Solubilization of binding sites for IAPP and CGRP from rat lung

Bhogal, Ranjev; Smith, David M.; Bloom, Stephen R.

doi:10.1016/0196-9781(94)90113-9

Cited by 6 publications

(3 citation statements)

References 35 publications

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“…Chemical crosslinking with BSOCOES followed by SDS\PAGE was performed as previously described [17,26,27]. Membranes were incubated with 12 000 Bq (1.9 nM) of "#&I-rat adrenomedullin or 10 000 Bq (278 pM) of ["#&I]CGRP [28]. Autoradiographs were exposed to Kodak X-Omat film (IBI Ltd, Cambridge, Cambs., U.K.) at k80 mC for 1-10 days before development.…”

Section: Receptor Binding Assays and Chemical Cross-linking Of Receptmentioning

confidence: 99%

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

Coppock

Owji

Bloom

et al. 1996

Biochemical Journal

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We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22 +/- 0.04 nM (mean +/- S.E.M.) and a concentration of binding sites (Bmax) of 0.95 +/- 0.19 pmol/mg of protein (mean +/- S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8-37) competed weakly at this site (IC50 > 10 and 601 +/- 298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13 +/- 0.01 nM; Bmax = 0.83 +/- 0.10 pmol/mg of protein) where both CGRP(8-37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15 +/- 0.12 and 8.68 +/- 0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site-ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8-37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

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Section: Receptor Binding Assays and Chemical Cross-linking Of Receptmentioning

confidence: 99%

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

Coppock

Owji

Bloom

et al. 1996

Biochemical Journal

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“…Low-affinity binding sites have been studied in the liver, skeletal muscle [26] and lung [27]. Using in vitro autoradiographic techniques, high-…”

Section: Introductionmentioning

confidence: 99%

Interaction of the Renal Amylin and Renin-Angiotensin Systems in Animal Models of Diabetes and Hypertension

Wookey

Cao²,

Cooper

1998

Mineral Electrolyte Metab

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The range of known actions of amylin are reviewed together with the proposal that an important role for amylin may be the hormonal integration of diverse physiological systems activated with feeding. Major targets for the action of amylin are found within the kidney. Components of the amylin system (AS) have been shown to influence the activity of components of the renin-angiotensin system (RAS), and vice versa, in normal, hypertensive and diabetic models. For instance, amylin injected into humans and rats elicits a rapid rise in plasma renin activity. Furthermore, in two models of hypertension (the spontaneously hypertensive rat (SHR) and the model with subtotal nephrectomy (STNx)), the density of amylin-binding sites in the renal cortex associated with the proximal tubules, was associated with elevation of blood pressure. In normotensive controls and in the STNx model, but not in the SHR model, treatment with angiotensin-converting enzyme (ACE) inhibitors reduced blood pressure and the density of amylin binding in the renal cortex. In Sprague-Dawley rats, angiotensin II (Ang II) infusion was associated with increased density of amylin-binding sites as well as elevated blood pressure. Thus, there appears to be a direct relationship between the activity of Ang II and the binding sites for amylin in the renal cortex. From these studies it has been postulated that the activation of the AS in the kidney may play a role in the genesis and/or development of hypertension in certain contexts. The transient expression of amylin mRNA has been detected perinatally, using in situ hybridization, in the subnephrogenic zone of the metanephros and is associated with proximal tubules of the developing nephron. These cells situated close to the glomeruli, represent a subset of brush border epithelial cells. Amylin immunoreactivity (IR) is also found in these cells and colocalizes with angiotensinogen IR. Thus a second important role for amylin is described in which it plays a role as a growth factor in the developing kidney and in renal regrowth in the adult kidney. In a model of IDDM (streptozotocin diabetes), amylin and angiotensinogen IR are both restricted to a subset of brush border epithelial cells close to glomeruli which, in the developing kidney, expressed amylin mRNA. Thus in this IDDM model, we hypothesize that amylin mRNA transcription which is normally downregulated in the adult, is upregulated in this subset of these brush border epithelial cells, and that it stimulates the activity of a local RAS by an intracellular mechanism, leading to the biosynthesis of Ang II. It remains to be determined that if amylin is playing a role in stimulating local Ang II production at these sites, this provides a mechanism for activation of TGF-β, ultimately leading to interstitial fibrosis.

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“…20 Plasma membranes from rat lung tissue were prepared and used as previously described. 21 Exendin-4 was used as label due to the instability of GLP- …”

Section: Glp-1 Receptor Binding Assaymentioning

confidence: 99%

Subcutaneous oxyntomodulin analogue administration reduces body weight in lean and obese rodents

et al. 2010

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Objective: To determine the efficacy of a long-acting oxyntomodulin (OXM) analogue, OXM6421, in inhibiting food intake and decreasing body weight in lean and diet-induced obese (DIO) rodents. Research design and methods: The glucagon-like peptide-1 (GLP-1) receptor binding affinity and efficacy, sensitivity to enzymatic degradation in vitro and persistence in the circulation after peripheral administration were investigated for OXM6421 and compared with native OXM. The chronic effect of OXM6421 on food intake, body weight and energy expenditure was examined in lean rats, and its anti-obesity potential was evaluated in DIO mice. Results: OXM6421 showed enhanced GLP-1 receptor binding affinity and cyclic adenosine monophosphate (cAMP) stimulation, and higher resistance to enzymatic degradation by dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidase (NEP) compared with native OXM. OXM6421 persisted longer in the circulation than OXM after peripheral administration. Acute administration of OXM6421 potently inhibited food intake in lean rodents, with cumulative effects lasting up to 24 h. In lean rats, daily subcutaneous (s.c.) administration of OXM6421 caused greater weight loss than the pair-fed animals, and a higher rate of oxygen consumption than both the pair-fed and the saline controls. In DIO mice, continuous s.c. infusion of OXM6421 resulted in a significant weight loss, accompanied by an improvement in glucose homeostasis and an increase in circulating adiponectin levels. Once-daily s.c. administration of OXM6421 for 21 days caused sustained weight loss in DIO mice. Conclusion: OXM6421 induces negative energy balance in both lean and obese rodents, suggesting that long-acting OXM analogues may represent a potential therapy for obesity.

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Solubilization of binding sites for IAPP and CGRP from rat lung

Cited by 6 publications

References 35 publications

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

Interaction of the Renal Amylin and Renin-Angiotensin Systems in Animal Models of Diabetes and Hypertension

Subcutaneous oxyntomodulin analogue administration reduces body weight in lean and obese rodents

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