Akio Shino

Pathophysiological role of endothelin in acute renal failure

In conscious rats, the i.v. injection of endothelin (ET) caused an increase in blood urea nitrogen (BUN), an index of renal dysfunction. In the model of acute renal failure which was induced by occlusion of the bilateral renal arteries of rats followed by reperfusion, ET-monoclonal antibody improved the renal function. In this model, ET-antibody also protected the kidneys from renal proximal tubular necrosis and suppressed Ca++-accumulation in necrotic tissues. Plasma ET level increased 5 min and 5 hr and renal ET content did 5 and 20 hr after reperfusion. BUN level increased 5 and 20 hr after reperfusion. These results strongly suggest that the endogenously increased ET may be one of the important deleterious mediators in the pathogenesis of ischemic acute renal failure.

A New Genetically Obese-Hyperglycemic Rat (Wistar Fatty)

The fa-gene was transferred from the Zucker rat (13 M strain) to the Wistar Kyoto (WKY) rat. The survey, performed at the 10th generation of backcrossing, showed that Wistar fatty rats (fa/fa), a congenic strain of WKY, developed obesity and obesity-related features, such as hyperinsulinemia and hyperlipemia, in the same manner as Zucker fatty rats. Males, but not females, showed hyperglycemia, glucosuria, and polyuria as early as 8 wk of age. Tolerance and insulin response to oral glucose were decreased with advancing age in males. The diabetic changes appeared to be caused by an interaction between predisposition to develop diabetes in the WKY rat and fa-induced obesity. This is because WKY rats were found to be less sensitive to insulin than Zucker rats by both the glucose tolerance test and the steady-state blood glucose method which estimates overall insulin sensitivity.

Effect of ipriflavone on glucocorticoid-induced osteoporosis in rats.

Ipriflavone, 7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one, was administered orally for 12 weeks to male rats with prednisolone-induced osteoporosis. Microdensitometric analysis of a roentgenograph of the femurs revealed that ipriflavone increased the density of the distal metaphysis dose-dependently and tended to increase the density of the diaphysis. It also inhibited dose-dependently the decrease in the mechanical strength of the tibia, breaking strain and breaking energy, and the fractional content of ash in femurs. These results indicate that ipriflavone markedly suppresses bone resorption at the metaphysis where the content of trabecular bone with a rapid turnover rate is high, and possibly inhibits bone reduction at the diaphysis.

Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia

Recently, we demonstrated that transient forebrain ischemia in rats leads to an early and strong induction of basic fibroblast growth factor (bFGF) synthesis in astrocytes in the injured brain regions. In this study, in order to clarify the targets of such raised endogenous bFGF levels, the messenger RNA (mRNA) expression of its receptors (flg and bek) at in the hippocampus following transient forebrain ischemia induced by four-vessel occlusion for 20 min was investigated using an in situ hybridization technique. Transient forebrain ischemia induced an increase in the number of flg mRNA-positive cells from an early stage (24 h after ischemia) in the hippocampal CA1 subfield where delayed neuronal death occurred later (48–72 h after ischemia). This increase became more marked with the progression of neuronal death and was still evident in the same area 30 days later. The time course of the appearance and distribution pattern of flg mRNA-positive cells in the CA1 subfield were quite similar to those of bFGF mRNA-positive cells. On the other hand, in situ hybridization for bek mRNA showed only slight and transient (observed 72 h and 5 days after ischemia) increases in the number of mRNA-positive cells in the CA1 subfield following ischemia. The use of in situ hybridization and glial fibrillary acidic protein immunohistochemistry in combination demonstrated that the cells in the CA1 subfield that exhibited ischemia-induced flg or bek mRNA expression were astrocytes. These data indicate that transient forebrain ischemia induces upregulation of fibroblast growth factor-receptor expression, accompanied by increased bFGF expression in astrocytes, and suggest that the increased astrocytic bFGF levels in injured brain regions act on the astrocytes via autocrine systems and are involved in the development and maintenance of astrocytosis.

Structural changes of pancreatic islets in genetically obese rats.

SummaryLight and electron microscopic observations were performed on pancreatic islets from genetically obese rats, (Zucker, “fatty”), from 5 to 52 weeks of age. At 5 weeks of age, islets were moderately hypertrophied. After that age, hypertrophy of islets became more prominent, until 24 weeks of age, with accompanying degranulation of B cells. The plasma insulin level also continued to increase during this period, but the glucose level was normal. Degranulated B cells contained a highly developed Golgi complex, numerous vesiculated, granular, endoplasmic reticulum and a small number of secretory granules, but no glycogen deposits. Emiocytosis and microtubule formation were very remarkable with these B cells. Frequently, mixed or intermediate cells, such as exocrine-endocrine or ductural-endocrine cell, were observed in pancreas with hypertrophied islets. At 52 weeks of age, both the plasma insulin and triglyceride levels decreased. In the pancreas, there were observed proliferation of fibrous tissue and well granulated B cells in hypertrophied islets. Hence, in fatty rats, pancreatic islets were in an active state during the period of development of obesity and hyperlipaemia (from 5 to 24 weeks of age). These correlates of obesity and hyperinsulinism disappeared at 52 weeks of age.

Targeted disruption of the neurotrophin-3 gene with lacZ induces loss of trkC-positive neurons in sensory ganglia but not in spinal cords

We have replaced the NT-3 gene with Escherichia coli-derived lacZ gene by means of homologous recombination in embryonic stem cells and thus produced null mutant mice. Mice homozygous for this mutation developed to birth, but most of them could not suck well and died within 2 days after birth. The surviving homozygous mutant mice displayed movement disorder similar to ataxia. The expression of lacZ was widely distributed in the target tissues of peripheral nerves, spinal motor neurons, lumbar dorsal root ganglia and trigeminal ganglia during the prenatal periods. A neuroanatomical examination revealed that there was marked cell reduction present in trigeminal and lumbar dorsal root ganglia in the developing homozygous mutant mice. In these tissues, the expression of trkC, a high-affinity receptor for NT-3, was markedly reduced. In contrast, we did not find any morphological abnormalities, significant cell loss or decreased levels of trkC expression in the motor neurons present in the ventral horn of the spinal cord. These results indicate that the absence of the NT-3 gene leads to a defect in the sensory nervous system, but it may be complemented by other neurotrophins in the motor nervous system during the development.

Epithelial cell components immunoreact with antiserum thymic factor (FTS) antibodies: Possible association with intermediate-sized filaments

Abstract By indirect immunofluorescence microscopy, an antiserum raised in rabbit against serum thymic factor (FTS) was found to decorate the epithelial cells not only in the thymus, but also in the kidney, uterus, urinary bladder, prostatic glands, stomach, ileum, colon, submaxillary glands, trachea, epidermis and epidermal appendages of mouse. The staining ability was completely absorbed with an FTS-binding immunoabsorbent, and affinity-purified anti-FTS IgG showed the same staining patterns as the original antiserum. The staining profiles resembled those described for tissues stained with antiprekeratin and antikeratin antibodies in both distribution in tissue and localization in the epithelial cells. In primary-cultured cells from mouse kidney medullae, the anti-FTS antibodies decorated the cytoplasmic fiber network. The fibers were wavy, bundled together and branched. They were dense in the perinuclear cytoplasm and spread in the cytoplasm toward the cell periphery. This decoration was resistant to colchicine and cytochalasin B, but sensitive to pretreatment with formaldehyde. The organization and shape of the fiber network were similar to those of the networks of intermediate-sized filaments containing cytokeratins, keratins and vimentin. However, the antiserum did not give a precipitin band in immunodiffusion test with prekeratin from bovine muzzle, keratin from human epidermis or 3T3 vimentin. Neither tubulin nor actin formed precipitin bands with the antiserum. These results show that the epithelial cells of various mouse tissues contain FTS or substances close to FTS in chemical structure and suggest that they are associated with the intermediate-sized filaments.

EM574, an erythromycin derivative, is a motilin receptor agonist in the rabbit

This study was performed to examine whether an erythromycin derivative, de(N-methyl)-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574) is a motilin receptor agonist in the rabbit gastrointestinal tract. EM574 and porcine motilin induced contractions in segments of isolated rabbit intestine with pEC50 values of 8.26 +/- 0.04 and 8.69 +/- 0.07, respectively, but not in rat or guinea pig preparations. The sensitivity and efficacy of the response to both compounds in rabbits decreased aborally and was insensitive to pretreatment with atropine or tetrodotoxin, but was markedly suppressed under Ca(2+)-free conditions. EM574 and porcine motilin specifically displaced [125I-Tyr23]canine motilin bound to gastric antral smooth muscle homogenates with plC50 values of 8.21 +/- 0.13 and 9.20 +/- 0.11, respectively. The pEC50 value for the contractile response and plC50 value for the receptor binding for motilin, EM574, erythromycin A and three other derivatives correlated well (r = 0.94, P < 0.01). Tissue section autoradiography in the antrum revealed that specific labeled motilin binding sites were localized in the circular muscle layer and myenteric plexus, and could be reduced in the presence of an excess of EM574. These results indicate that EM574 is a potent motilin receptor agonist in the rabbit gastrointestinal tract.

Stimulation of Thrombopoiesis in Mice by Fibroblast Growth Factor 9

Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9-treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes.

Inhibitory effect of TCV-309, a novel platelet activating factor (PAF) antagonist, on endotoxin-induced disseminated intravascular coagulation in rats: possible role of PAF in tissue factor generation.

The possible involvement of platelet activating factor (PAF) in the pathogenesis of endotoxin-induced disseminated intravascular coagulation (DIC) was investigated in rats using a novel potent PAF antagonist, TCV-309. TCV-309 (> 1 mg/kg, i.v.) showed beneficial effects in rats with experimental DIC induced by a 4-hour sustained infusion of endotoxin (1 mg/kg) in a dose-dependent manner. TCV-309 (1 mg/kg) significantly ameliorated the decrease in platelet count and plasma fibrinogen, the prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) and the increase in fibrin and fibrinogen degradation products (FDP) and inhibited glomerular fibrin deposition. Furthermore, plasma tissue factor (TF) activity was greatly increased in the DIC rats, and this was also significantly decreased by TCV-309 (1 mg/kg). TCV-309 (1 mg/kg) did not affect these parameters in normal rats. A 4-hour sustained infusion of PAF (60 micrograms/kg) caused mild but significant changes in some DIC parameters such as PT, fibrinogen and FDP concentration and increased the plasma TF activity. TCV-309 (1 mg/kg) inhibited all these PAF-induced changes. TCV-309 (0.1 mM) itself had no direct in vitro effects on the blood coagulation system including TF activity. These results strongly suggest that PAF plays a role in the pathogenesis of endotoxin-induced DIC via the generation of TF. Prophylactic use of PAF antagonists may therefore be useful for the treatment of DIC with sepsis.