Takako Moriyama

Differential regulation of CREB and ERK phosphorylation through corticotropin-releasing factor receptors type 1 and 2 in AtT-20 and A7r5 cells.

The corticotropin-releasing factor (CRF) family of peptides generally exerts its biological actions by binding to two major subtypes of CRF receptors: CRF receptor type 1 (CRF1 receptor) and CRF receptor type 2 (CRF2 receptor). In this study, we investigated the mechanism by which three ligands altered phosphorylation of CREB and ERK 1/2, using AtT-20 cells (expressing CRF1 receptor) and A7r5 cells (expressing CRF2 receptor). Incubation with 100 nM of CRF, urocortin 1 (UCN 1), or UCN 2 increased CREB phosphorylation. The protein kinase A pathway was involved in the CRF- or UCN-mediated increase in CREB phosphorylation in both cell lines. Bisindolylmaleimide partially inhibited the CRF-mediated increase in CREB phosphorylation, but only in AtT-20 cells, suggesting that the protein kinase C pathway is involved in regulation of CREB phosphorylation via CRF1 receptor but not CRF2 receptor. CRF increased ERK phosphorylation in AtT-20 cells, whereas the UCNs decreased it in A7r5 cells. Bisindolylmaleimide partially inhibited the UCN-mediated decrease in ERK phosphorylation in A7r5 cells, suggesting that the protein kinase C pathway is partially involved in CRF2 receptor signal transduction. In AtT-20 cells, the mitogen-activated protein kinase kinase pathway regulated ERK phosphorylation following CRF1 receptor activation. These findings suggest differential regulation of CREB and ERK 1/2 phosphorylation through CRF receptors.

Differential regulation of corticotropin-releasing factor receptor type 1 (CRF1 receptor) mRNA via protein kinase A and mitogen-activated protein kinase pathways in rat anterior pituitary cells.

Corticotropin-releasing factor (CRF) receptor type 1 (CRF(1) receptor) mRNA levels are down-regulated by CRF via the cyclic AMP-protein kinase A (PKA) pathway. In this study, we focused on the involvement of both the mitogen-activated protein (MAP) kinase pathway and PKA in this regulation. Real-time PCR (RT-PCR) revealed that a MAP kinase, extracellular signal-regulated kinases 1/2, pathway was also involved in the down-regulation of CRF(1) receptor mRNA levels by CRF in the rat anterior pituitary (AP). Down-regulation of CRF(1) receptor mRNA levels was caused by a post-transcriptional system such as mRNA degradation, as incubation with CRF significantly decreased the half-life of CRF(1) receptor mRNA. Furthermore, pre-treatment with a PKA inhibitor completely blocked CRF-induced CRF(1) receptor mRNA destabilization, while pre-treatment with an extracellular signal-regulated kinases 1/2 inhibitor had no inhibitory effect. These results suggested that in the rat AP, down-regulation of CRF(1) receptor mRNA levels is caused by mRNA degradation via PKA, but not by the MAP kinase pathway.

Evaluation of the (1–24) adrenocorticotropin stimulation test for the diagnosis of primary aldosteronism

Objective: The purpose of this study was to investigate the diagnostic power of the adrenocorticotropin (ACTH) stimulation test in patients with primary aldosteronism (PA) and those with aldosterone-producing adenoma (APA). Design: This study was based on a retrospective database analysis. Subjects and methods: We assessed 158 hypertensive patients with a high plasma aldosterone-to-renin ratio (ARR) including 97 with at least one positive confirmatory test result who did not undergo surgery and comprised a “possible PA” group, 19 with negative results in all tests who were the “non-PA” group, and 41 diagnosed with APA following surgery who were the APA group. The “confirmed PA group” included APA patients and patients from the possible PA group showing both high ARR and hypokalemia. One case was diagnosed as a metastasis. Results: Receiver-operating characteristic (ROC) analysis showed that the diagnostic accuracy of ACTH test was not very effective in differentiating between APA patients and possible PA and non-PA patients. The optimal cut-off value of maximal plasma aldosterone concentration for differentiating between patient in the confirmed PA group and other patients showed moderate accuracy. Conclusions: The ACTH test may not be useful as a screening or confirmatory test, but the test may be useful for differentiating between patients with confirmed PA and the rest of the cohort. The positive finding of the ACTH test may at least support a higher likelihood of lateralizing on adrenal venous sampling.

Gene analysis of the calcium channel 1 subunit and clinical studies for two patients with hypokalemic periodic paralysis

Hypokalemic periodic paralysis (HypoPP) is a skeletal muscle disorder in which episodic attacks of muscle weakness occur; they are associated with decreased serum potassium (K+) levels. Recent molecular approaches have clarified that the condition is caused by mutations in the skeletal muscle voltage-gated calcium channel 1 subunit (CACNA1S). We describe two unrelated patients with HypoPP, followed by their relevant clinical studies and gene analysis. Clinical studies included an oral glucose tolerance test (OGTT), food-loading and insulin tolerance tests (ITT). For Case 1, serum K+ levels were extremely decreased following insulin tolerance testing compared with levels for controls. These results support the hypothesis that no efflux of K+ ion occurs in patients because of low activity of adenosine triphosphate (ATP)-sensitive K+ channel (KATP) channels. Mutational analysis of the CACNA1S gene showed a duplicate insertion of 14 base pairs (bp) from 52 to 65 in intron 26, present in the heterozygous state in both patients. No other mutations were detected in the CACNA1S gene, the muscle sodium channel gene (SCN4A) or the voltage-gated K+ channel gene (KCN3) of either patient. Further analysis showed that this duplicate insertion of 14 bp in intron 26 of the CACNA1S gene was found in 23.7% of healthy subjects. K+ dynamics studies are useful for confirming this syndrome, while further gene analysis for various ion channels using amplification and direct sequencing are required to evaluate the molecular basis of the disorder in the individual patient.

Diagnosis of a case of Gitelman’s syndrome based on renal clearance studies and gene analysis of a novel mutation of the thiazide-sensitive Na-Cl cotransporter

Gitelman’s syndrome is a recessively inherited renal tubular disorder characterized by low plasma potassium and magnesium levels, reduced calcium excretion, metabolic alkalosis, and increased plasma renin activity and plasma aldosterone concentration with normal blood pressure levels. A 23-yr-old man was referred to our department for further evaluation of hypokalemia. The patient also had hypomagnesemia and markedly reduced urinary calcium excretion. Renal clearance studies and gene analysis of the thiazide-sensitive Na-Cl cotransporter (TSC) were performed in the patient. In response to an iv injection of furosemide, chloride clearance (CCl) increased markedly, while distal fractional chloride reabsorption CH2O/ (CH2O+CCl) was considerably reduced. In contrast, thiazide ingestion had no significant effects on these parameters. The patient had compound heterozygous mutations in the alleles encoding the TSC gene, one of which has not been formerly reported. Renal clearance studies and TSC gene analysis by amplification and direct sequencing are useful diagnostic tools for confirming a diagnosis of Gitelman’s syndrome.