Regulation of corticotropin releasing hormone receptor (CRH-R) in the rat anterior pituitary as assessed by radioimmunoassay.

Since the cloning of corticotropin releasing hormone receptor type 1 (CRH-R1), an essential component of the hypothalamo-pituitary-adrenal (HPA) axis, numerous studies have been conducted to monitor its changes in transcription levels under various conditions. However, the precise dynamics at the protein levels are yet to be elucidated. In the present study we aimed at establishing an RIA system for CRH-R1 protein, with an antiserum against the C-terminal fragment of human/rat CRH-R1. The generated antiserum showed a moderate cross-reactivity with CRH-R2. We examined the in vivo effect of adrenalectomy (ADX) on immunoreactive CRH-R (irCRH-R) levels in the rat AP, and the in vitro profile of irCRH-R levels in cultured rat AP cells after administration of CRH. The irCRH-R in the AP membrane of intact rats was 51.8 +/- 6.8 fmol/mg protein, which is comparable to those reported in binding studies. ADX elicited a significant decrease of irCRH-R to approximately 50% of the control level one day after ADX, which returned to the baseline level the following day. Addition of CRH to cultured AP cells resulted in a significant decrease of irCRH-R in the membrane fraction to 18% of the control level at 4 h, and it returned rapidly to 70% at 8 h. These experiments together with our previous study implicate that irCRH-R makes a different profile, with an earlier recovery than that of mRNA. Although this system cannot precisely discriminate between CRH-R1 and CRH-R2, our findings may serve to demonstrate differing CRH receptor regulations at the synthesis level and at the protein level in the rat AP.

Novel nonsense mutation in the Na+/HCO3- cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma.

Permanent isolated proximal renal tubular acidosis (pRTA) with ocular abnormalities is a systemic disease involving short stature, isolated pRTA, mental retardation, and ocular abnormalities. Kidney Na+/HCO3- cotransporter (kNBC1) cDNA from peripheral lymphocytes from a patient with permanent isolated pRTA and bilateral glaucoma was screened, and a novel homozygous mutation, namely a cytosine-to-thymine transition at nucleotide 234, which resulted in the formation of a stop codon at codon 29, was identified. This homozygous mutation, Q29X, was identified in the unique 5'-end of the kNBC1 gene (SLC4A4) of the patient. Cosegregation of this Q29X mutation with the disease and heterozygosity in the parents of the affected patient were observed. The absence of this mutation in 156 alleles from 78 Japanese individuals indicates that this mutation is directly related to the disease and is not a common DNA sequence polymorphism. This nonsense mutation predicts a truncated kNBC1 protein that lacks the 1007 amino acids of the carboxyl-terminus, and the effect on kNBC1 cotransport activity is likely to be a loss of function. In contrast, the pancreatic Na+/HCO3- cotransporter of the patient is not likely to be affected by this nonsense mutation. These results have implications for understanding the role of kNBC1 in the pathophysiologic processes of pRTA associated with ocular abnormalities and mental retardation.