Masashi Yanagisawa

Induction of endothelin-1 gene by angiotensin and vasopressin in endothelial cells

To elucidate the cellular mechanism of endothelin-1 biosynthesis induced by angiotensin and vasopressin, we first cloned and sequenced full-length bovine preproendothelin-1 complementary DNA (cDNA) from a cultured bovine carotid artery endothelial cell cDNA library. The predicted bovine preproendothelin-1 consists of 202 amino acid residues and has a high percentage of homology to human, porcine, and rat preproendothelin-1 (70%, 81%, and 77%, respectively). Big endothelin-1, an intermediate form, consists of 39 residues differing only at position Val28 from porcine (He28) and His27 from rat (Arg27). The predicted 21-residue mature endothelin-1 is identical to human, porcine, rat, canine, and mouse endothelin-1. Northern blot analysis with the cloned cDNA as a probe demonstrated that a single 2.3-kb preproendothelin- 1 messenger RNA (mRNA) is expressed not only in endothelial cells, but also in various bovine tissues, including lung, brain, heart, intestine, kidney, ovary, and urinary bladder. Angiotensin II and arginine vasopressin immediately and dose-dependently induced expression of preproendothelin-1 mRNA, whose effects were abolished by specific receptor antagonists. These findings suggest that stimulation of endothelin-1 secretion from endothelial cells by both agonists may be principally due to induction of preproendothelin-1 mRNA.

Concomitant expression of receptor subtype and isopeptide of endothelin by human adrenal gland.

We studied whether specific receptors for endothelin (ET) isopeptide exist in human aldosterone-producing adenoma and normal adrenal cortex, and whether ET isopeptides are produced by human adrenal gland. Competitive binding studies using [125I]ET-1 as a radioligand revealed the presence of a single class of high-affinity binding sites for ET-1 with the apparent KD of 70 +/- 31 pM and Bmax of 226 +/- 139 fmol/mg protein in adenoma membranes almost comparable to those in adjacent normal cortex. The apparent Ki for ET-2 and ET-3 were 89 +/- 33 pM and 82 +/- 16 pM, respectively. Northern blot analysis of poly(A)+ RNA of adenoma and adjacent normal cortex using cDNAs for ET receptor subtype (ETA, ETB) and ET isopeptide (ET-1, ET-3) as probes revealed that ETA and ETB receptors as well as ET isopeptides (preproET-1, preproET-3) are concomitantly expressed in both tissues. Our data demonstrate for the first time that ET receptor subtype (ETA and ETB) and ET isopeptide (ET-1 and ET-3) are concomitantly expressed by human adrenal cortex, suggesting the potential role of ETs as a local mediator in human adrenal gland.

Endothelin receptors in human parathyroid gland

We studied whether specific receptors for endothelins (ETs) exist in human parathyroid tissues and whether ETs may have any effect on secretion of PTH from parathyroid cells. Binding studies using [125I]ET-1 to the parathyroid membranes obtained from patients with hyperparathyroidism (2 adenomas, 2 hyperplasias) revealed that ET-1 competitively inhibited the binding of [125I]ET-1 to the membranes (the apparent Kd: 62 +/- 18 pM), whereas ET-3 showed biphasic and less steep inhibition curve than ET-1 in all tissue membranes examined. Northern blot analysis of poly(A)+ RNA from the parathyroid adenoma clearly demonstrated gene expression of both ETA and ETB receptors as well as preproET-1. ET-1 inhibited basal PTH secretion from dispersed adenoma cells more potently than ET-3. The present study clearly demonstrates the presence of both ETA and ETB receptor subtypes in human parathyroid tissues through which ETs may modulate PTH secretion in an autocrine and/or paracrine manner.

Endothelin-1 in luteal tissue

The aim of this study was to immunologically and biologically detect endothelin-1 (ET-1) in rat corpora lutea (CL). Recently, we established a highly sensitive and specific sandwich-enzyme immunoassay (EIA) for ET-1. Using this assay, the presence of ET-1 was investigated in superovulated ovaries, induced with pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and ovaries from pseudopregnant rats, induced by cervical stimulation. A high concentration of immunoreactive endothelin-1 (ir-ET-1) was found in the CL. On reverse phase-high performance liquid chromatography (RP-HPLC) coupled with EIA, ir-ET-1 was exclusively eluted at the same position as synthetic ET-1, indicating that ir-ET-1 is identical to ET-1. The level of ir-ET-1 was significantly (P less than 0.001) higher in the CL 7 days after hCG injection than it was 4 days after hCG injection. On day 7 of pseudopregnancy (PSP), the ir-ET-1 level was also significantly (P less than 0.001) higher than on day 4 of PSP. These results demonstrated that ET-1 is present in a high concentration in the CL, suggesting a new intraovarian peptide which may have a physiological function in the ovary and which may vary in quantity according to the age of the CL.

Chapter V Orexin receptors

Publisher Summary In accordance with the diffuse projection of orexin neurons, orexin receptors have a widespread distribution in the brain. This suggests diverse and complex physiological roles of the orexins in brain function. The expression patterns of the two orexin receptors are quite different and usually complementary, suggesting differential roles for each subtype. Structures of orexins were chemically determined by biochemical purification and sequence analysis by Edman sequencing and mass spectrometry. Orexin-A is a 33-amino-acid peptide of 3562 Da, with an N-terminal pyroglutamyl residue and C-terminal amidation. Molecular mass of the purified peptide, as well as its sequencing analyses, indicated that the four Cys residues of orexin-A formed two sets of intrachain disulfide bonds.