Antisense and Sense RNA Probe Hybridization to Immobilized Crude Cellular Lysates: A Tool to Screen Growth Hormone Antagonists

Abstract

The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor-1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human α1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds. (Journal of Biomolecular Screening 2005:260-269)

In vivo evaluation of a novel, orally bioavailable, small molecule growth hormone receptor antagonist

“…In animals treated with 0.48 mg kg À1 day À1 GH and the same doses of BVT-A the concentration was 3.3 ± 1.2 and 6.2 ± 1.5 lM, respectively. These levels are in the same range as concentrations found to have an effect in vitro [16]. Of note is that increased dose of GH lowered the plasma concentration of BVT-A administered at the same dose.…”

“…The most marked induction was seen for IGF-I mRNA, which is in line with its high hepatic levels and tight regulation by GH. The level of induction was similar to the effect of GH in cultured hepatocytes in vitro [16]. The suppression of GH-induction of IGF-I mRNA by BVT-A in vivo was however less pronounced than previously observed in vitro.…”

“…Studies on the effect of inhibitors of GH action using normal experimental animals are difficult to evaluate because of the complex relationship and feed back mechanisms of the GH/IGF-I system and the hypothalamo-pituitary axis. Therefore we have explored the use of hypophysectomized (HX) rats treated with GH, to circumvent the GH/IGF-I feedback mechanisms, for investigation of in vivo effects of a low molecular weight compound, BVT-A, shown to be a GH receptor (GHR) antagonist in cultured hepatocytes [16]. To evaluate the in vivo effects we measured body weight, serum IGF-I levels and hepatic expression of components of the GH/IGF-I system.…”

Growth hormone receptor modulators

“…The small molecular weight compound down-regulates GH-stimulated IGF-I expression [49]. Administration of BVT-A suppresses serum IGF-I, hepatic mRNA levels of IGF-I, IGF-BP3, ALS, and the IGF-I and GH receptors in hypophysectomized rats [50].…”

Targeting growth hormone function: strategies and therapeutic applications

“…In two studies, BVT-A suppressed GH induction of IGF1 expression in hepatocytes in vitro and reduced GH stimulation of IGF1 secretion and body weight in hypophysectomized rats. 138,139 However, it is unclear where in the GH signaling pathway this compound acts, and no subsequent activity has been reported in this area since the original publication.…”