Rat kidney thromboxane receptor: molecular cloning, signal transduction, and intrarenal expression localization.

Thromboxane (TX) plays important roles in control of renal hemodynamics and water and electrolyte metabolism, and is involved in the pathophysiology of many renal diseases. The aim of the present study is to isolate a rat kidney cDNA encoding functional TX receptor, and to reveal its intrarenal expression localization. A clone (rTXR2) was isolated from a rat kidney cDNA library by a homology screening approach. rTXR2 was shown to encode the amino acid sequence containing seven transmembrane spanning domains representing rat (r) TX receptor. The membrane from COS-7 cells transiently transfected with rTXR2 cDNA was shown to be specifically bound by a thromboxane receptor antagonist, SQ29548. Either in Xenopus oocyte expression or in transfected COS-7 cells, rTX receptor was shown to be linked with Ca2+ messenger system. TX receptor-mediated increase in cytosolic Ca2+ was also observed in cultured glomerular mesangial cells. In situ hybridization showed that rTX receptor mRNA was detected in renal glomeruli, smooth muscle cells in renal arterioles, and transitional cell epithelium of renal pelvis. Reverse transcription linked to PCR applied to microdissected nephron segments indicated the presence of rTX receptor mRNA exclusively in the glomerulus. In conclusion, we have cloned a functional rat kidney TX receptor, which is expressed specifically in renal glomerulus, arterial smooth muscle cells, and transitional cell epithelium of renal pelvis. The present study will provide important insights into the etiology and pathophysiology of renal diseases with relation to TX metabolism.

Biliary elimination of diazepam in man.

The metabolism of 14C-5-diazepam has been studied in 5 patients with T tube biliary drainage. A single bolus of 40 to 50 muCi was given intravenously and blood, urine, and bile were analyzed from 5 to 14 days. The mean half-life of elimination from blood was 93.2 hr; the major metabolite noted in blood was N-desmethyl-diazepam. In urine the average recovery of radioactivity was 48.9% and consisted of 3 OH-diazepam, 4'OH-diazepam, and oxazepam. In bile the average recovery of radioactivity was 5.35% (corrected to a bile flow of 700 ml was 15.0%) and consisted of the same metabolites as in the urine. Essentially no diazepam or N-desmethyl-diazepam was found, and therefore an enterohepatic circulation cannot be held to account for the prolonged half-life of these substances in man.

Functional analysis and chromosomal gene assignment of rat kidney prostaglandin EP3 receptor.

We have reported two isoformes of rat prostaglandin EP3 receptor with their different carboxyl-terminal tails (rEP3A and rEP3B receptors), which are derived by alternative RNA splicing, and both receptors have been shown to be localized to renal distal tubules. In the present study, we characterized the signal transduction system of rat kidney EP3 receptors either in a renal cell line mimicking renal distal tubule cells, TKC2, or in COS-7 cells by functional expression of these receptors. We also examined the chromosomal localization of the EP3 receptor gene by fluorescence in situ hybridization (FISH). In TKC2 cells, vasopressin (AVP, 10(-7) M), prostaglandin (PG) E2 (10(-7) M), or forskolin (10(-8) M) markedly stimulated cyclic AMP formation. Overexpression of the rEP3A receptor significantly attenuated the AVP-, PGE2- or forskolin-induced cyclic AMP formation, whereas there was no change with rEP3B receptor expression. On the other hand, in COS-7 cells transfected with rEP3A receptor cDNA, PGE2 (10(-7) M) did not affect cytosolic free calcium concentration ([Ca2+]i), whereas transfection of rEP3B receptor cDNA evoked PGE2-induced increases in [Ca2+]i. Moreover, we have revealed that the rEP3 receptor gene is localized to rat chromosome 2q44-45. In conclusion, rEP3A or rEP3B receptor is suggested as a mediator of the natriuretic/diuretic action of PGE2 in renal distal tubules via a decrease in cyclic AMP formation or an increase in [Ca2+]i, respectively. Information of the gene assignment of rat EP3 receptor to rat chromosome 2q44-45 is useful for further analysis of the role of EP3 receptor in genetically hypertensive rat models.

Effect of furosemide on serum clearance and renal excretion of digoxin.

Serum turnover and urinary excretion of digoxin with or without oral furosemide were studied in six healthy subjects who received 0.006 mg/kg body weight digoxin intravenously. During furosemide treatment, the total amount of urinary digoxin did not change but the digoxin clearance during the diuretic phase and the digoxin excretion after the diuresis decreased significantly. The average serum half-life was prolonged from 37 hours in the control period to 86 hours in the furosemide period. Decreased glomerular filtration rate by volume depletion might have been responsible for the decreased excretion of digoxin, but there was no significant difference in urine volume after diuresis between the two periods, suggesting the possibility of inhibition of tubular secretion of digoxin by furosemide. It is also possible that serum digoxin concentration may be elevated if furosemide were given more frequently.

Activated astrocytes with glycogen accumulation in ischemic penumbra during the early stage of brain infarction: immunohistochemical and electron microscopic studies.

Brain infarction was induced in rats by injection of microspheres through the right internal carotid artery, and structural changes in the astrocytes were observed during the early period following the infarction. Necrotic foci, varying in size and shape, were found in the right hemisphere. After immunohistochemical staining for GFAP, GFAP-positive astrocytes in the perinecrotic area known as the ischemic penumbra had distinctly increased in number and size with elongation of cytoplasmic processes 3 days after infarction. Electron microscopic observation revealed that glycogen granules had markedly accumulated in the cytoplasm of astrocytes located in the ischemic penumbra 3 and 5 days after infarction. Seven days after infarction, however, the glycogen granules disappeared from the astrocytes. Intermediate filaments increasingly appeared in the protoplasmic astrocytes after 3 days and were abundant in the activated and hypertrophied astrocytes after 7 days. As a result of our present study, we conclude that: (1) the function of glucose uptake from blood vessels was not impaired in the astrocytes under hypoxic conditions; (2) the astrocytes actively ingested blood glucose through the endothelial cells and accumulated it as glycogen for activation of their functions, and the cell volume increased under hypoxic conditions; (3) the depression of energy metabolism and the decrease in the uptake of energy sources in the nerve cells promoted glycogen accumulation in the astrocytes under hypoxic conditions; (4) intermediate filaments (GFAP filaments) increased in number, coincident with the activation and enlargement of the astrocytes; and (5) protoplasmic astrocytes were transformed into fibrous astrocytes in the ischemic penumbra of the brain infarction.

Preventive effects of a traditional Chinese medicine (sho-saiko-to) against oxygen toxicity and membrane damage during endotoxemia.

The preventive effects of a traditional Chinese medicine Sho-saiko-to (Kampo prescription, TJ-9) were determined from oxygen toxicity and membrane damage in liver during endotoxemia. The liver lipid peroxide level and xanthine oxidase activity 18 h after administration of endotoxin (6 mg/kg, i.p.) to TJ-9 (500 mg/kg/d, p.o.)-pretreated mice were markedly lower than that in endotoxin-treated mice, whereas the administration of TJ-9 significantly increased superoxide dismutase and glutathione peroxide activities in liver of endotoxin-injected mice. In the mice pretreated with a TJ-9, the levels of alpha-tocopherol and nonprotein SH in liver tissue 18 h after endotoxin injection were markedly increased as compared to those in endotoxin-treated mice. Leakages of acid phosphatase and lactate dehydrogenase isozyme in serum were markedly lower in endotoxin-TJ-9-treated mice than those in mice given endotoxin. The administration of TJ-9 clearly prevented the membrane protein damage arising from endotoxin challenge. Kampo prescription Sho-saiko-to thus appears to protect the liver plasma membrane from injury by free radicals which occur in a tissue ischemic state during endotoxemia.

Decline in plasma membrane Ca(2+)-ATPase activity and increase in cytosolic-free Ca2+ concentration of endotoxin-injected mice livers.

The present study describes the activity of Ca(2+)-ATPase in liver plasma membrane and cytosolic-free Ca2+ concentration ([Ca2+]i) in an individual vital cell in mouse liver 18 h after endotoxin administration. Ca(2+)-ATPase activity in liver plasma membrane in the poisoned mice was markedly decreased to 28% of that in the control. The membrane protein damage in liver was found mostly in the molecular weight (M.W.) regions near 60,000-150,000 in endotoxemic mice, and was markedly injured near 140,000 (M. W. of Ca(2+)-ATPase in liver plasma membrane). The level of [Ca2+]i in liver cells in endotoxin-poisoned mice was greater at 18 h postintoxication than that of the control. These findings suggest the possibility that the depression of Ca(2+)-ATPase activity in liver plasma membrane in mice may contribute to membrane damage caused by the endotoxin, and that the increase of [Ca2+]i in liver cytoplasm may partially explain various endotoxin-induced metabolic disorders.

Defense effects of a traditional Chinese medicine (sho-saiko-to) against metabolic disorders during endotoxemia; approached from the behavior of the calcium ion.

The present study was carried out as an approach from intracellular Ca2+ to clarify the preventive effects of a traditional Chinese medicine, Sho-saiko-to (Kampo prescription, TJ-9), against various metabolic disorders during endotoxemia. In this experiment, we estimated the cytosolic-free Ca2+ concentration ([Ca2+]i) in liver single cells using a photonic microscope system. The [Ca2+]i in a single liver cell of endotoxin (6 mg/kg, i.p.)-injected mice was greater at 18 h post-intoxication than that in the control, whereas the administration of endotoxinin to TJ-9 (500 mg/kg/d, p.o.)-pretreated mice resulted in a markedly lower level of [Ca2+]i than that in endotoxin-treated mice. In the mice pretreated with TJ-9, the CA(2+)-ATPase activity in liver plasma membrane 18 h after endotoxin injection was markedly increased as compared to that in the endotoxin-treated mice. By contrast, the Ca(2+)-ATPase activity in liver mitochondria was lower in endotoxemic mice pretreated with TJ-9 than in mice given endotoxin alone. State 3 respiration and the respiratory control index (RCI), which are the parameters of mitochondrial function, were 41% and 35% lower, respectively, in the liver of mice given endotoxin than those levels in the control. However, the levels of state 3 and RCI in endotoxin-TJ-9-treated mice were markedly increased as compared to those of the endotoxin-treated mice. These findings suggest the protective effect of TJ-9 in the damage of liver mitochondrial function in endotoxin-poisoned mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of oxisuran on differential inhibition of cell-mediated immunity.

Oxisuran, 2-[(methylsulfinyl) acetyl] pyridine suppressed allogeneic skin graft rejection in rats. In PHA responses and one way mixed lymphocyte culture, suppression of cell-mediated immunity was indicated. However, little inhibition of PFCs was observed with Oxisuran, and unlike Azathioprine or Neocarzinostatin, Oxisuran did not concomitantly suppress antibody-producing system in rats. These data suggest that selective suppression of cell-mediated immunological responses by Oxisuran is possible in rats, and Oxisuran may be a better immunosuppressant for clinical organ transplantation.

Different cellular mechanisms of vasopressin receptor V1 and V2 subtype in vasopressin-induced adenosine 3', 5'-monophosphate formation in an immortalized renal tubule cell line, TKC2.

Vasopressin (VP) stimulates adenosine 3',5'-monophosphate (cAMP) formation in an immortalized renal tubule cell line, TKC2, which is derived from transgenic mouse harboring temperature-sensitive SV40 T-antigen gene. VP (10(-8) M)-induced cAMP formation was significantly attenuated by either non-peptide vasopressin receptor V1 or V2 subtype antagonist, OPC-21268 (10(-8) and 10(-6) M) or OPC-31260 (10(-8) and 10(-6) M), respectively, and it was completely abolished by combination of both agents (10(-6) M). VP (10(-8) M) also induced an increase in cytosolic free Ca2+ and prostaglandin (PG) E2 synthesis, both of which were significantly inhibited by OPC-21268 (10(-8) M), but not by OPC-31260 (10(-6) M). Either OPC-21268 (10(-8) M), depletion of extracellular Ca2+ or inhibition of cyclooxygenase attenuated both VP-induced PGE2 synthesis and cAMP formation. In conclusion, both V1 and V2 receptors can stimulate cAMP formation. V1 receptor, however, stimulates cAMP formation via Ca(2+)-dependent PGE2 synthesis, whereas V2 receptor may stimulate it directly.

Functional difference between two isoforms of rat kidney prostaglandin receptor EP3 subtype.

We have cloned two isoforms of rat kidney prostaglandin E2 receptor EP3 subtype (rEP3A and rEP3B), which differ only in their cytosolic carboxyl-terminal tails (30 and 29 amino acids, respectively). The aim is to clarify the functional difference between two rEP3 receptor isoforms by examining formation of adenosine 3',5'-monophosphate (cAMP) and change in cytosolic free calcium ([Ca2+]i) in cultured cells transiently transfected with cloned rEP3A or rEP3B receptor cDNA. In immortalized renal distal tubule cells (TKC2), vasopressin (VP) stimulated cAMP formation, and the cAMP formation was significantly attenuated by a non-peptide VP receptor antagonist, OPC-31260. The VP-induced increase in cAMP formation was also attenuated by over-expression of rEP3A receptor but not that of rEP3B receptor. On the other hand, in COS-7 cells transfected with rEP3B receptor cDNA, PGE2 induced an increase in [Ca2+]i, but no increase in [Ca2+]i was observed in the cells transfected with rEP3A cDNA. In conclusion, rEP3A receptor is suggested to antagonize VP (V2) receptor by inhibiting cAMP formation, whereas rEP3B receptor is linked with Ca2+ messenger system.

Effect of aging on renal cytochrome P450-dependent arachidonic acid metabolism in Dahl rats.

We investigated the age-related changes of renal cytochrome P450-dependent arachidonic acid metabolism in 3-, 5-, 7-, 9-, 11-, 13- and 20-week-old male Dahl salt-sensitive (DS) and -resistant (DR) rats on a low sodium diet (0.3% NaCl). NADPH-dependent arachidonic acid metabolism was separated and measured by a radio-HPLC system. The formation of 19-hydroxyeicosatetraenoic acid (HETE), 20 HETE, 1,20-dioic acid, epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acid (DHET) was age dependent in both DS and DR rats. omega-Hydroxylase (20-HETE and 1,20-dioic acid formation) and (omega-1)-hydroxylase (19-HETE formation) were increased from 3 to 5 weeks age, then decreased with aging in DR rats. Whilst omega/(omega-1)-hydroxylase activities were increased from 3- to 9-week-old rats, they decreased with aging in DS rats. omega/(omega-1)-Hydroxylase activities were higher in 3-5-week-old DR than DS rats. Epoxygenase activities (EETs and DHET formations) were highest in 3-week-old DS and DR rats, and showed no significant differences between two strains of rats at any ages tested. Renal cytochrome P450-dependent arachidonic acid metabolites have a wide and contrasting spectrum of biological and renal effects, and their relative rates of production may influence not only renal hemodynamics but also pro- and antihypertensive mechanisms of hypertension in Dahl rats.

Approximate surface development of the left side half-trunk by a free-formed model.

The approximate surface development, skin length, and surface area of the left side of the trunk of 51 female students were compared with regard to static and stretched postures. The data for each subject were obtained from geometrical models generated by moiré topography with a computer. When the chest was stretched, the anterior surface, the shoulder line, and the arm-base line were transformed from concave to convex, and a gap oriented toward the nipple widened out. The skin elongated vertically and transversely, except at the side of the waistline, where the skin contracted. The area at the top of the trunk decreased about 25%, while the other parts of the trunk increased 8-15%. The total anterior area was 1.20 m2 for the static posture and 1.29 m2 for the stretched posture. When the posterior surface was stretched, the shoulder line changed from convex to concave, the side line from quasi-straight to concave, and gaps oriented toward the chest line disappeared. The skin elongated most at the infrascapular region (20-35%), while the neck base line contracted (-11%). The center of the back and the lower arm base areas enlarged the most (25%) and the lumbar area enlarged the least (12%). The total posterior area was 1.26 m2 in the static posture and 1.37 m2 in the back-stretched posture. In conclusion, the back skin elongated and enlarged more when stretched than the frontal skin.