Masatora Iwashina

Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues.

Background Urotensin-II (UII), a cyclic dodecapeptide originally isolated from fish urophysis that has potent cardiovascular effects, has recently been identified as an endogenous ligand for the orphan G protein-coupled receptor, GPR14. The physiological roles of endogenous UII and its receptor in humans remain unknown. Objective To investigate the presence of human (h) UII-like immunoreactivity (hUII-LI) in human biological fluids, and the expression of hUII and GPR14 genes in human tissues. Methods We have established a specific radioimmunoassay for hUII and the real-time quantitative reverse transcriptase polymerase chain reaction method using LightCycler for the quantification of hUII and GPR14 mRNAs. Results Gel filtration and reverse-phase high performance liquid chromatography of human urine extracts revealed a single major peak of hUII-LI co-eluting with known hUII. The concentrations of hUII-LI in urine from normal individuals were 7.4 ± 0.9 μg/g creatinine, whereas its plasma concentration was undetectable (< 50 pg/ml). Urinary hUII concentrations from patients with essential hypertension and those with renal tubular abnormality, but not with glomerular diseases, were significantly greater than those from normal individuals. The resulting fractional excretion of hUII, exceeding the glomerular filtration rate, suggests a renal origin of urinary UII-LI. hUII mRNAs were abundantly expressed in the kidney and the right atrium, but far less so in the vasculature, whereas GPR14 mRNAs were equally and abundantly expressed in both cardiovascular and renal tissues. Conclusions These data suggest that urinary hUII is derived mainly from a renal source, and that hUII functions as an autocrine/paracrine vasoactive factor not only in the cardiovascular system, but also in the kidney, with an as yet unspecified function.

Characterization of immunoreactive adrenomedullin in human plasma and urine

Adrenomedullin(AM) is a novel vasodilator peptide recently isolated from pheochromocytoma. Using a specific and sensitive radioimmunoassay for human AM, we have characterized immunoreactive AM in human plasma and urine. Patients with chronic renal failure had about five-fold higher plasma immunoreactive AM levels than normal subjects, which did not change before and after hemodialysis. Immunoreactive AM was present in normal human urine, whose concentrations were about six-fold greater than those in human plasma. Reverse-phase HPLC of human plasma and urine revealed that immunoreactive AM emerged as a single peak at a position identical to that of authentic human AM(1-52). These data suggest that circulating AM is cleared by the kidney and urinary excretion of AM may be derived from glomerular filtration and/or its renal production.

Natriuretic Peptides and Nitric Oxide Induce Endothelial Apoptosis via a cGMP–Dependent Mechanism

Apoptosis is a mode of cell death in which the cell participates in its own demise. We studied whether endothelium-derived relaxing factor, nitric oxide (NO), and natriuretic peptides affect apoptosis of rat vascular endothelial cells via a cGMP-dependent pathway and whether such effects are antagonized by an endothelium-derived vasoconstrictor, endothelin-1 (ET-1). Three natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide) induced endothelial apoptosis as demonstrated by nucleosomal laddering on agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling method. This dose-dependent relation was assessed by quantifying the fragmented and intact DNA contents by the diphenylamine method. The atrial natriuretic peptide-induced endothelial apoptosis was completely blocked by a guanylate cyclase-coupled receptor antagonist (HS-142-1) and an inhibitor of cGMP-dependent protein kinase (KT5823). An NO donor, NOR3 ((+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide; FK409) also induced endothelial apoptosis; the effect of this compound was abrogated by KT5823 and an inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). A cGMP derivative, 8-bromo-cGMP, but not the cAMP derivative 8-bromo-cAMP, caused endothelial apoptosis; the effect of ODQ was also abrogated by KT5823. Endothelial apoptosis induced by ANP, NOR3, and 8-bromo-cGMP was similarly antagonized by ET-1. ANP, NOR3, and 8-bromo-cGMP caused marked accumulations of the tumor suppressor gene product p53 but not of bcl-2, as determined by Western blot analysis. These results demonstrate for the first time that endothelium-derived NO and natriuretic peptides are proapoptotic factors for endothelial cells, whereas the endothelium-derived vasoconstrictor ET-1 is an antiapoptotic factor, suggesting that the countervailing balance between these vasodilators and vasoconstrictors, in addition to regulation of vascular tonus, may contribute to endothelial cell integrity.

Transfection of Inducible Nitric Oxide Synthase Gene Causes Apoptosis in Vascular Smooth Muscle Cells

BACKGROUND Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.

Secretion of Adrenomedullin by Renal Tubular Cell Lines

Adrenomedullin (AM) is a novel and potent vasodilator peptide originally isolated from human pheochromocytoma. The present study was designed to study whether AM is produced by and secreted from renal tubular cell lines and whether arginine vasopressin (AVP) affects AM secretion from these cell lines. Three renal tubular cell lines derived from different species (LLCPK1, MDCK, and MDBK) secrete AM-like immunoreactivity (AM-LI) into culture medium, the immunological and physicochemical properties of which are similar to that of synthetic human AM as evaluated by reverse-phase high-performance liquid chromatography. Among the three cell lines, AVP in combination with a phosphodiesterase inhibitor (isobutylmethylxanthine) stimulated AM-LI secretion most potently from MDCK cells in a time- and dose-dependent manner. In MDCK cells, a V2 receptor agonist (deamino-D-Arg8-vasopressin) dose-dependently stimulated AM-LI secretion in the same manner as AVP. Furthermore, the AVP-induced AM-LI secretion was blocked by a V2 receptor antagonist (OPC31260), but not by a V1 receptor antagonist (OPC21268). These data indicate that AM is secreted from renal tubular cell lines and that AVP stimulates AM secretion via V2 receptors, suggesting its autocrine/paracrine role in renal function.