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Hypoxia-inducible factor 1 a (Hif1α) plays a crucial role in brain development. To study the function of Hif1α in early brain development, we generated neuroepithelial cell-specific Hif1α-knockout mice. Hif1α-knockout mice died soon after birth; these mice exhibited an abnormal head shape, indicating the presence of brain defects. Morphological analysis revealed that Hif1α ablation reduced the overall size of the brain, especially affecting the telencephalon. Neuronal apoptosis predominantly occurred in deep-layer neurons, consequently the alignment of cortical layers was severely disorganized in Hif1α knockout mice. Furthermore, we demonstrated that Vegf signaling contributes to the survival of deep-layer neurons as a downstream effector of Hif1α-dependent hypoxia signaling. Taken together, our findings demonstrate that Hif1α plays a critical role in the early stages of telencephalon development.

Here, we studied the involvement of glutamate accumulation in the synaptic cleft in the distinctive defects in the brain of SAMP8. First, we demonstrated the developmental changes in glutamate transporter activities in the cerebral cortex of SAMP8 and SAMR1 at ages 4, 10, 16 and 32 weeks. Glutamate transporter activity in SAMR1 reached a maximum at 10 weeks of age. Thereafter, there was no significant change observed up to at least 32 weeks of age. Although the same activities were observed in two strains at ages of 4 and 32 weeks, the activities in 10- to 16-week-old SAMP8 were markedly lowered in comparison with those in age-matched SAMR1. Next, we showed the developmental changes in glutamate transporter proteins, a neuronal EAAC1 and two glial GLT-1 and GLAST, by immunoblotting analysis in the cerebral cortex of SAMP8 and SAMR1 at the above indicated ages. In the two strains, developmental changes in the content of GLT-1 protein were extremely similar to those in glutamate transporter activities. Additionally, the concentration of EAAC1 protein in the two strains showed a slight similarity to the developmental change in transporter activity, while there was no difference between the two in the expression of GLAST protein. These results suggest that the reduction of glutamate transporter activity in 10- to 16-week-old SAMP8 compared with that in age-matched SAMR1 is mainly caused by the developmentally retarded expression of GLT-1 protein.

In this paper, we showed that the oxidative stress in brain of senescence-accelerated prone mouse 8 (SAMP8) at earlier stages was increased compared with that of senescence-accelerated resistant mouse 1 (SAMR1) irrespective of the breeding conditions. Furthermore, we found that manganese superoxide dismutase (Mn-SOD) activity in the cerebral cortex of 10-week-old SAMP8 was decreased by about 50% compared with that in age-matched SAMR1. These results indicate that the decrease of Mn-SOD activity may be involved in the increased oxidative stress in the brain of SAMP8 at younger stages. However, there was no difference in the expression of this protein between the two strains at 10 weeks of age, suggesting that Mn-SOD protein in SAMP8 was post-translationally modified to reduce its enzymatic activity.

We investigated the effect of two isomers of retinoic acid (RA), all-trans RA and 9-cis RA, on the proliferation of Y79 human retinoblastoma cells. The two isomers inhibited the cell proliferation in a concentration-dependent manner. The IC50 for this inhibition by all-trans RA and 9-cis RA was 1.50 and 0.15 microM, respectively. The inhibitory effect of 9-cis RA on Y79 cell growth was observed within 24 hr, thereafter the cell number was gradually decreased. In contrast, no inhibition by all-trans RA of Y79 cell growth was observed within 24 hr, thereafter the cell number was slightly increased. In these cases, the cell viability at 4 days after the addition of 9-cis RA and all-trans RA was more than 90% and 95%, respectively. These results indicate that the two RA inhibit the proliferation of Y79 human retinoblastoma cells without inducing the cell death and that the effect of 9-cis RA on the inhibition of Y79 cell growth is much greater than that of all-trans RA.

Age-related changes in glycogen concentration in the heart of SAMP8, a substrain of senescence-accelerated prone mouse, were compared with that in SAMR1, a substrain of senescence-accelerated resistant mouse. The decrease by about 50% in the glycogen concentration in the hearts of 4-8 weeks old SAMP8 was observed in comparison with that in age-matched SAMR1, whereas there was no difference in adult hearts between the two substrains. Additionally, glycogen phosphorylase activity was significantly increased (1.3-fold) in the heart of 4–8 weeks old SAMP8 without the change in glycogen synthase activity between the two substrains. At the same age, the lipid peroxide concentration was also increased (1.4-fold) in the hearts of SAMP8, suggesting that oxidative stress was involved in the early damage to the hearts of SAMP8. Furthermore, the present findings that the cardiac glycogen concentration in SAMP8 decreases more quickly than that in SAMR1 may indicate the possibility that an accelerated aging of SAMP8 is related to the early changes in the energy sources in the heart.

We investigated the expression of glucose transporter (GLUT) protein isoforms in two human retinoblastoma cell lines, Y79 and WERI-Rb1, by Western blotting analysis with anti-GLUT1, 2, 3, and 4 antibodies. GLUT1 and GLUT4 proteins were detected in Y79, whereas GLUT1 and GLUT3 proteins were found in WERI-Rb1. GLUT2 protein was not detected in Y79 or WERI-Rb1. Our findings are of interest because (1) the expression of GLUT protein isoforms in the two retinoblastoma cell lines was different, and (2) GLUT4 protein, the insulin-sensitive GLUT isoform, was detected in Y79. This suggests that these cell lines have different mechanisms of glucose transport.

The expression of rho family along the crypt-villus axis of rat small intestine was investigated. The immunoblot analysis showed that the content of rac1 protein gradually increased from tip-villus to crypt-villus enterocytes without changes in the contents of rhoA and rac2 proteins. Furthermore, the contents of rac1 protein in the entire enterocytes were markedly decreased in streptozotocin-induced diabetic rats and restored by short-term treatment with insulin. These results suggest that differentiation and proliferation in the enterocytes of rat small intestine are possibly related to the expression of rac1 protein through insulin action.

The effects of Panax ginseng extract, ginseng saponins, and some other triterpenoid saponins on glucose uptake were examined by using sheep erythrocytes. Initial rates of glucose transport were determined by measurements of 2-deoxy-D-glucose (2-DG) uptake. From kinetic analysis apparent Km and Vmax values of facilitated glucose transport in sheep erythrocytes were calculated as 2.3 +/- 0.08 mM and 1.4 +/- 0.05 nmol/min/10(9) cells. The results showed that ginseng extract stimulated glucose uptake in sheep erythrocytes dose-dependently. Ginseng saponins, in general, also stimulated glucose transport. The maximum effect was observed at 1 microM of ginsenoside Rb1 showing an increase of 24 +/- 5% above basal activity. However, ginsenoside Rg3, chikusetsusaponin Ia, and glycyrrhetic acid induced significant inhibitory effects on glucose transport in sheep erythrocytes.

The rate of 6-[14C]D-glucose oxidation in cerebral cells of SAMP8, a substrain of senescence accelerated mouse, was investigated in vitro. The production of 14CO2 in dissociated intact brain cells prepared from the cerebrum of 4-8-week-old SAMP8 was higher than that of age-matched SAMR2 as a control mouse, while no difference between these two strains was observed in the production of 14CO2 in the cerebral homogenates. These results indicated that the increased metabolism of glucose in SAMP8 might be associated with the glucose transport system across the cell membrane. Therefore, 2-deoxy-D-glucose (2-DG) uptake into the brain cells and cytochalasin B (CB) binding to cerebral crude membranes were examined. Both the 2-DG uptake and the CB binding in SAMP8 were much greater than in SAMR2. Furthermore, the increased CB binding in SAMP8 was seen only in the cerebral cortex of 4- to 8-week-old mice, and neither in other regions of the cerebrum nor in other aged mice (2-week- and 40- to 48-week-old mice). These results suggest that the transient overproduction of the glucose transporter protein in the cerebral cortex is involved in the increased glucose metabolism in 4- to 8-week-old SAMP8.

The effect of standardized Panax ginseng extract (G115 Pharmaton) on D-glucose uptake by Ehrlich ascites tumour cells from mouse was examined. Measurements were carried out using [ 3 H]-2-deoxy-D-glucose, a non-metabolizable glucose analogue. [ 3 H]-2-deoxy-D-glucose was transported from the medium to cells and phosphorylated but was neither further metabolized nor eliminated. Thus, the amount taken in the cells was a measure of D-glucose uptake. The results showed that G115 stimulated D-glucose transport and the maximum effect was obtained at a G115 concentration of 2.0 μg/mL representing an increase of about 35% above basal activity

The effect of insulin on the translocation of GLUT4 (glucose transporter isoform 4) from the intracellular membranes to the plasma membranes was compared in the hearts of 4-8 week old SAMP8, a substrain of senescence-accelerated prone mouse and those of SAMR1, a substrain of senescence-accelerated resistant mouse. After 20 min of the intravenous injection of insulin, the blood glucose levels in SAMR1 and SAMP8 were decreased by 50 and 68%, respectively. Under this condition, the concentrations of GLUT4 protein in the plasma membranes in the hearts of SAMR1 and SAMP8 were increased 1.8- and 2.1-fold, respectively. Concomitantly, the concentrations of the GLUT4 protein in the intracellular membranes in the hearts of SAMR1 and SAMP8 were decreased by about 70 and 50%, respectively. These results suggest that the heart of 4-8 week old SAMP8 is more sensitive to insulin than that of age-matched SAMR1.

SAMP8, a substrain of senescence-accelerated mouse (SAM), has been characterized by several age-related deficits in the brain. Previously, we reported that the contents of lipid peroxides and protein carbonyl, and net generation of H2O2 were increased in the cerebral cortex of SAMP8 at 4–8 weeks of age in comparison with those in age-matched SAMR1 substrain used as a control. To study the cause of these increases, we compared the activities of antioxidative enzymes in the cerebral cortex between the two substrains. The catalase activity was decreased by 75% in SAMP8 at 4–8 weeks of age, whereas neither superoxide dismutase nor glutathione peroxidase activities were changed. The change in the catalase activity was seen only in the cerebral cortex where oxidative stress was increased in SAMP8. On the contrary, the activity of acyl-CoA oxidase, a microperoxisomal H2O2-producing enzyme, in the cerebral cortex of SAMP8 was increased 1.6 fold in comparison with that in age-matched SAMR1 without change in the activity of D-amino acid oxidase. Furthermore, the changes in the activities of catalase and acyl-CoA oxidase with age were paralleled with those observed in oxidative stress in SAMP8. These results suggest that the abnormality of activities in two microperoxisomal enzymes, catalase and acyl-CoA oxidase, may be one of the causes of the early increase in oxidative stress observed in the cerebral cortex of 4–8 weeks old SAMP8.

d-Glucose metabolism in cerebral cells prepared from aged senescence-accelerated mouse (SAM), was investigated in consideration of a sex difference. The production of 14CO2 from 6-[14C]D-glucose was reduced in female senescence-accelerated-prone mouse (SAMP) 8, a prone substrain, in comparison with that in female senescence-accelerated-resistant mouse (SAMR) 2, a control substrain, whereas there was no difference in males. The 2-deoxy-D-glucose uptake into cerebral cells from female SAMP8 was also lower than that of control mice. But, the 3-O-methyl-D-glucose uptake in SAMP8 was higher than that of SAMR2, suggesting that the low hexokinase activity was involved in the decreased glucose metabolism in cerebrum of SAMP8 females irrespective of glucose transporter. This possibility was supported by the finding that the contents of glucose 6-phosphate produced from glucose added to cerebral cells from SAMP8 was lower than that in ICR mice.

Age-related changes in oxidative stress in the cerebral cortex of SAMP8, a substrain of senescence-accelerated mouse (SAM), were investigated in comparison with those in SAMR1, which were used as a control. The lipid peroxide and protein carbonyl contents were transiently increased in SAMP8 from 4- to 8-weeks of age. The increases in lipid peroxide were seen only in the cerebral cortex and not in other regions of the cerebrum. Furthermore, the net generation of reactive oxygen species in cerebral cells was also increased in SAMP8. In addition, the activity of glutamine synthetase, which is known as an enzyme-highly sensitive to reactive oxygen, was decreased in the cerebral cortex of SAMP8 from 4- to 8-weeks of age. These results suggest that oxidative stress may be induced in the cerebral cortex of SAMP8 from 4- to 8-weeks of age, preceding the appearance of distinctive deficits in the brain of SAMP8.

Mono(ADP-ribose) synthetase activity in the nuclear fraction of immature rat testis was investigated in reference to the effect of testosterone. When the nuclear fraction was incubated with [ 32 P] NAD in the presence of nicotinamide, an inhibitor of poly (ADP-ribose) synthetase, some proteins with a molecular mass of 14.4∼21.5 kDa were ADP-ribosylated. In these proteins, H2B and H3 histones were found by sequencing 20 amino acids in the N-terminus. Their incorporations of ADP-ribose moiety were significantly decreased in immature rats at 4 hr after the subcutaneous injection of testosterone. Furthermore, the treatments with LH and FSH of the immature rats reduced mono(ADP-ribosyl)ations of H2B and H3 histones in a time-dependent manner. These results suggest that the testicular mono(ADP-ribose) synthetase activity is under the control of gonadotropin-testosterone system and is possibly related to differentiation of the testis.

The effect of plant extracts on D-glucose uptake by Ehrlich ascites tumour cells was examined. Among the 23 extracts of medicinal plants, five samples inhibited, and six samples activated, the uptake significantly. From one of the active plants, Lagerstroemia speciosa, two triterpenoids, colosolic acid and maslinic acid were isolated. Colosolic acid was shown to be a glucose transport activator. Since this compound was known to have hypoglycemic activity, our simple in vitro bioassay method can at least be used as a first screening for anti-diabetic activity.

An aqueous extract of mesocarps of the fruits of Balanites aegyptiaca exhibited a prominent antidiabetic activity by oral administration in streptozotocin induced diabetic mice. From one of the active fractions of this extract, two new steroidal saponins were isolated, and their structures were determined as 26-O-beta-D-glucopyranosyl-(25R)-furost-5-ene-3 beta,22,26-triol 3-O-[alpha-L-rhamnopyranosyl-(1----2)]-[beta-D-xylopyranosyl-(1---- 3)]-[alpha-L-rhamnopyranosyl-(1----4)]-beta-D-glucopyranoside and its 22-methyl ether. In addition, two known saponins, 26-O-beta-D-glucopyranosyl-(25R)-furost-5-ene-3 beta,22,26-triol 3-O-(2,4-di-O-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside and its methyl ether were isolated and identified. It was revealed that the individual saponins did not show antidiabetic activity, while the recombination of these saponins resulted in significant activity. From an ethanolic extract of the epicarps, two known flavonol glycosides, isorhamnetin-3-O-robinobioside and isorhamnetin-3-O-rutinoside were isolated and identified.

In order to examine the involvement of insulin in the activity of Na+/glucose cotransporter in rat small intestine, we compared Na+-dependent uptake of d -glucose by brush-border membrane vesicles prepared from control, streptozotocin-induced diabetic, insulin-treated diabetic and starved diabetic rats. In four groups, the uptake of d -glucose showed a transient overshoot in the presence of Na+ gradient between medium and vesicles (medium > vesicles). The overshoot magnitude was increased (1.8-fold of controls) in diabetic brush border membrane vesicles and recovered to the control level by the treatment of diabetic rats with insulin. In contrast, increased uptake of d -glucose in diabetic rats was not recovered by the starvation of diabetic rats although the blood glucose level was the same as that of controls. Furthermore, we attempted to examine phlorizin binding activities among four groups. Scatchard analysis indicated that phlorizin binding to diabetic brush border membrane vesicles was increased (1.6-fold of controls) without a change of the affinity for phlorizin as compared with controls. Increased binding of phlorizin to diabetic brush border membrane vesicles was also recovered to the control level by the treatment of diabetic rats with insulin, but not by starvation. These results suggested that the increased activity of Na+/glucose cotransporter in diabetic rats was due to the increase of the number of cotransporter and that intestinal cotransporter was physiologically controlled by insulin, but not by blood glucose levels.

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.

The effect of alkaline phosphatase (3.1.3.1) on desensitization of beta-adrenoceptor-responsive adenylate cyclase and the role of phosphorylation in desensitization were examined. Treatment of rat reticulocytes with isoproterenol, dibutyryl cAMP and tetradecanoyl phorbol acetate (TPA) caused the desensitization of beta-adrenoceptor-coupled adenylate cyclase. When the membranes from dibutyryl cAMP- and TPA-desensitized cells were incubated with alkaline phosphatase for 60 min at 30 degrees C, pH 8.0, the desensitization of isoproterenol-stimulated adenylate cyclase was markedly attenuated in both preparations. When the membranes from isoproterenol-desensitized cells were treated with alkaline phosphatase under the same conditions, the attenuation of the desensitization of alkaline phosphatase was less than in the case of treatment with dibutyryl cAMP or TPA. In other words, isoproterenol-induced desensitization was more resistant to alkaline phosphatase treatment. Isoproterenol- and dibutyryl cAMP-induced desensitization of NaF-stimulated adenylate cyclase were also attenuated by alkaline phosphatase treatment. Although the stability of the Gs-catalytic unit complex of adenylate cyclase was reduced by isoproterenol treatment, the reduction of stability was also decreased by alkaline phosphatase treatment.

The uptake of Na+-dependent d -glucose by renal brush-border membrane vesicles (BBMV) isolated from streptozotocin-induced diabetic rats was decreased as compared with controls. Since a V max of 4.8 nmol/mg protein per 30 s in diabetic BBMV was significantly decreased as compared with that of controls ( V max = 7.0 nmol/mg protein per 30 s) without changing an apparent affinity for d -glucose, the decrease in the Na+-dependent d -glucose uptake in diabetic rats is likely to be due to the reduction in the number of the transporter. These results are also confirmed by the binding study of [3H]phlorizin to diabetic BBMV. When the blood glucose level is lowered in diabetic rats by both the treatment with insulin and starvation, the decreased Na+-dependent d -glucose uptake is returned to control level. These results suggest that Na+-dependent d -glucose reabsorption through the apical membrane in proximal tubular kidney cells is dynamically regulated by the change in blood glucose level.

Treatment of rat reticulocytes with isopreterenol resulted in dose- and time-dependent desensitization of adenylate cyclase to β-adrenoceptor agonist stimulation. Cyclic AMP-dependent protein kinase was possibly involved in this desensitization. Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), also induced desensitization to β-adrenoceptor agonists, indicating the possible involvement of protein kinase C. The addition of a β-adrenoceptor agonist enhanced the desensitizing effect of dibutyryl cyclic AMP or TPA. T 1 2 (time for induction of half maximal desensitization) was decreased from 30 to 2.5 min in accordance with an increase in the concentration of the agonist. The extent of the desensitization was also increased by addition of the agonist. The potency with which the increase was induced was compatible with the potency for binding of the agonist to β-adrenoceptors (KD values). These results suggest that cyclic AMP- or phorbol ester-induced desensitization is enhanced by receptor occupation by β-adrenoceptor agonists.

Guanylate cyclase activity was examined in inflammatory granuloma tissue induced by subcutaneous injection of carrageenin solution in the rat. Enzymological properties of guanylate cyclase in this tissue, such as pH optimum, metal ion requirement, etc., were examined, with special reference to the intracellular distribution of the enzyme. Differences were found between guanylate cyclase in the soluble fraction and that in the particulate fraction in the response to Mn2+ and Triton X-100 in this tissue.

Treatment of rat reticulocytes with isoproterenol caused about 50, 25, and 25% decreases in beta-adrenergic agonist-, fluoride-, and guanine nucleotide-stimulated adenylate cyclase activities, respectively. The desensitization was also induced by dibutyryl adenosine 3',5'-cyclic monophosphate (cyclic AMP) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) prevented the isoproterenol-induced desensitization, suggesting the involvement of cyclic AMP in the desensitization. Time course studies revealed that the desensitization to NaF-AlCl3 occurred faster than that to isoproterenol. Furthermore, the rate of the resensitization to NaF-AlCl3 by removal of isoproterenol was also faster than that to isoproterenol. Thus, it is likely that both guanine nucleotide-binding stimulatory regulatory protein, Ns, and beta-adrenergic receptor are sequentially involved in both desensitization and resensitization of the adenylate cyclase system in rat reticulocytes to isoproterenol.

The effect of dan-shen extract, the root of Salvia miltiorrhiza, on adenylate cyclase was investigated in both rat brain and rat erythrocytes. The EtOAc fraction of the MeOH extract was proved to have significant inhibitory activity. Potent inhibitory principles in the EtOAc fraction were isolated and identified as 4 polyphenolic acids, rosemarinic acid, lithospermic acid, and their methyl ester derivatives.

Guanine nucleotide-binding stimulatory regulatory protein of adenylate cyclase system, Ns, in rat erythrocytes was activated by the treatment with guanylyl 5′-imidodiphosphate or NaFAlCl3 in the presence of Mg2+. The activation was counterbalanced to the basal state either by the removal of Mg2+ or by the addition of β(γ)-subunit of N protein of this system. The depression from the activated state was markedly protected by the coexistence of forskolin at the time of the deactivation depending on the dose of forskolin. EC50 of forskolin for the stabilizing effect was much lower than that for the stimulation of adenylate cyclase activity. These data indicate that forskolin has an effect on the interaction between Ns and catalytic unit of adenylate cyclase system in addition to the direct effect on the catalytic unit.

The transport function and orientation of the reconstituted human erythrocyte glucose transporter was studied with liposomes made with bovine brain lipid or Escherichia coli lipid. Reconstitution was achieved by a simple octyl glucoside dilution method. The reconstituted transporters with either lipid showed identical counterflow transport activity, the same response to various inhibitors, and characteristic cytochalasin B (CB) labeling. Functional location and purification of the glucose transporter was performed by using gel-permeation high-performance liquid chromatography with octyl glucoside-containing buffer. The reconstituted transport activity was associated only with band 4.5 protein (preactin) and not with band 3 protein. Both CB binding and transport function of the reconstituted transporters were resistant to trypsin but susceptible to chymotrypsin digestion. However, both trypsin and chymotrypsin treatment of unsealed ghosts completely eliminated the CB labeling and transport function of the glucose transporter. In our reconstitution system the glucose transporters maintained a normal asymmetrical (right-side-out) orientation and good transport function. A specific monoclonal antibody against the glucose transporter inhibited CB labeling of the transporters on unsealed ghosts. This was not found with the reconstituted system; however, after freeze-thawing there was a significant inhibition of CB binding by the antibody. These findings suggest that the CB-binding site of the reconstituted transporter is on the inner side of the proteoliposomes.

Treatment of rat reticulocytes with tetradecanoyl phorbol acetate (TPA), a tumor-promoting phorbol ester which activates protein kinase C, resulted in an about 50% decrease in the stimulation of adenylate cyclase activity by a subsequent challenge with a beta-adrenoceptor agonist. This phenomenon mimics agonist-induced desensitization. This decline is due to a reduction in the Vmax of the adenylate cyclase system rather than to a change in affinity to the agonist. The beta-adrenoceptor number was not changed while the KD for an agonist but not for an antagonist was increased by TPA treatment. Prostaglandin E1 (PGE1) plus GTP, NaF plus AlCl3, and guanylyl-5'-imidodiphosphate (GppNHp) regulated adenylate cyclase activity in a biphasic manner, i.e. stimulation at lower concentrations and inhibition at higher concentrations. The same treatment also caused a dose- and time-dependent reduction of the inhibitory phase of the PGE1/GTP action but did not affect the inhibitory phase of GppNHp and NaF/AlCl3 actions. Pertussis toxin (IAP) treatment caused a reduction of the inhibitory phase of PGE1/GTP action similar to that caused by TPA treatment. No synergistic effect was observed when the cells were treated with TPA and IAP simultaneously. These results suggest that TPA treatment impairs the coupling between PGE1 receptor and Gi rather than enhances that between PGE1 receptor and Gs. Protein kinase C was involved in the regulation of hormone-sensitive adenylate cyclase, the beta-agonist-induced stimulatory pathway and the PGE1-induced inhibitory pathway in rat reticulocytes, since other phorbol esters and diacylglycerol, which activate this kinase, caused the same response.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the effects of a forskolin suspension on intraocular pressure (IOP) in normal albino and pigmented rabbits and in alpha-chymotrypsin induced ocular hypertensive rabbits. Experimental-induced ocular hypertensive rabbits were produced by injecting of alpha-chymotrypsin (167 units) into the posterior chamber of the eye of albino rabbits. Ocular hypertensive rabbits were classified into 3 groups according to the IOP (Group A; 15-19 mmHg, B; 20-24 mmHg, C; 25-29 mmHg). Topical application of 1% forskolin caused a significant decrease in IOP in Groups B and C, as well as in normal albino and pigmented rabbits. The hypotensive effects were lower in pigmented rabbits than in albino rabbits, although the duration was longer. Subconjunctival injection of 1% forskolin reduced IOP 1 to 5 hrs after treatment in albino rabbits. However, in pigmented rabbits, a slight increase in IOP was observed at 30 min, followed by a significant decrease 5 to 10 hrs after injection. Furthermore, the binding ability of forskolin to melanin granules was determined in vitro. Forskolin exhibited specific affinity towards melanin granules obtained from bovine eyes, with the binding reaching a plateau after 5 min of incubation.

Ehrlich ascites tumor-bearing mice were treated with colchi-cine or mitomycin C. Under colchicine, adenylate cyclase activity in tumor cells decreased to about a third of the untreated control level, whereas 5'-nucleotidase activity increased to almost twice the control level. Colchicine treatment resulted in about a 30 % reduction of 3H-thymidine incorporation into DNA. Mitomycin C inhibited the incorporation of 3H-thymidine much more mark-edly but changes in the two enzyme activities were less than under colchicine. The two enzyme activities and 3H-thymidine incorporation into DNA showed parallel changes with lapse of time after inoculation of tumor cells into mice. Thus, the colchicine effects on the two enzyme activities seemed not due simply to inhibition of cell proliferation. The two enzymes seemed to be associated differently with the microtubular system in Ehrlich ascites tumor cells.

Involvement of divalent cations was examined in the activation of catecholamine sensitive adenylate cyclase in Ehrlich ascites tumor cells. In the presence of various concentrations of either Mg2+ or Mn2+, both basal and norepinephrine-stimulated, adenylate cyclase activities were measured. Both activities were gradually increased with the increase in Mg2+ concentration. On the other hand, with Mn2+, both activities were maximum at the low (2.5 mM) concentration, and decreased at the highest concentrations, the decrease being marked in the norepinephrine-stimulated activity. Similar difference between the two divalent cations was also observed in the effect on the binding of the beta-adrenergic ligand to the membrane receptor.

The effect of barbiturates on an adenylate cyclase system in rat brain was examined. The activity of the catalytic unit of this system isolated from the synaptic membrane was inhibited by phenobarbital in dose- and time-dependent manners. The mode of the inhibition was non-competitive with respect to Mg-adenosine triphosphate (ATP). The activity of the synaptic membrane-bound adenylate cyclase was also inhibited by phenobarbital in a similar manner. The inhibitory effect of phenobarbital was more potent on the activation of the enzyme by 5'-guanylylimidodiphosphate (GppNHp) than on the basal enzyme activity. The inhibitory effect, however, was not observed in the synaptic membrane preparation in which the guanine nucleotide-binding stimulatory regulatory protein (Ns) and the catalytic unit of adenylate cyclase system had been functionally coupled by pretreatment with GppNHp. Similar results were obtained with other pharmacologically active barbiturates. These findings indicate that barbiturates primarily affect the activation of the catalytic unit by an interaction with Ns resulting in the inhibition of the enzyme activity.

In vitro incubation of Ehrlich ascites tumor cells in the presence of norepinephrine induced desensitization of adenylate cyclase to the later norepinephrine stimulation. Such a desensitization was not accompanied by a decrease in the number of receptor sites. Formation of actin filaments from actin monomers was not changed in the desensitized cells, whereas polymerization of tubulin was significantly increased. The increase in the polymerizaton was dependent on the concentration of norepinephrine.

It is well known that the adenylate cyclase system in the cell membrane consists of the ligand receptor, the guanine nucleotide-binding regulatory unit (G/F), and the catalytic unit which catalyzes the formation of cyclic AMP from ATP (1,2). As compared with the former two, the catalytic unit has been less characterized, principally because of instability of this protein after solubilization with the use of detergents. Nevertheless, many efforts have gradually clarified the nature of this protein. Among them, success in separation of the catalytic unit from G/F (3) and use of the cyc mutant of S49 lymphoma cell which apparently lacks a functional G/F protein (4) have facilitated the clarification. As a result, the molecular size of the catalytic unit has been estimated to be 170,000–220,000 in several tissues (5,6).

The effect of barbiturate on adenylate cyclase system was examined in rat brain. Pentobarbital inhibited the enzyme activities in both synaptic membrane and solubilized catalytic unit of the system in dose and time-dependent manners. The inhibitory effect of pentobarbital was more potent on the activation of the system by NaF-AlCl 3 than on the basal activity. The inhibitory effect, however, was less in the synaptic membrane in which the catalytic unit was prestimulated through coupling with N s by the treatment with NaF-AlCl 3 . Similar results were obtained with the solubilized preparation which was pretreated with guanylyl-5′-imidodiphosphate before solubilization. On the other hand, the effect of pentobarbital was not modified by the treatment of the synaptic membrane with pertussis toxin. These findings indicate that barbiturates suppress primarily the activation of the catalytic unit through the coupling with guanine nucleotide-binding stimulatory protein (N s ) without affecting the inhibitory protein (N i ).

Adenylate cyclase activity of the homogenate of Ehrlich ascites tumor cells pretreated with catecholamine at 37 degrees C was not stimulated by the addition of the same catecholamine, whereas that of the cells without the pretreatment was stimulated. Such a desensitization was induced hardly at all when the pretreatment was performed at low temperature. The desensitization of adenylate cyclase activity to catecholamine stimulation was prevented by pre-pretreatment of the cells with colchicine prior to the catecholamine pretreatment. The effect of colchicine was dependent on the period of the treatment and concentration of colchicine. Vinblastine had a similar effect, whereas cytochalasin B was without effect. Thus, involvement of microtubules was suggested in the desensitization of the membrane-associated enzyme to external stimulation.

Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in the desensitization of adenylate cyclase to the beta-adrenergic agonist stimulation depending on the dose and period of the TPA treatment. Treatment of the reticulocytes with TPA caused approximately 40% reduction in the stimulation by beta-adrenergic agonists of adenylate cyclase activity, whereas the treatment had little effect on the basal activity and the activation by fluoride and guanine nucleotide of the enzyme system. No change in the number of beta-adrenergic receptors was observed after the TPA treatment. Treatment with 1-oleoyl-2-acetyl-glycerol (OAG), an activator of protein kinase C, also caused the desensitization of reticulocyte adenylate cyclase to isoproterenol. On the other hand, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C, prevented the desensitization induced by TPA. These results suggest the involvement of protein kinase C in a process of desensitization of adenylate cyclase system to beta-adrenergic agonists in rat reticulocytes.

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.

Forskolin-activated adenylate cyclase activity in rat erythrocyte membranes was markedly inhibited by methanol in a concentration dependent manner. On the contrary, no change was observed in the basal cyclase activity in the presence of methanol. The methanol inhibition forskolin-activated cyclase activity was protected by the stimulation of Gs in the presence of NaF in a concentration dependent manner. These data indicate that low- and high-affinity binding sites of forskolin in rat erythrocyte membranes have different sensitivities to methanol.

Transmembrane signalling function of adenylate cyclase system in the cell membranes is discharged and regulated by interactions of the component proteins; receptor protein (either stimulatory (Rs) or inhibitory (Ri)), guanine nucleotide-binding regulatory trimeric protein (either stimulatory (Gs) or inhibitory (Gi)), and catalytic protein (C) for cyclic AMP production (1,2). GTP and its analogs such as guanosine 5′-(β,γ-imino)triphosphate (GppNHp) and guanosine 5′-(γ-thio)triphosphate, regulate the function of G proteins in the presence of Mg2+ (3,4), while F- does in the presence of Al3+ (5). On the other hand, divalent cations, such as Mg2+ and Mn2+, and forskolin directly activate C (6,7). It is also known that cholera and pertussis toxins affect the functions of Gs and Gi, respectively, through ADP-ribosylation of their α-subunits (8, 9).

The effect of pentobarbital on the adenylate cyclase system was examined in synaptosomal membranes from rat brain. Pentobarbital inhibited forskolin-stimulated enzyme activity more effectively than the basal and Mn2(+)-stimulated enzyme activities. The degree of inhibition of the enzyme activity by pentobarbital was increased by the presence of forskolin in a concentration-dependent manner. No significant difference is observed in the degree of the inhibition by pentobarbital between the basal and forskolin-stimulated activities in the membranes prepared from the peripheral tissues.

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5′-imidodiphosphate (GppNHp) or NaFAlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaFAlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.

Treatment of rat reticulocytes with isoproterenol resulted in the heterologous desensitization of adenylate cyclase, causing a reduction in NaF/AlCl3- and guanylyl 5'-imidodiphosphate (Gpp(NH)p)-stimulated activities as well as in activity stimulated by beta-adrenoceptor agonists. Desensitization was also induced by dibutyryl cyclic AMP and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of cyclic AMP-dependent protein kinase (protein kinase A), prevented the isoproterenol-induced desensitization, suggesting the involvement of protein kinase A in the desensitization. Gs in the desensitized cell membrane was activated by treatment with Gpp(NH)p or NaF/AlCl3 in the presence of Mg2+. The activated state, the Gs-C complex, was gradually shifted to the basal state, i.e. the Gs-C complex was dissociated by removal of Mg2+. The rate constant of the dissociation was increased in the desensitized cell membranes (0.074 min-1) as compared with that in the control cell membranes (0.022 min-1). The half life of the Gs-C complex, calculated from the rate constant, was decreased during the process of desensitization. The results indicate that reduction in the stability of the Gs-C complex is related to heterologous desensitization. Agents which increase the level of cyclic AMP in the cell also reduced the stability of the complex. The potency of such an effect was in the following order; isoproterenol approximately NaF/AlCl3 (k = 0.071 min-1) greater than forskolin (0.039 min-1) greater than Mn2+ approximately dibutyryl cyclic AMP (0.030 min-1). The former two activate Gs and form Gs-C complex, while the latter two cause intracellular cyclic AMP accumulation without activation of Gs. It may be deduced from this order that the reduction of the stability depends not only on the increase in the cyclic AMP level but also on the level of Gs-C complex formed.

Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.