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Biomedical subjects

T Inagami

Publications and source records attributed to T Inagami.

At least 181 records · Page 10Linked to original sources
PubMed1993

Endothelin as a neuropeptide. Cardiovascular effects in the brainstem of normotensive rats.

The relevance of endothelin in central cardiovascular function was studied in urethane-anesthetized Sprague-Dawley rats. Blood pressure (BP) was monitored intra-arterially, and cerebrospinal fluid (CSF) was collected through an intracisternal catheter for radioimmunoassay of endothelin-1 (ET-1). Endothelin levels in the CSF were significantly higher (39 +/- 3 pg/ml) than in plasma (10 +/- 3 pg/ml, n = 11). ET-1 in CSF or plasma was not affected by systemic infusion of saline, but its levels significantly decreased when a sustained increase in BP was elicited with phenylephrine (14 +/- 7 pg/ml in the CSF and 6 +/- 4 pg/ml in plasma, n = 5). In sinoaortic-denervated animals, phenylephrine failed to reduce CSF endothelin levels. In different experiments, intracisternal administration of ET-1 (10 pmol) evoked an initial decrease in BP and heart rate (HR), followed by pronounced hypertension, bradycardia, and, in 70% of the animals, death from cardiorespiratory failure. Intracisternal administration of endothelin-3 (ET-3, 80 pmol, n = 11) evoked only a modest hypotensive and bradycardic response without cardiorespiratory impairment. Microinjection of ET-1 (0.5, 1, 2, 4, and 6 pmol/60 nl) into the nucleus of the solitary tract or area postrema produced a decrease in BP and HR. On the other hand, injection of low concentrations of ET-3 into the nucleus of the solitary tract increased BP and HR (at 2 pmol, 17 +/- 3 mm Hg, 14 +/- 6 beats per minute, n = 7), whereas ET-3 in the area postrema produced a prominent dose-related decrease in BP and HR. In the rostroventrolateral medulla, the lowest doses of ET-1 first modestly increased BP and renal sympathetic nerve activity. These effects were followed by hypotension, bradycardia, increase in respiratory frequency, and further enhancement of sympathetic nerve traffic. In 29% of the animals, these effects were followed by cardiorespiratory arrest. The specificity of the cardiovascular response to endothelin was demonstrated by the inhibitory effects of the receptor antagonist BQ-123. These results demonstrate that endothelin has specific cardiovascular effects in the brainstem of the rat and support a role for endothelin in cardiovascular regulation.

Animals↗
PubMed1993

Hyporeninemic normoaldosteronism in severe autonomic failure.

Sympathetic mechanisms play an important role in the acute regulation of renin release in humans. On the other hand, the effect of chronic sympathetic deprivation on the renin-aldosterone system has not been fully evaluated. We, therefore, studied 24 patients with severe autonomic failure due to pure autonomic failure (n = 14) or to multiple system atrophy (n = 10). Supine plasma renin activity (PRA) was low (0.09 +/- 0.01 ng/(L*s)) and remained virtually unchanged in the upright posture (0.10 +/- 0.01 ng/(L*s)) despite profound orthostatic hypotension to levels that should activate renal baroreceptors. Isoproterenol, at doses that produced significant tachycardia and hypotension, also failed to stimulate PRA. Furthermore, renin-producing cells were absent in the kidneys of two autopsy cases. Norepinephrine depletion alone could not explain these findings because no correlation was found between plasma norepinephrine and PRA, and because PRA was normal in patients with intact sympathetic innervation but congenital absence of norepinephrine. In contrast, supine and upright plasma aldosterone were normal in autonomic failure patients (220 +/- 20 and 440 +/- 70 pmol/L). We speculate that direct sympathetic innervation is essential for the maintenance of renin, perhaps by providing trophic stimuli.

Aged↗
PubMed1993

A novel endogenous sodium-pump inhibitor in pig urine: purification and comparison with the inhibitor purified from bovine adrenal glands.

To clarify the chemical nature and pathophysiological significance of an endogenous, specific Na-pump inhibitor, extracts of various tissues have been examined. The activities were extracted by an Amberlite XAD-2 adsorbent and fractionated by high-performance liquid chromatography (HPLC) on an ODS column. Among many tissues and fractions tested, the extract of pig urine has been found to contain a large amount of 86Rb uptake inhibitory activities. The strongest inhibitory activity for 86Rb influx into erythrocytes also exhibited a cross-reactivity with anti-ouabain antibodies. This activity has been purified to homogeneity using five steps of reverse-phase HPLC. Although most of the dose-response curves for this purified Na-pump inhibitor, designated as uroxin, in the various assay systems paralleled those of ouabain and the inhibitor purified from bovine adrenal glands (designated as adrexin C), the cross-reactivity curve with anti-ouabain antibodies did not. The retention time of uroxin on an ODS HPLC column was also different from that of digoxin, ouabain, or adrexin C. 1H nuclear magnetic resonance spectroscopic study suggests that uroxin is a novel Na-pump inhibitor that is structurally different from any of the known cardiotonic steroids or the substances previously reported to exhibit Na-pump inhibitory activity. Thus, uroxin may be a new type of endogenous regulator for the Na pump.

Adrenal Glands↗
PubMed1993

New signaling mechanism of angiotensin II in neuroblastoma neuro-2A cells: activation of soluble guanylyl cyclase via nitric oxide synthesis.

We previously reported that angiotensin II (Ang II) increases cGMP content through a new Ang II receptor subtype that is distinct from both the AT1 and AT2 subtypes in differentiated Neuro-2A cells. In this study, the mechanism of the Ang II-stimulated cGMP increase was investigated in comparison with bradykinin- and atrial natriuretic factor (ANF)-stimulated cGMP increases in differentiated Neuro-2A cells. Ang II increased cGMP in differentiated Neuro-2A cells rapidly, with a maximal effect in 30 sec and a return to basal levels in 60 sec. Removal of extracellular Ca2+ or pretreatment with a membrane-permeable Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester] attenuated Ang II-stimulated cGMP accumulation. Both the time course and Ca2+ dependency of the effect of Ang II were similar to those of the effect of bradykinin, which activates soluble guanylyl cyclase, but distinct from those of the effect of ANF, which activates particulate guanylyl cyclase. Methylene blue, an inhibitor of soluble guanylyl cyclase, attenuated the effects of Ang II and bradykinin but not that of ANF. LaCl3, a nonspecific Ca2+ blocker, prevented Ang II-stimulated cGMP accumulation. L-type Ca2+ channel blockers, nifedipine and diltiazem, or an N-type Ca2+ channel blocker, omega-conotoxin, failed to inhibit the effect of Ang II. Ang II had no effect on formation of 1,4,5-inositol trisphosphate or cAMP content, whereas bradykinin stimulated 1,4,5-inositol trisphosphate formation in differentiated Neuro-2A cells. Further, the nitric oxide synthase inhibitors NG-monomethyl-L-arginine and NG-nitro-L-arginine attenuated Ang II- and bradykinin-stimulated elevation of cGMP content but not that stimulated by ANF. The Ca2+ ionophore A23187 also stimulated cGMP formation and the effect was inhibited by the nitric oxide synthase inhibitors. These results indicate that the newly found Ang II receptor mediates cGMP formation through activation of soluble guanylyl cyclase and that the activation is mediated by nitric oxide, which is increased by Ca2+ influx via an ion channel distinct from the L-type and N-type Ca2+ channels.

Amino Acid Oxidoreductases↗
PubMed1993

Angiotensin II receptors: cloning and regulation.

Angiotensin II subtype 1 (AT1) receptor cDNA was cloned by expression cloning from bovine adrenal cortical cells. Human AT1 receptor was also cloned. These receptors were found to have a seven transmembrane structure. The receptor seems to interact with more than one type of G-proteins. AT1 consists of subtypes. cDNA for AT1A was cloned from rat kidney and that for AT1B was cloned from rat adrenal by plaque hybridization. They have similar base sequences in the coding region but are different in non-coding regions. Their functional implication is not clear. The regulation of the receptors occur at many stages. Expression of mRNA is studied in cultured rat mesangial cells. It was down regulated by angiotensin II and cAMP. On the other hand in whole body experiments, chronic infusion of angiotensin II was shown to upregulate adrenal AT1, and bilateral nephrectomy or losartan (CAS 124750-99-8) administration reduced AT1 mRNA expression. In addition to AT1 and AT2 the presence of a new subtype AT3 has been shown.

Angiotensin II↗
PubMed1992

Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis.

To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor.

Angiotensin II↗
PubMed1992

A Na pump inhibitor from bovine posterior pituitary: purification, structure determination and its cardiovascular effect in rat.

We examined the hypothesis that hypothalamo-hypophysial tissue contains an endogenous Na pump inhibitor. From bovine posterior pituitary, we purified a substance which inhibits Rb uptake by human erythrocytes. This inhibitory activity was found in the eluate of 10% acetonitrile from a C18 flash column and purified by subsequent three steps of reversed-phase high-performance liquid chromatography (HPLC). Sequence analysis revealed that this substance was identical to joining peptide, one of the major products of proopiomelanocortin (POMC). This peptide had hypertensive and tachycardiac effects in spontaneously hypertensive rats (SHR) after central administration, with weak Na,K-ATPase inhibitory activity (IC50 = 0.5 mM).

Amino Acid Sequence↗
PubMed1992

Phospholipase D mimics platelet-derived growth factor as a competence factor in vascular smooth muscle cells.

Recent studies have suggested the importance of phosphatidylcholine (PC) metabolism in growth factor-stimulated cells. In these cells, PC is hydrolyzed not only by PC-specific phospholipase C but also by phospholipase D (PLD). In the present investigation, we show that the simple addition of PC-hydrolyzing PLD from Streptomyces chromofuscus to the culture medium of vascular smooth muscle cells elicits choline release into the medium accompanied by the formation of phosphatidic acid. In the presence of ethanol, this treatment elicits a formation of phosphatidylethanol (PEt) at the expense of phosphatidic acid. Furthermore, we show here that exogenous addition of S. chromofuscus PLD induces a marked DNA synthesis in quiescent vascular smooth muscle cells. This DNA synthesis induced by S. chromofuscus PLD is, like platelet-derived growth factor (PDGF)-elicited DNA synthesis, largely dependent on the presence of insulin. In addition, S. chromofuscus PLD-induced PEt formation and DNA synthesis were not affected by protein kinase C down-regulation, whereas PDGF-induced PEt formation and DNA synthesis were significantly inhibited. These observations strongly suggest that protein kinase-dependent activation of PLD is involved in mitogenic signal in PDGF-stimulated cells and that exogenously added PLD acts as a competence factor in the same way as PDGF.

Animals↗
PubMed1992

Platelet-derived growth factor AA homodimer stimulates protein synthesis rather than DNA synthesis in vascular smooth muscle cells from spontaneously hypertensive rats but not from normotensive rats.

Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-gamma 1 in SHR cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response.

Animals↗
PubMed1992

Further evidence of the SA gene as a candidate gene contributing to the hypertension in spontaneously hypertensive rat.

We have recently reported that the allele of the SA gene of the Spontaneously hypertensive rat (SHR) has a capacity to influence blood pressure in a F2 rat population prepared from SHR and Wistar-Kyoto rat. In the present study, we have undertaken a similar genetic co-segregation analysis of the F2 rat population prepared from SHR and Lewis rat. The result indicated that, although overall effects of the SA gene genotypes on blood pressure were not significant, a correlation of the genotypes of the SA gene with blood pressure was significantly observed in the female rats of this population. The present results further strengthen our hypothesis that the SA gene, or a gene closely linked to this gene, has a capacity to influence blood pressure.

Animals↗
PubMed1992

Differential regulation of rat AT1a and AT1b receptor mRNA.

Rat type 1 angiotensin II receptor has two subtypes, namely type 1a and type 1b. The regulation of the expressions of these two subtype receptor mRNAs was studied by using a competitive polymerase chain reaction method. The expression of the type 1a mRNA in the liver was negatively and that of the type 1b mRNA in the adrenal was positively modulated by bilateral nephrectomy. In the ventricle of 16 week old Spontaneously hypertensive rat, the expression level of the AT1b receptor mRNA was higher than that in the ventricle of the age-matched Wistar-Kyoto rat, while the expression levels of the AT1a mRNA in the ventricle were almost similar between the two strains at this age. Although type 1a and type 1b have almost similar functional properties, the expressions of their mRNAs were differentially regulated.

Adrenal Glands↗
PubMed1992

Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.

To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.

Angiotensin II↗
PubMed1992

Diacylglycerol formation from phosphatidylcholine in angiotensin II-stimulated vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMC), angiotensin II (Ang II) induces a biphasic diacylglycerol (DAG) formation peaking at 15 sec and 5 min. Although it has been well established that the first peak is produced by the hydrolysis of inositol 4,5-bisphosphate (PIP2), the origin of the second DAG peak has never been examined in detail. In the present paper, we provide evidence that the second peak of DAG formation in Ang II-stimulated VSMC originates mainly from PC.

Angiotensin II↗
PubMed1992

The distribution of angiotensin II AT1 receptor subtype mRNA in the rat brain.

The present study demonstrates the existence and regional distribution of angiotensin II AT1 receptor subtype mRNA expression in the rat brain by the use of in situ hybridization and RNase protection assay. Substantial expression levels in the brain have only been detected in certain distinct areas, such as the subfornical organ, the parvocellular part of the paraventricular hypothalamic nucleus, and the median preoptic nucleus. The results give further evidence for the involvement of the angiotensin II AT1 receptor subtype in the classical functions of central angiotensin II, like blood pressure control, body fluid homeostasis and in corticotropin-releasing factor (CRF) secretion.

Angiotensin II↗
PubMed1992

Analysis of the evolution of angiotensin II type 1 receptor gene in mammals (mouse, rat, bovine and human).

The nucleotide and amino acid sequences for mouse angiotensin II (AII) type 1A and 1B receptors were deduced from their complementary and genomic DNAs. Evolutionary analyses based on the nucleotide sequences of the coding region of AII type 1 receptor genes indicated that the duplication event of the type 1 gene occurred 24 +/- 2 million years ago before the divergence between the rat and mouse but after the divergence between rodents and the human/artiodactyls couple. This conclusion was consistent with the results of genomic Southern blot analyses, which revealed that the mouse and rat possess 2 similar but separate genes, whereas the bovine and human have only a single class gene.

Amino Acid Sequence↗
PubMed1992

The rat angiotensin II AT1A receptor couples with three different signal transduction pathways.

To examine whether the subpopulation of the rat type 1 angiotensin II (AII) receptor (AT1A) couples with a single or multiple signal transduction pathways, we constructed Chinese hamster ovary (CHO) cell lines producing the recombinant receptor. The expressed AT1A receptor exhibits typical pharmacological characteristics of the AT1 receptor, known to mediate the main physiological function of AII. Addition of AII to the CHO cells induced a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+]i) followed by a lower, sustained phase. Nicardipine, a blocker of voltage-dependent L-type Ca2+ channels, attenuated the transient [Ca2+]i response and abolished the sustained phase. The transient phase was also reduced dose-dependently by the phospholipase C inhibitor neomycin. Furthermore, AII inhibited forskolin-evoked cAMP accumulation. These data suggest, although another subpopulation named AT1B is present, that the rat AT1A receptor can independently couple with all three signal transduction pathways known to be induced by AII: i.e., i) activation of phospholipase C resulting in InsP3 generation with a subsequent release of intracellularly stored Ca2+, ii) activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels, and iii) inhibition of adenylate cyclase activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

Two distinct pathways in the down-regulation of type-1 angiotension II receptor gene in rat glomerular mesangial cells.

The mRNA level of the type-1 angiotensin II receptor (AT1) was down-regulated by angiotensin II in cultured rat glomerular mesangial cells. The effect was maximum with 1 microM AII at 6 h, sensitive to cycloheximide, and specific to AT1 since this phenomenon was blocked by DuP753, an AT1 antagonist, but not by type-2 antagonist PD123319. Dibutyryl cAMP, forskolin, and cholera toxin also caused AT1 down-regulation. These effects were not altered by either the protein kinase A inhibitor H-8 or cycloheximide. Calcium ionophore A23187, pertussis toxin, protein kinase C inhibitor staurosporine, or prolonged incubation with phorbol ester were without effect. These results suggest that there are at least two pathways to down-regulate AT1 mRNA; one way is an angiotensin II-induced, protein kinase C-independent, and cycloheximide-sensitive pathway and the other is an angiotensin II-independent, cAMP-induced, and cycloheximide-insensitive pathway.

Angiotensin II↗