐car‐bamoyloxy)‐propyl‐2‐(2‐thiazolio)‐ethylphosphate. These results suggest that DBB primes the responses of human PMNs, and the priming effect is caused by the activation of phospholipase A2—and Ca2+ ‐calmodulin‐pathways, but not by the activation of lipoxygenase, cyclo‐oxygenase and protein kinase C or by the release of platelet activating factor.

Substance P-induced diacylglycerol formation in rat parotid acinar cells

The mechanisms underlying the ability of substance P, to stimulate the sn-1,2-diacylglycerol (DAG) formation were studied using rat parotid acinar cells. During a 60 s stimulation, 1 microM substance P caused a rapid rise in DAG accumulation at 5 s, whereas a low (0.1 microM) concentration of agonist did not. During long term stimulation for 30 min, DAG accumulation induced by 1 microM substance P reached near maximal levels at 5 min and remained elevated for at least 20 min. In contrast, DAG formation induced by 0.1 microM substance P exhibited a peak at 5 min, gradually declined and returned to near basal levels at 30 min. Furthermore, DAG accumulation in response to substance P at 5 and 20 min increased in a dose-dependent manner. The breakdown of both [32P]phosphatidylinositol 4-monophosphate ([32P]PIP) and [32P]phosphatidylinositol 4,5-bisphosphate ([32P]PIP2) stimulated by 1 microM substance P significantly increased from 5 to 20 min and returned to basal levels by 30 min; however, the breakdown of [32P]PIP2 was greater than that of [32P]PIP. At a low concentration of substance P, [32P]PIP2 breakdown reached maximal levels at 5 min followed by a progressive decrease and returned to basal levels at 30 min, whereas the breakdown of [32P]PIP reached maximal levels at 5 min and returned to near basal levels at 10 min. Both concentrations of substance P caused some [32P]phosphatidylinositol breakdown at 5 min. Changes in [3H]inositol trisphosphate induced by substance P were similar to those in [32P]PIP2. In addition, substance P (1 microM) did not stimulate the release of [3H]choline or [3H]ethanolamine metabolites into the medium. Substance P-induced DAG formation was not inhibited by staurosporine, a protein kinase C inhibitor. These results suggest that DAG formation caused by substance P is closely associated with the hydrolysis of phosphatidylinositides but not that of phosphatidylcholine or phosphatidylethanolamine, and is not regulated by protein kinase C-dependent mechanism(s).

Priming effect of 2,3-dibenzylbutane-1,4-diol (mammalian lignan) on superoxide production in human neutrophils

We investigated the effect of 2,3-dibenzylbutane-1,4-diol (DBB), a mammalian lignan, on superoxide production and [Ca2+]i mobilization in human neutrophils. DBB did not generate superoxide production by itself, but enhanced the FMLP or A23187-induced superoxide production in a dose dependent manner. DBB did not influence the OAG-induced superoxide production. The priming effect of DBB was inhibited by W-7 or trifluoroperazine, but not by H-7 or staurosporine. And the priming effect of DBB was observed in the presence or absence of extracellular Ca2+. DBB enhanced the low dose FMLP-induced [Ca2+]i mobilization. These results suggest that the priming effect of DBB in human neutrophils may be caused by the activation of the calcium-calmodulin pathway but not the protein kinase pathway.

In vivo adaptative regulation of muscarinic receptors and muscarinic stimulation-induced Ca2+ mobilization during short-term heat exposure in rat parotid glands.

1. Adaptation of muscarinic receptors (MR)--muscarinic stimulation--induced intracellular Ca2+ mobilization during short-heat exposure (33 degrees C). 2. Heat-exposure for 48 hr decreased the carbachol (CCh)-stimulated cytosolic Ca2+ concentration increase. 3. The number of MR on cell surface increased transiently at 24 hr with a subsequent decrease at 48 hr. 4. CCh-stimulated inositol triphosphate (IP3) formation decreased at 48 hr. 5. In saponin-permeabilized cells, 1,4,5-IP3-induced 45Ca2+ release decreased at 24 hr. 6. The data suggest that the adaptation for increased muscarinic stimulation occurs at IP3 generating sites as well as at intracellular IP3 receptor sites during heat exposure.

Ca2+ Potentiates Corticotropin-Induced, But Not Isoproterenol-Induced, (3H)Guanosine Diphosphate Release in Rat Adipocyte Membranes

EGTA abolished corticotropin (ACTH)-stimulated adenylate cyclase in rat adipocyte membranes. In contrast, the potency of guanosine triphosphate (GTP) stimulation of adenylate cyclase activated with ACTH was greater in the presence of Ca2+ (1 mmol/L). EGTA (1 mmol/L) powerfully inhibited ACTH-stimulated [3H]guanosine diphosphate (GDP) release from membranes prelabeled with [3H]GTP in the presence of isoproterenol (ISO) or ACTH, whereas Ca2+ significantly increased it. In contrast, neither EGTA nor Ca2+ affected ISO-stimulated [3H]GDP release. These data clearly show that Ca2+ is necessary for the binding of ACTH to its receptor, and that Ca2+ stimulates the interaction of the ACTH-occupied receptor with GTP-binding proteins.

In vivo adaptive control of β-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands

1. Adaptation of beta-adrenergic receptors (beta-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33 degrees C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of beta-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between beta-AR and GTP binding protein(s).