ABSTRACT
A 537 base pair cDNA clone for murine GIP has been isolated. The elucidated sequence encodes an open reading frame of 432 base pairs which codes for a 144 amino acid precursor. Murine GIP is predicted to differ from the human hormone by three amino acid substitutions: arginine for histidine at position 18, arginine for lysine at position 30 and serine for lysine at position 34. GIP mRNA levels in STC6-14 cells incubated in the presence of varying glucose concentrations was investigated using a competitive-PCR method. In the presence of a 5-mM glucose stimulus, 1 x 10(5) GIP cells were found to contain 3.9 +/- 0.59 amol of GIP mRNA while the same number of cells contained 11.6 +/- 1.4 amol when subjected to a high (25 mM) glucose stimulus.
Subject(s)
Gastric Inhibitory Polypeptide/biosynthesis , Gene Expression Regulation , Glucose/pharmacology , Protein Precursors/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Dose-Response Relationship, Drug , Gastric Inhibitory Polypeptide/genetics , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Neoplasm/analysis , Sequence Homology, Amino Acid , Tumor Cells, CulturedABSTRACT
This study describes the development of an oncorhynchid growth hormone (GH) radioimmunoassay using recombinant chum salmon GH (rsGH) and a rabbit antiserum (TJK-1) raised against this recombinant material. The assay was designed to measure the wide range of circulating immunoreactive GH (IRGH) levels in Pacific salmonids, resulting in a standard curve capable of accurately determining plasma levels of IRGH from 0.5 to 250 ng/mL without dilution. The assay ED50 and ED90 values averaged 13.1 and 0.5 ng/mL, respectively. This radioimmunoassay specifically recognizes oncorhynchid IRGH, showing no cross-reactivity with recombinant porcine and bovine GH, or natural chum salmon prolactin at concentrations up to 10 micrograms/mL. Curves approximately parallel to the standard curve were obtained with purified natural coho salmon GH and plasma from chinook salmon. Recovery of rsGH from plasma was complete over the full range of the standard curve. Intra- and inter-assay coefficients of variation were 6.0 and 12.9%, respectively. Plasma IRGH levels in fed coho salmon were 30.6 +/- 5.3 ng/mL, while those in fish starved for 2 weeks were 132.9 +/- 53.9 ng/mL. Starvation for an additional 4 weeks had no significant effect. Plasma IRGH levels in control rainbow trout injected with saline were significantly higher 45 min post-injection. In contrast, fish injected with recombinant porcine GH exhibited no elevation in IRGH. It is speculated that exogenous GH inhibits the production of endogenous GH.
