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)

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.

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.

Effect of dibutyryl adenosine 3',5'-monophosphate administration on plasma concentrations of 1,25-dihydroxyvitamin D in pseudohypoparathyroidism type I.

The effect of dibutyryl cAMP on plasma concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D] was studied in two patients with pseudohypoparathyroidism (PHP) type I, five normal adults, and three normal children as controls. In normal adults, plasma 1,25-(OH)2D tended to increase 3 h after the infusion of 2.5 mg/kg dbcAMP and was significantly increased after 6 h. In normal children, plasma 1,25-(OH)2D increased 3 h after the infusion, then gradually decreased; in the patients with PHP type I, it increased greatly from 6.8 and 11.2 pg/ml to 264.6 and 128.2 pg/ml 3 h after the infusion. These results suggest that the renal mechanism for the response to parathyroid hormone is intact distal to the renal adenylate cyclase in patients with PHP type I.

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.

Cellular and subcellular distribution of alpha 2A-adrenergic receptors in the visual cortex of neonatal and adult rats.

Activation of alpha 2-adrenergic receptors (alpha 2AR) in the cerebral cortex has been shown to modulate visually guided delayed response tasks as well as anxiety and depression. We used an antiserum directed specifically against the A subtype of alpha 2AR (alpha 2AAR) to determine the cell types and subcellular sites for noradrenergic reception mediated by this receptor in the adult and the developing rat visual cortices. Light microscopic examination of adult tissue revealed numerous labeled perikarya in layers II-VI, many of which appeared distinctly pyramidal. A few perikarya in layer I also were immunoreactive. In all layers, alpha 2AAR immunoreactivity (alpha 2AAR-ir) was present within proximal dendrites and fine processes. In neonatal tissue, there was an intense, distinct band of immunoreactivity spanning the layer composed of tightly packed immature cell bodies, i.e., the cortical plate. The band dissipated as this tier differentiated postnatally into the supragranular layers. Electron microscopy showed that the supragranular layers, which contain the highest density of noradrenergic fibers, also contain the highest areal density of labeled postsynaptic junctions beyond 2 weeks of age. Throughout the ages, the majority of immunoreactivity occurred at sites which, in single ultrathin sections, appeared to be nonjunctional sites of axons, dendrites, and in glial processes. Our observations indicate that (1) both pyramidal and nonpyramidal neurons are receptive to norepinephrine via alpha 2AAR, (2) alpha 2AAR synthesis is robust prior to synaptogenesis, and (3) alpha 2AAR operates both pre- and postsynaptically.

Adrenergic alpha2C-receptors reside in rat striatal GABAergic projection neurons: comparison of radioligand binding and immunohistochemistry.

We have investigated the distribution of alpha2c-adrenergic receptors in the rat striatum and characterized the striatal neuron types expressing these receptors. Sequential double-labelled immunocytochemistry was performed with a polyclonal antibody against rat alpha2c-adrenoceptors and antibodies against GABA, Calbindin-D28k, parvalbumin and calretinin. The subregional distribution of alpha2c-adrenoceptor binding sites in the striatum was also quantitatively investigated using selective radioligands. Almost all lightly stained striatal GABAergic neurons, with the morphological characteristics of medium-sized spiny projection neurons (94% of GABAergic cells counted), contained alpha2c-adrenoceptor-immunoreactive structures. Intensely labelled GABAergic inteneurons (6%) were devoid of alpha2c-adrenoceptor immunoreactivity. The co-localization of calbindin- and alpha2c-adrenoceptor immunoreactivity in the majority of the cells confirmed the presence of alpha2c-adrenoceptors in the population of medium-sized spiny neurons. Furthermore, the alpha2c-adrenoceptor/calbindin double-labelling disclosed the existence of three neuronal subsets in the matrix compartment of the striatum: a large proportion (83%) of double-labelled neurons, a population of neurons (8%) that exhibited only alpha2c-adrenoceptor immunoreactivity without calbindin immunoreactivity, and a population of neurons (9%) immunoreactive for calbindin, but lacking alpha2c-adrenoceptors. In addition, alpha2c-adrenoceptor immunolabelled neurons were observed in calbindin-free striatal patches. Parvalbumin- and calretinin-positive neurons never displayed alpha2c-adrenoceptor immunoreactivity, confirming that striatal GABAergic interneurons are devoid of alpha2c-adrenoceptors. The present findings indicate that alpha2c-adrenoceptors are localized in GABAergic medium-sized spiny projection neurons but not in interneurons of the rat striatum, and that they may modulate both the direct and indirect pathways of the basal ganglia, as well as participate in the regulation of mesencephalic dopaminergic neurons.

Ser203 as well as Ser204 and Ser207 in fifth transmembrane domain of the human beta2-adrenoceptor contributes to agonist binding and receptor activation.

We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.

Differential contribution of two serine residues of wild type and constitutively active beta2-adrenoceptors to the interaction with beta2-selective agonists.

1. We have studied the difference in receptor binding activity between partial and full beta2-adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the beta2-adrenoceptor, amino acid residues that are important for activation of the beta2-adrenoceptor. 2. In the binding study with [125I]-iodocyanopindolol, the Ki values of (+/-)-salbutamol, (+/-)-salmeterol, TA-2005 and (-)-isoprenaline for the beta2-adrenoceptor expressed in COS-7 cell membranes were 3340, 21.0, 12.0 and 904 nM, respectively. The beta1/beta2 selectivity of these agonists was in the order of (+/-)-salmeterol (332 fold) > TA-2005 (52.8) > (+/-)-salbutamol (6.8) > (-)-isoprenaline (1.1), and the beta3-/beta2-adrenoceptor selectivity of these agonists was in the order of TA-2005 (150 fold) > (+/-)-salmeterol (88.6) > (+/-)-salbutamol (10.4) > (-)-isoprenaline (3.2). 3. The maximal activation of adenylyl cyclase by stimulation of the beta1-, beta2- and beta3-adrenoceptors by TA-2005 was 32, 100 and 100% of that by (-)-isoprenaline, respectively, indicating that TA-2005 is a full agonist at the beta2- and beta3-adrenoceptors and a partial agonist at the beta1-adrenoceptor. (+/-)-Salbutamol and (+/-)-salmeterol were partial agonists at both beta1- (8% and 9% of (-)-isoprenaline) and beta2- (83% and 74% of (-)-isoprenaline) adrenoceptors. 4. The affinities of full agonists, TA-2005 and (-)-isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the beta2-adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (+/-)-salbutamol and (+/-)-salmeterol. Although the affinities of full agonists for the S207A-beta2-adrenoceptor were decreased, those of partial agonists for the S207A-beta2-adrenoceptor were essentially the same as for the wild type receptor. 5. The constitutively active mutant (L266S, L272A) of the beta2-adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the S207A-beta2-adrenoceptor were essentially the same as those for the wild type beta2-adrenoceptor, the affinities of (+/-)-salbutamol and (+/-)-salmeterol for the constitutively active beta2-adrenoceptor were decreased by substitution of Ser207. 6. These results suggest that Ser204 and Ser207 of the wild type and constitutively active beta2-adrenoceptors differentially interacted with beta2-selective agonists.

Perikaryal and synaptic localization of alpha 2A-adrenergic receptor-like immunoreactivity.

Through molecular cloning, the existence of three distinct subtypes of alpha 2-adrenergic receptors (alpha 2AR)--A, B and C--has been established and are referred to as alpha 2A AR, alpha 2B AR and alpha 2CAR. Due to limitations in pharmacological tools, it has been difficult to ascribe the role of each subtype to the central functions of alpha 2AR. In situ hybridization studies have provided valuable information regarding their distribution within brain. However, little is known about their subcellular distribution, and in particular, their pre- versus postsynaptic localization or their relation to noradrenergic neurons in the CNS. We used an antiserum that selectively recognizes the A-subtype of alpha 2AR to determine: (1) the regional distribution of the receptor within brains of rat and monkey; (2) the subcellular distribution of the receptor in locus coeruleus (LC) of rats and prefrontal cortex of monkeys; and (3) the ultrastructural relation of the receptor to noradrenergic processes in LC. Light microscopic immunocytochemistry revealed prominent immunoreactivity in LC, the brainstem regions modulating the baroreflex, the granule cell layer of the cerebellar cortex, the paraventricular and supraoptic nuclei of the hypothalamus (PVN, SON), the basal ganglia, all thalamic nuclei, the hippocampal formation and throughout cerebral cortical areas. Comparison of results obtained from rat and monkey brains revealed no apparent interspecies-differences in the regional distribution of immunoreactivity. Immunoreactivity occurred as small puncta, less than 1 micron in diameter, that cluster over neuronal perikarya. Besides these puncta, cell bodies, proximal dendrites and fine varicose processes--most likely to be axonal--of the PVN and SON and the hippocampal granule cells also exhibited homogeneously intense distribution of immunoreactivity. Subcellularly, alpha 2AAR-ir in LC and prefrontal cortex were associated with synaptic and non-synaptic plasma membrane of dendrites and perikarya as well as perikaryal membranous organelles. In addition, cortical tissue, but not LC, exhibited prominent immunoreactivity within spine heads. Rat brainstem tissue immunolabeled dually for alpha 2AAR and dopamine beta-hydroxylase (D beta H, the noradrenaline-synthesizing enzyme) revealed that alpha 2AAR-li occurs in catecholaminergic terminals but is also prevalent within non-catecholaminergic terminals. Terminals exhibiting alpha 2AAR-li formed symmetric and asymmetric types of synapses onto dendrites with and without D beta H-immunoreactivity. These results indicate that: (1) the A-subtype of alpha 2AR is distributed widely within brain; (2) alpha 2AAR-li reflects the presence of newly synthesized alph 2AAR in perikarya as well as those receptors along the plasma membrane of perikarya, dendritic trunks and spines; and (3) alpha 2AAR in LC may operate as heteroreceptors on non-catecholaminergic terminals as well as autoreceptors on noradrenergic terminals.

Molecular characterization of pharmacological properties of T-0509 for beta-adrenoceptors.

The pharmacological properties of T-0509, (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, were compared with those of isoproterenol. In the radioligand binding studies of [125I]iodocyanopindolol with COS-7 cell membranes that transiently expressed beta-adrenoceptor subtypes, T-0509 exhibited 11- and 97-fold greater Ki values for beta 2- and beta 3-adrenoceptors, respectively, compared with beta 1-adrenoceptors. Affinities of beta 2- and beta 3-adrenoceptors to isoproterenol were 1.4- and 28-fold lower than that of beta 1-adrenoceptors, respectively. The maximal stimulatory effects of T-0509 on adenylyl cyclase of CHO-K1 (chinese hamster ovary K1) cell membranes expressing beta 1- or beta 2-adrenoceptors were 85% or 96% of those produced by isoproterenol, respectively. These results indicate that T-0509 is a relatively specific beta 1-adrenoceptor agonist with a high intrinsic activity as compared with isoproterenol.

Similar ligand binding in recombinant human alpha 2 C2-adrenoceptors produced in mammalian, insect and yeast cells.

Ligand binding properties were investigated in recombinant human alpha 2C2-adrenoceptors expressed in three different host systems: Shionogi S115 mouse mammary tumour cells, Spodoptera frugiperda Sf9 insect cells and Saccharomyces cerevisiae yeast cells. The expected 43 kDa alpha 2C2 protein was visualized with immunoblotting using a polyclonal alpha 2C2-receptor antibody. [3H]Rauwolscine binding in cell homogenates or membranes (Bmax 3-11 pmol/mg protein; Kd approximately 5.5 nM) was inhibited by prazosin, oxymetazoline, RX821002, chlorpromazine and (-)-noradrenaline with and without the GTP-analogue Gpp(NH)p with similar Ki values in the different host systems. This indicates that alpha 2C2-adrenoceptors retain their binding characteristics irrespective of the host environment.

Low affinity of beta1-adrenergic receptor for beta-arrestins explains the resistance to agonist-induced internalization.

It has been reported that beta-arrestin is essential for the internalization of many G protein-coupled receptors. Since beta1-adrenergic receptor (beta1AR) shows the resistance to agonist-induced internalization, we examine the interaction of beta-arrestin with beta1AR with three different approaches: translocation of beta-arrestin to the plasma membrane, direct binding of in vitro translated beta-arrestin to intracellular domains of beta1- and beta2ARs, inhibition of beta1- and beta2AR-stimulated adenylyl cyclase activities by beta-arrestin. The enhanced green fluorescent protein (EGFP)-tagged beta-arrestin 2 (beta-arrestin 2-GFP) translocates to and stays at the plasma membrane by beta2AR stimulation. Beta-arrestin 2-GFP also translocates to the plasma membrane upon beta1AR stimulation. However, it returns to the cytoplasm 10 - 30 min after agonist stimulation. The amount of beta-arrestin bound to the third intracellular loop and the carboxyl tail of beta1AR is lower than that of beta2AR. The fusion protein of beta-arrestin 1 with glutathione-S-transferase inhibits the beta1- and beta2AR-stimulated adenylyl cyclase activities. However, inhibition of the beta1AR-stimulated activity requires a higher amount of the fusion protein than that of the beta2AR-stimulated activity. These results suggest that affinity of beta1AR for beta-arrestins is lower than that of beta2AR, and explains the resistance to agonist-induced internalization. This conclusion is further supported by the finding that beta-arrestin can induce internalization of beta1AR when beta-arrestin 1 fused to the carboxyl tail of beta1AR.

Domains of beta1 and beta2 adrenergic receptors to bind subtype selective agonists.

We studied the binding region of several beta1 and beta2 selective agonists by using chimeric beta1 and beta2ARs, and point-mutated beta2 adrenergic receptors (ARs). By replacing a single transmembrane domain (TMD) of beta1AR (or beta2AR) with the corresponding region of beta2AR (or beta1AR), we found that beta2 or beta1 selectivities were determined by TMD2 and TMD7 of beta2AR or by TMD2 of beta1AR, respectively. Alanine-substituted beta2AR mutants showed that tyrosine at position 308 in TMD7 played an important role in binding of beta2 selective agonists with high affinity. These data also suggested that the substituent on the amine portion was important for subtype selective agonist binding.