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Biomedical subjects

H Kurose

Publications and source records attributed to H Kurose.

At least 55 records · Page 3Linked to original sources
PubMed1992

Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism.

To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.

Adenosine Diphosphate Ribose↗
PubMed1991

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.

Animals↗
PubMed1990

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increases insulin-like growth factor I (IGF-I) receptors in clonal osteoblastic cells. Study on interaction of IGF-I and 1,25-(OH)2D3.

We previously reported a cooperative effect between insulin-like growth factor I (IGF-I) and 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] in murine clonal osteoblastic cells, MCT3T3-E1. In the present study, the possible mechanism of interaction between these hormones was investigated. The effect of IGF-I on 1,25-(OH)2D3 receptors in MC3T3-E1 cells was examined. The affinity and hormone binding capacity of 1,25-(OH)2D3 receptors were not altered by IGF-I. Immunoblot analysis showed about 54 kilodaltons (kDa) 1,25-(OH)2D3 receptors, similar to that observed for mouse fibroblasts. The synthesis of IGF-I by the cells under a serum-free condition was determined by RIA. The assay revealed immunoreactive IGF-I secreted by MC3T3-E1 cells (1.79 +/- 0.04 x 10(-9) M, mean +/- SE, n = 5). Rat GH significantly increased the concentration of IGF-I, but 1,25-(OH)2D3 did not. IGF-I radioligand-receptor assay revealed specific binding of IGF-I to MC3T3-E1 cells. The relative potency of IGF-I-related peptides to bind with the cells was in the order of IGF-I much greater than multiplication-stimulating activity (the rat homologue of IGF-II) greater than insulin, and the receptor protein migrated as a 130-kDa band in autoradiography. Scatchard analysis showed a significant increase in IGF-I binding sites by 50% after 3-day treatment with 5 x 10(-11) M 1,25-(OH)2D3, without any change in affinity. These results indicate that the interaction of IGF-I and 1,25-(OH)2D3 in the culture of MC3T3-E1 cells may be mediated by the effect of 1,25-(OH)2D3 on IGF-I receptors.

Calcitriol↗
PubMed1989

Cooperation of synthetic insulin-like growth factor I/somatomedin C and 1,25-dihydroxyvitamin D3 on regulation of function in clonal osteoblastic cells.

We investigated the effects of insulin-like growth factor I/somatomedin C (IGF-I/SM-C), and the interaction of IGF-I and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on mouse clonal osteoblasts, MC3T3-E1. IGF-I stimulated [3H]thymidine incorporation into the DNA of the cells at concentrations of 1.3-130 X 10(-9) M. The alkaline phosphatase (ALP) activity in cultures was also raised by the hormone at the same concentrations. The optimal dose of IGF-I was 13 X 10(-9) M. Co-addition of IGF-I (1.3-130 X 10(-9) M) and 1,25(OH)2D3 (10(-11) to 10(-10) M) to the culture of MC3T3-E1 cells caused a synergistic increase in ALP activity. 25(OH)D3 and 24,25(OH)2D3 showed a similar effect with IGF-I at 1000-2000 times higher concentrations than 1,25(OH)2D3. [3H]Proline incorporation into collagenase digestible protein (CDP) in media was stimulated dose-dependently by IGF-I up to 2.2-fold over the control levels at 130 X 10(-9) M. Addition of 1,25(OH)2D3 (5 X 10(-11) M) and IGF-I further elevated the proline incorporation into CDP. However, the increment in CDP synthesis, induced by the two hormones was less than the increment in ALP activity. Thus, we conclude (1) that IGF-I stimulates both cell replication and differentiated functions in cultured murine osteoblasts and (2) that IGF-I and 1,25(OH)2D3 have the synergistic effect on ALP activity and the additive effect on collagen synthesis in MC3T3-E1 cells.

Alkaline Phosphatase↗
PubMed1989

Effect of single oral phosphate loading on vitamin D metabolites in normal subjects and in X-linked hypophosphatemic rickets.

There have been several reports that document abnormal vitamin D metabolism in X-linked hypophosphatemic rickets (XLH). Those reports indicate a blunted renal 25-hydroxyvitamin D-1 alpha-hydroxylase response to a potent stimulator, phosphorus restriction. We examined here its response to phosphate supplementation. Seven normal volunteers and 12 patients with XLH were submitted to single oral phosphate loading. This treatment produced a marked elevation of the serum phosphorus level, with a mild reduction in the serum calcium level. In normal subjects, although the concentrations of intact parathyroid hormone and mid-region parathyroid hormone were increased, with two peaks at 2 and 8 h after treatment, there were no significant changes in vitamin D metabolites including 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D (24,25(OH)2D) and 1,25-dihydroxyvitamin D (1,25(OH)2D). On the other hand, in the patients with XLH, the serum 1,25(OH)2D level increased from 23.4 +/- 12.0 (mean +/- SD) pg/ml to 44.3 +/- 33.6 pg/ml 6 h after ingestion without any significant change in 25(OH)D or 24,25(OH)2D.

24,25-Dihydroxyvitamin D 3↗
PubMed1988

Microcarriers facilitate mineralization in MC3T3-E1 cells.

MC3T3-E1 cells showed mineral deposits after about 1 week of culture when incubated in the presence of microcarrier beads. These deposits appeared as white spots on the dish surface, and under light microscopy the cells showed multiple cell layers and mineralization around the microcarriers. The deposits stained positive with calcium-specific Von Kossa's method. Using conventional assay, alkaline phosphatase activity (ALP) and parathyroid hormone-stimulated intracellular cAMP production were lower in the microcarrier cultures than in the control, but using cytochemical methods, high alkaline phosphatase activity was found around the microcarriers. These results indicate that microcarriers facilitated the formation of multiple cell layers and provided a culture environment for mineralization.

Alkaline Phosphatase↗
PubMed1988

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 uptake by duodenal mucosal cells of Hyp mice.

Effects of the administration of phosphate on nuclear 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] uptake by duodenal mucosal cells of Hyp mice were investigated. In Hyp mice fed a high phosphate diet (1.1% Ca and 2.0% phosphate) for 2 weeks, maximal nuclear 1,25-(OH)2D3 binding by duodenal mucosal cells is significantly increased from 5.01 +/- 0.49 x 10(3) to 8.23 +/- 1.10 x 10(3) sites/cell (P less than 0.05). No significant change was observed in normal mice fed the same diet. The serum phosphate concentration of Hyp mice increased significantly (P less than 0.01), whereas no significant change was found in normal mice. On this regimen, serum calcium, urinary cAMP to creatinine ratio, and cytosolic 1,25-(OH)2D3 receptor number in Hyp mice were not changed significantly. On the basis of these data, we speculate that the recovery of serum phosphate in Hyp mice fed a high phosphate diet affects the recovery of nuclear 1,25-(OH)2D3 uptake by duodenal mucosal cells. The mechanism for this recovery is not related to either the secondary hyperparathyroidism or the change in cytosolic 1,25-(OH)2D3 receptor content but, rather, to increased binding of 1,25-(OH)2D3-receptor complex to nuclei. Hypophosphatemia, therefore, appears to play a role in the vitamin D resistance in Hyp mice.

Animals↗
PubMed1987

Participation of adenosine 5'-triphosphate in the activation of membrane-bound guanylate cyclase by the atrial natriuretic factor.

The addition of ANF to Percoll-purified liver plasma membranes produced a slight activation of guanylate cyclase; the ANF-stimulated cyclase activity was further increased upon the addition of ATP to the enzyme assay mixture. The effect of ATP to potentiate the cyclase activation was concentration-dependent, required Mg2+ as a divalent cation, and was seen with membranes from various tissues and cells. ATP increased the maximal velocity of the cyclase without a change in the affinity for GTP or ANF. Phosphorylation by ATP might not be involved since ANF-stimulated guanylate cyclase was enhanced by non-phosphorylating ATP analogues as well. Thus, an allosteric ATP binding site is suggested to participate in ANF-induced regulation of membrane-bound guanylate cyclase.

Adenine Nucleotides↗
PubMed1987

Intranasal absorption of salmon calcitonin.

Eight normal subjects and 4 children with osteogenesis imperfecta were administered salmon calcitonin (S-CT) intranasally, and the pharmacokinetics of S-CT were studied. In the normal subjects, the plasma S-CT concentration showed a dose-dependent increase over a dosage range of 200-400 IU. Maximal plasma concentrations were reached 20-60 min after intranasal administration of S-CT. The plasma calcium concentration was significantly decreased 60 min after the administration. In the children, S-CT was also absorbed through the nasal mucosa. This suggests that nasal spraying may be an efficient method for administration of S-CT.

Administration, Intranasal↗
PubMed1986

Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the receptor-Ni interaction, i.e. carbachol stimulation of the activities of Ni and the GTP gamma S effect on carbachol binding, were no longer observed, when this IAP-treated Ni, instead of the nontreated Ni, was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated Ni in their basal activities observable without carbachol. No, the protein with a character very similar to Ni in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.

Adenylate Cyclase Toxin↗
PubMed1985

Dual pathways of receptor-mediated cyclic GMP generation in NG108-15 cells as differentiated by susceptibility to islet-activating protein, pertussis toxin.

The cellular cGMP content increased in response to a variety of receptor agonists, which activate [e.g., prostaglandin (PG) E1, E2, and F2 alpha] or inhibit (e.g., alpha-adrenergic, muscarinic, and opiate agonists) adenylate cyclase in neuroblastoma X glioma hybrid NG108-15 cells. The responses were additive when PGF2 alpha and enkephalin were mixed. The inhibitory guanine nucleotide regulatory protein (Ni) is involved in adenylate cyclase inhibition; this function of Ni is lost when it is ADP-ribosylated by islet-activating protein (IAP), pertussis toxin [H. Kurose, T. Katada, T. Amano, and M. Ui (1983) J. Biol. Chem. 258, 4870-4875]. The cGMP rise induced by stimulation of the receptors linked to adenylate cyclase inhibition was also diminished by IAP; the time course and dose response for the IAP-induced diminution were the same between adenylate cyclase inhibition and cGMP generation. Ni thus appears to mediate guanylate cyclase activation as well as adenylate cyclase inhibition initiated via the same receptors. Melittin also increased cGMP. No additivity was shown when enkephalin and melittin were combined, suggesting that phospholipase A2 might play a role in Ni-mediated guanylate cyclase activation. On the other hand, the PGF2 alpha-induced cGMP rise was associated with increased incorporation of 32Pi into phosphatidylinositol; was not affected by cholera toxin, IAP or forskolin; and showed no additivity when combined with A23187, which increased cGMP by itself. PGs would occupy receptors linked to phosphatidylinositol breakdown, thereby increasing the availability of intracellular Ca2+, which is responsible for guanylate cyclase activation. Thus, dual pathways are proposed for a receptor-mediated cGMP rise in NG108-15 cells.

Adenylate Cyclase Toxin↗
PubMed1985

Effects of prostaglandin E2 and indomethacin on 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in isolated kidney cells of normal and streptozotocin-induced diabetic rats.

The effects of prostaglandin E2 (PGE2) (1 microM) and indomethacin (IN) (20 microM) on 1,25 dihydroxyvitamin D3 production were studied in renal cell suspensions isolated from control, streptozotocin-induced diabetic, and insulin-treated diabetic rats. Renal cortex cells were isolated by enzymatic digestion, and preincubated for 30 min at 37 degrees C with the appropriate additive(s) followed by a 1 h incubation with 8 nM 25-hydroxyvitamin D3 in serum-free medium. Radioactivity incorporated into that fraction of the cell suspension extract co-eluting with synthetic 1,25-dihydroxyvitamin D3 on high pressure liquid chromatography was determined. All animals were raised for 5 weeks on a vitamin D-deficient diet. Isolated kidney cells from vitamin D-deficient rats showed dose-dependent response of 1,25 dihydroxyvitamin D3 production to PGE2. Cells from control animals demonstrated a stimulatory effect of PGE2 (P less than 0.05) and a suppressive effect of IN (P less than 0.01) on 1,25 dihydroxyvitamin D3 production. In contrast, cells from diabetic rat kidneys failed to respond to these agents, alterations which were reversed by insulin treatment. The accumulated data suggest that depressed synthesis of 1,25 dihydroxyvitamin D3 previously observed in the experimental diabetic rat is due, at least in part, to an impaired production and response to PGE2-like prostaglandins.

Animals↗
PubMed1985

Bone calcification and calcium homeostasis in rats with non-insulin-dependent diabetes induced by streptozocin.

The effect of mild, non-insulin-dependent diabetes (NIDDM) on bone calcification and calcium (Ca) homeostasis was studied in growing rats (males and females). The diabetic state was characterized by mild insulin deficiency, plasma levels being 73% of controls, and mild hyperglycemia, with nonfasting plasma glucose levels of 1.5 times normal. There was no difference in plasma levels of Ca, phosphate (Pi), magnesium (Mg), alkaline phosphatase, immunoreactive parathyroid hormone (iPTH), calcitonin, 25-(OH)vitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25[OH]2D), and 24,25-dihydroxyvitamin D (24,25[OH]2D) between the NIDDM rats and their controls of either sex. Metabolic Ca and Pi balance studies revealed that the experimental animals of both sexes were in positive Ca and Pi balance similar to that of their controls. Histologic studies of the kidney and intestinal slices from the experimental group were normal. Ca and Pi bone content calculated per gram bone ash of the femur, mandible, and second and fourth caudal vertebrae, and the organic content in the bones of the NIDDM animals showed no difference from their controls. Femur bone density and tibial epiphyseal growth plate width and morphology were similar histologically in the experimental and control rats. No decreased osteoid content in the tibial bone was found in the diabetic rats compared with controls. Physiologic sex differences, consisting of lower plasma Pi, higher plasma calcitonin levels, increased ratio of femur dry bone weight to total body weight, and increased percentage of mineralized and total bone volume at the tibial metaphysis seen in female compared with male control rats were also seen in the diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells.

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.

Adenosine Diphosphate Ribose↗
PubMed1983

Functional uncoupling of muscarinic receptors from adenylate cyclase in rat cardiac membranes by the active component of islet-activating protein, pertussis toxin.

Direct effects of islet-activating protein (IAP), pertussis toxin, on membrane preparations from rat heart tissues were studied. The native IAP was without effect, but its A-protomer, an active subunit, was effective after reduction of disulfide bonds in the peptide chain; it catalyzed ADP-ribosylation of the membrane Mr = 41,000 protein. Simultaneously, muscarinic receptor-mediated inhibition of adenylate cyclase was abolished. Carbachol, an agonist of muscarinic receptors, bound to membranes with the Hill coefficient smaller than unity. The affinity for the carbachol binding was lowered and the Hill coefficient was increased by guanylylimidodiphosphate (Gpp(NH)p), reflecting the muscarinic receptor coupling to the guanine nucleotide regulatory protein (N). Carbachol bound to the A-promoter-treated membranes with a lower affinity and a higher Hill coefficient, and these kinetic values were not altered by Gpp(NH)p, indicating that treatment of membranes with the A-protomer of IAP uncoupled muscarinic receptors from N. This IAP-sensitive N is Ni involved in the cyclase inhibition. Neither beta-adrenergic activation of adenylate cyclase nor beta-agonist binding to membranes was affected by the A-protomer of IAP. Thus, N (Ns) coupled to beta-receptors is not the site of its action. Although the affinity and the Hill coefficient for beta-agonist binding was not affected by preactivated cholera toxin either, the effect of Gpp(NH)p to alter these kinetic parameters was much smaller in the cholera toxin-treated membranes than in non-treated membranes. Thus, cholera toxin modified beta-receptor coupling to Ns in a manner quite different from IAP-induced modification of muscarinic receptor coupling to Ni.

Adenosine Diphosphate Ribose↗