Biomedical subjects

G Tsujimoto

Publications and source records attributed to G Tsujimoto.

At least 109 records · Page 6Linked to original sources

PubMed1995

Blockade of the renin-angiotensin system in heart failure in conscious dogs.

OBJECTIVE: To study the different cardiac and renal hemodynamic effects of an angiotensin converting enzyme inhibitor and an angiotensin II receptor antagonist in experimental heart failure in conscious dogs. DESIGN AND METHODS: We compared the effects of the angiotensin converting enzyme inhibitor, captopril, with those of the angiotensin II (Ang II) subtype-1 receptor antagonist, losartan, on hemodynamics and hormonal changes in congestive heart failure by rapid ventricular pacing on conscious dogs. Furthermore, we characterized the Ang II receptors in canine heart, using the canine cardiac membrane fraction in heart failure. RESULTS: Acute intravenous administration of captopril improved the cardiac output by 19% (P < 0.01) but losartan did not, although blockade of the renin-angiotensin system by losartan (1.1 mumol/kg) or captopril (0.69 mumol/kg) induced similar changes in the plasma renin activity, norepinephrine and arginine vasopressin, and a similar decrease in mean arterial pressure (-10 mmHg). Renal blood flow was increased by either losartan or captopril. In the binding study, losartan produced a single displacement curve (IC50 = 0.25 mumol/l), while the Ang II subtype-2 (AT2) receptor antagonist PD123319 did not, indicating that the predominant Ang II receptor is type-1 (AT1) in canine heart. Neither the ratio of AT1 to AT2 receptors nor the receptor density changed with the development of heart failure. CONCLUSIONS: The lack of effect of losartan on cardiac output may be the result of its inability to block non-AT1 receptor-mediated Ang II activities adequately. Captopril may improve cardiac output by means of mechanisms not mediated by Ang II, such as locally increasing bradykinin.

Angiotensin II↗

PubMed1994

Evidence for the existence of vasopressin V2 receptor mRNA in rat hippocampus.

Arginine-vasopressin (AVP) in the septum and hippocampus are thought to be involved in memory and learning, and their receptors have been classified into three subtypes, V1a, V1b and V2. In this study, we tested for AVP V1a- and V2-receptor mRNAs in the hippocampus of adult rats by a reverse transcription-polymerase chain reaction (RT-PCR) method. PCR product of the V2 receptor mRNA was detected in the hippocampus, but that of V1a receptor mRNA was not. This is the first demonstration of the existence of the V2 receptor mRNA in the hippocampus.

Animals↗

PubMed1994

Cloning, functional expression and tissue distribution of human cDNA for the vascular-type vasopressin receptor.

We have cloned a human vasopressin receptor from human mesenteric artery using RACE (Rapid Amplification of cDNA Ends) methods. The deduced amino acid sequence of the clone (HV-RACE) encodes a protein of 418 amino acids that showed a strong sequence homology to the previously cloned rat V1A vasopressin receptor. The [3H] arginine vasopressin (AVP) binding to HV-RACE expressed in COS-7 cells was potently inhibited by AVP (Ki = 2.9 nM). Interestingly, a new non-peptide "V1-selective" antagonist OPC-21268 exhibited markedly higher affinity for rat V1A receptor (Ki = 57 nM) rather than for HV-RACE (Ki = 56 microM). With the reverse-transcription polymerase chain reaction assay, we observed a large amount of HV-RACE transcripts in the mesenteric artery, while a small amount in a variety of other tissues. The data show that the clone HV-RACE encodes a human vascular-type vasopressin receptor cDNA.

Amino Acid Sequence↗

PubMed1994

The pharmacological profile of cloned and stably expressed alpha 1b-adrenoceptor in CHO cells.

Using Chinese hamster ovary (CHO) cells stably expressing the alpha 1B-adrenoceptor (CHO alpha 1B cells) as a model, we investigated whether the transfected cells that express alpha 1B subtype of adrenoceptor can show the pharmacologic characteristics as previously defined in native tissues. Radioligand binding studies with 2-[beta-(4-hydroxy-3-[125I]iodophenyl)ethylamino-methyl]tetralone ([125I]HEAT) in CHO alpha 1B cells showed the similar Ki values of the alpha 1-adrenoceptor selective drugs as previously observed in rat liver and spleen, and that pretreatment with chlorethylclonidine markedly inactivated the binding sites (94.7-98.6%). In CHO alpha 1B cells alpha 1-adrenoceptor agonists caused a dose-dependent increase in transients of cytosolic Ca2+ concentrations ([Ca2+]i), and the potency order of antagonists in inhibiting norepinephrine-induced [Ca2+]i response was similar to that observed in radioligand binding assays. In summary, the present study shows that the ligand binding property, the pharmacological characteristics and the intracellular transduction mechanisms of alpha 1B-adrenoceptors stably expressed in CHO cells appear to be the same as those defined in native tissues. Thus they can be a useful model system for further characterization of the receptor as well as for the development of specific ligands.

Adrenergic alpha-Agonists↗

PubMed1994

Endothelin-A receptor mediates cardiac inhibition by regulating calcium and potassium currents.

Voltage-sensitive ion channels play fundamental roles in the regulation of cardiac function by various neurotransmitters. Endothelins have strong positive inotropic and chronotropic effects, for which recent studies have implicated various intracellular mechanisms. However, very little is known about the underlying ion-channel regulation by the peptide. We report here that endothelin-1 consistently hyperpolarizes the membrane and shortens the duration of the action potential in mammalian atrial myocytes, leading to suppression of electrical excitability of the heart. Endothelin-1, but not endothelin-3, inhibited the L-type calcium current by decreasing cyclic AMP accumulation and activated the muscarinic potassium current by stimulating a pertussis toxin-sensitive GTP-binding protein. Consistent with these results, endothelin-1 strongly reduced the heart rate when it was increased by beta-adrenoceptor stimulation. These effects were blocked by an ETA (endothelin-1-selective) receptor-selective antagonist, BQ123 (refs 8-11). The ETA receptor-mediated regulation of cardiac ion channels gives new insight into our understanding of the physiological and pathophysiological roles of endothelins in the control of cardiac function.

Action Potentials↗

PubMed1994

Pseudo-noncompetitive antagonism by BQ123 of intracellular calcium transients mediated by human ETA endothelin receptor.

The family of endothelins, endothelin-1 (ET-1), ET-2 and ET-3, act on two subtypes of receptors called ETA and ETB receptors. BQ123 is an ETA-selective competitive antagonist. In this study, however, BQ123 inhibited ET-1-induced transients of cytosolic Ca2+ concentrations ([Ca2+]i) in an apparently noncompetitive manner in mouse L cell stably expressing cloned human ETA receptor. BQ123 (3-30 nM) did not change the EC50 of ET-1 (6.6 nM), but reduced the maximum responses down to 15.0%. In an non-equilibrium binding assay which mimicks the conditions of [Ca2+]i transient assay (cells were incubated with [125I]ET-1 only for 30 s), BQ123 (30 nM) constantly decreased the specific [125I]ET-1 binding to approximately 13% of that without the antagonist. Thus, the apparent noncompetitive antagonism by BQ123 of ETA receptor-mediated [Ca2+]i transient is due to the assay condition where the interactions of the agonist, antagonist and receptor remained non-equilibrium state.

Animals↗

PubMed1994

Distribution and developmental change of vasopressin V1A and V2 receptor mRNA in rats.

With the reverse transcription-polymerase chain reaction (RT-PCR) method, we examined the tissue distribution of vasopressin V1A and V2 receptor transcripts in newborn and adult rats. In adult rats, vasopressin V1A receptor mRNA was detected in the brain, lung, liver and kidney, whereas vasopressin V2 receptor mRNA was found only in the kidney. In newborn rats, vasopressin V1A receptor mRNA was detected in the brain, liver, heart and kidney, whereas vasopressin V2 receptor mRNA in the kidney and brain. In the newborn rat brain the level of vasopressin V2 receptor mRNA decreased with age, and could not be detected in rats older than 2 weeks. Our results first demonstrated the extrarenal expression of vasopressin V2 receptor in the newborn rat brain. Also, the study showed that expressions of vasopressin V1A and V2 receptor mRNA transcripts are dynamically altered in the process of development.

Age Factors↗

PubMed1994

Effect of GnRH antagonists on phorbol ester-induced LH release from rat pituitary gonadotrophs.

We previously reported that a blockade of GnRH receptor activation inhibited the already-initiated C-kinase pathway(s). We tried to investigate whether this finding is a general phenomenon or not. In this study, we employed three GnRH antagonists, [D-Phe2,Pro3,D-Phe6]-GnRH, [Ac-D-Nal-Ala1,D-pCl-Phe2,D-Ser(Rha)6]-GnRH, and [Ac-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]-GnRH (referred to as #1-, #2-, #3-GnRH antag., respectively). Each antagonist was examined for its potency against GnRH by analyzing its inhibitory effect on LH release from pituitary gonadotrophs as well as on the increase in the cytosolic Ca2+ concentration. As a result, the #1-GnRH antag. was found to be weaker than the other two compounds. Consistent with a previous study, the #3-GnRH antag. inhibited the action of TPA on LH release. However, independently of their potency as GnRH-antagonists, the two other antagonists had no inhibitory effect on TPA-induced LH release. While it is generally accepted that the C kinase pathway plays a major role in the GnRH-induced LH release, not all GnRH antagonists can inhibit LH release by blocking the already-activated C kinase system.

Animals↗

PubMed1993

Cloning, functional expression and tissue distribution of human cDNA for the alpha 1C-adrenergic receptor.

We have cloned human alpha 1C-adrenergic receptor from human prostate cDNA library. The deduced amino acid sequence of the clone (P2C4) encodes a protein of 466 amino acids that showed strong sequence homology to the previously cloned bovine alpha 1C-adrenergic receptor. The radioligand binding properties of P2C4 expressed in COS-7 cells were very similar to those of bovine alpha 1C-adrenergic receptor. With reverse-transcription polymerase chain reaction assay, we observed alpha 1C-adrenergic receptor transcripts in heart, brain, liver and prostate, but not in kidney, lung, adrenal, aorta and pituitary. The data show that the clone P2C4 encodes a human alpha 1C-adrenergic receptor cDNA, and the receptor subtype is expressed not widely but localized in several human tissues.

Amino Acid Sequence↗

PubMed1993

Angiotensin II receptors in the rat urinary bladder smooth muscle: type 1 subtype receptors mediate contractile responses.

Angiotensin II (Ang II) receptors in the rat urinary bladder smooth muscle were investigated by in vitro responses of smooth muscle strips to exogenous Ang II stimulation and in radioligand binding assays. Ang II (10(-10) M. to 10(-5) M.) caused a potent contractile response in a concentration-dependent manner. Using the recently developed nonpeptide subtype-selective antagonists, the Ang II-induced contractile response was further characterized. The Ang II-induced contractile response was inhibited weakly by the type 2 subtype (AT2)-selective antagonist PD123319 but was potently inhibited by the type 1 subtype (AT1)-selective antagonist DuP 753 with a pA2 value of 9.03, suggesting that the response is mediated predominantly by AT1 receptors. [125I]Ang II was used to specifically label a single class of binding sites with a dissociation constant of 0.31 nM. and a maximal binding capacity of 41.5 fmol./mg. of protein. DuP 753 could completely antagonize the binding of Ang II in a particulate fraction of rat bladder (Ki = 14 nM), whereas PD123319 did not have any effect in the concentration range of 10(-9) to 10(-5) M. The results suggest that AT1 receptors rather than AT2 receptors predominantly mediate Ang II-induced contraction in the rat urinary bladder.

Angiotensin II↗

PubMed1993

Identification of alpha 1C-adrenergic receptor mRNA in bovine retinal pigment epithelium.

PURPOSE: To examine the localization of a novel alpha 1-adrenergic receptor subtype of alpha 1C receptor in the eye, we compared the amount of alpha 1C receptor transcripts in bovine retinal pigment epithelium (RPE) and in neural retina by employing reverse transcription of mRNA and the polymerase chain reaction (RT-PCR) assay. METHODS: RT-PCR assay with specific primers for the alpha 1C-adrenergic receptor and for beta-actin showed linear relationships between input quantity of RNA and the amount of amplified PCR products for the alpha 1C-adrenergic receptor and also for beta-actin, when PCR was conducted for 24 and 15 cycles, respectively. RESULTS: The RT-PCR assay demonstrated that the spontaneous expression level of the alpha 1C-adrenergic receptor mRNA was much higher in bovine RPE than in neural retina; the alpha 1C-adrenergic receptor/beta-actin ratio from RPE was 0.33 +/- 0.15 (n = 4), whereas that from neural retina was virtually zero. CONCLUSIONS: The RT-PCR assay is a sensitive semiquantitative method for a low abundance mRNA in a limited number of cells. Using the alpha 1C receptor as a model, we demonstrated the usefulness of this assay by showing the uneven distribution of the alpha 1C receptor transcripts in bovine RPE cells and neural retina.

Actins↗

PubMed1992

[Molecular structure and function of renal adrenergic receptors].

The kidney is the principal organ in the body that mediates the excretion of water and minerals, and also functions as an endocrine organ in producing hormones, enzymes (e.g., renin), and vasoactive molecules (e.g., prostaglandins). Circulating catecholamines and autonomic innervation of the kidney can influence those important functional responses as well as vascular constriction via renal adrenergic receptors. The adrenergic receptors are members of a large family of G protein-coupled receptors. Recent advance of molecular cloning has revealed a growing heterogeneity of adrenergic receptor subtypes. They share a common topographical motif consisting of seven putative transmembrane regions, where. They are thought to be essential for ligand binding. Multiple subtypes of adrenergic receptors may exist in the kidney. Defining the precise role of renal adrenergic receptors in disease states, particularly hypertension, will certainly require the application of new tools of biochemistry and molecular biology.

Glomerular Filtration Rate↗

PubMed1990

Different desensitization mechanisms of two alpha 1-adrenoceptor subtypes in the contraction of rabbit aorta.

Using alpha 1-adrenoceptor selective antagonists chlorethylclonidine (CEC) and WB4101, both functional and radioligand binding studies showed that rabbit aorta contains at least two pharmacologically distinct alpha 1-adrenoceptor subtypes of approximately 10% alpha 1a and 90% alpha 1b receptors, and that each receptor subtype has a distinct role in the alpha 1-adrenoceptor-mediated vasoconstrictive response through different biochemical mechanisms for increasing intracellular Ca2+; alpha 1a receptors cause tonic response predominantly dependent on the influx of extracellular Ca2+, while alpha 1b receptors stimulate phosphoinositides (PI) hydrolysis/intracellular Ca2+ mobilization and cause phasic response mainly independent of extracellular Ca2+. Incubation of rabbit aorta with 10 microM noradrenaline (NA) for 2 h resulted in a reduction in the phasic response and PI hydrolysis to NA with no change in the tonic response. Similar to the NA incubation, the protein kinase C stimulator PMA (1 microM) selectively attenuated alpha 1b-receptor mediated PI hydrolysis and phasic contraction but had little effect on alpha 1a-receptor-mediated tonic response. The protein kinase C inhibitor H-7 (10 microM) blocked these inhibitory effects of PMA. Treatment with H-7 (10 microM) prevented the NA-induced alpha 1b receptor desensitization in inositol monophosphate (IP) formation and phasic response. The results suggest that activation of C kinase may be involved in the development of selective desensitization of alpha 1b receptors by a short-time in vitro incubation of NA.

Adrenergic alpha-Antagonists↗

PubMed1990

Characterization of alpha 1-adrenergic receptor subtypes linked to iodide efflux in rat FRTL cells.

We have characterized alpha 1-adrenergic receptor subtypes in functional rat thyroid cells, FRTL, with relation to iodide efflux, and have also examined the effect of TSH on alpha 1 receptor subtypes. FRTL cells grown in a medium containing 5 mU TSH/ml (6H cells) had five times the number of alpha 1 receptors of those maintained in TSH-free medium (5H cells) (11.2 fmol/10(6) cells compared with 2.0 fmol/10(6) cells). Pretreatment with chlorethylclonidine (CEC; 10 mumols/l), which inactivates only alpha 1b receptors, caused 98.8% and 97.0% decreases in the density of specific [3H]prazosin-binding sites in 5H and 6H cells respectively. LIGAND computer program analysis of the displacement curves for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane (WB4101) showed that FRTL cells contained mostly low-affinity WB4101 sites. Using the phenoxybenzamine inactivation method, we found a linear relationship between alpha 1 receptor density and the cytosolic free Ca2+ concentration response in FRTL cells. Pre-exposure of intact FRTL cells to CEC caused a 98.7% decrease in noradrenaline-stimulated maximal increase in cytosolic free Ca2+. Also, CEC and 3,4,5-trimethoxy-benzoic acid 8-(diethylamino) octyl ester (TMB-8), but not nicardipine, inhibited noradrenaline-stimulated iodine efflux. The results suggest that FRTL cells contain mostly the alpha 1b-adrenergic receptor subtype; that the alpha 1b receptors mediate cytosolic free Ca2+ and iodide efflux responses, and that TSH enhances these responses by increasing the alpha 1b receptor density without affecting the post-receptor mechanism.

Animals↗

PubMed1990

Two pharmacologically distinct alpha 1-adrenoceptor subtypes in the contraction of rabbit aorta: each subtype couples with a different Ca2+ signalling mechanism and plays a different physiological role.

Using the alpha 1-adrenoceptor subtype-selective antagonists chlorethylclonidine (CEC), WB4101, and 5-methyl-urapidil, we have examined the possible heterogeneity in the alpha 1-adrenoceptor populations in rabbit aorta. The alpha 1-adrenoceptor alkylating agent CEC selectively inhibited the phasic component of the norepinephrine-induced contractile response, with little effect on the tonic component. The alpha 1-adrenoceptor occupancy-response relationship defined by the phenoxybenzamine inactivation method was rectangular hyperbolic for the tonic response, whereas that for the phasic response was linear, indicating the different degree of receptor reserve for the two responses. Radioligand binding studies with the nonselective alpha 1-adrenoceptor antagonist radioligand 125I-BE2254 showed that 73-87% of the binding sites in rabbit aorta are CEC sensitive and they are predominantly low affinity sites both for WB4101 (pKd = 8.1) and for 5-methylurapidil (pKd = 7.1). Moreover, alpha 1-adrenoceptor-mediated phosphatidylinositol (PI) hydrolysis was CEC sensitive, and fractional inactivation of alpha 1 receptors with CEC showed equivalent increments in the reduction of PI hydrolysis and phasic contractile response, suggesting that both responses are linearly related to the CEC-sensitive receptor sites. The Schild plots for the competitive antagonists WB4101 and 5-methyl-urapidil against alpha 1a-adrenoceptor-selective agonist methoxamine-induced contraction were linear and had slopes not significantly different from unity, with a pA2 of 9.07 +/- 0.07 (n = 5) for WB4101 and 9.09 +/- 0.05 (n = 3) for 5-methyl-urapidil. However, the Schilod plots for these antagonists against norepinephrine were curvilinear. Computer-assisted analysis of these curvilinear Schild plots in a two-receptor system indicated that alpha 1-adrenoceptor populations responsible for the constrictive response are predominantly (approximately 80-90%) low affinity sites for the two antagonists (pKd approximately 8.1 for WB4101 and pKd approximately 7.1 for 5-methyl-urapidil) and a small population (approximately 10-20%) are high affinity sites (pKd approximately 9.1 for both WB4101 and 5-methyl-urapidil), which was in good agreement with radioligand binding studies.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Antagonists↗

PubMed1989

Desensitization of alpha 1 adrenoceptor-stimulated glycogen phosphorylase activity in vascular smooth muscle.

Desensitization of alpha 1-adrenoceptor-mediated activation of glycogen phosphorylase was investigated in rabbit aorta. Activation of glycogen phosphorylase by epinephrine was antagonized by the alpha 1-receptor selective antagonist prazosin but not by yohimbine (alpha 2-receptor selective) or by propranolol (beta-receptor antagonist). Preincubation of rabbit aortic ring segments for 5 h with norepinephrine (NE, 10(-5) M) led to a 30-fold loss in sensitivity and a 55% decrease in maximal activation of the enzyme by alpha agonists. Preincubation of aortic ring segments with phenylephrine (10(-5) M) in the presence of propranolol (10(-6) M) also caused desensitization of glycogen phosphorylase activation. The desensitization was heterologous since maximal activation of the enzyme by histamine or KCl was also markedly diminished in segments preincubated with NE. In contrast to these results, catecholamine-induced desensitization to alpha 1-adrenoceptor-mediated smooth muscle contraction in aortic ring segments resulted in loss in sensitivity but not maximal force of contraction on subsequent stimulation by alpha 1 agonists. These results suggest that the mechanism responsible for desensitization of glycogen phosphorylase is distal to receptor activation and may involve attenuation of responses to intracellular Ca2+-dependent enzymes which have limited reserve.

Animals↗

PubMed1989

The first demonstration of CGRP-immunoreactive fibers in canine hearts: coronary vasodilator, inotropic and chronotropic effects of CGRP in canine isolated, blood-perfused heart preparations.

CGRP-immunoreactive nerve fibers were histologically stained in the endocardium and perivascular layer of coronary vessels of canine hearts. To examine the physiological role of the CGRP in the heart function, effects of exogeneous CGRP on the hearts were studied using canine isolated, blood-perfused heart preparations. CGRP exerted dose-related potent vasodilator effects with a minimal increase in the developed tension of the papillary muscle, but slightly decreased the sinoatrial rate. The vasodilator effects were unaffected by the pretreatment of either atropine or propranolol. These specific functional effects on the coronary artery are well in accordance with the anatomical localization of CGRP. Taken together, CGRP seems to play an important role in the regulation of coronary vascular tone, while it has only a small functional role in inotropism and chronotropism in canine hearts.

Animals↗

PubMed1989

Glycogen phosphorylase activation by two different alpha 1-adrenergic receptor subtypes: methoxamine selectively stimulates a putative alpha 1-adrenergic receptor subtype (alpha 1a) that couples with Ca2+ influx.

We compared the effects of methoxamine on alpha 1-adrenergic receptor-mediated phosphorylase activation in rat hepatocytes and rabbit aorta. Although methoxamine is a potent agonist in activating phosphorylase of rabbit aorta, it had little effect in rat hepatocytes. Using the phenoxybenzamine inactivation method, we found that the quantitative relationship between 125I-BE2254 (125I-BE) binding capacity and maximal norepinephrine-stimulated phosphorylase activation was nonlinear in rabbit aorta, whereas it was linear in rat hepatocytes. The potency of methoxamine in inhibiting specific 125I-BE binding is significantly (p less than 0.05) higher in rabbit aorta (Kd, 96.4 +/- 7.7 microM), compared with rat hepatocytes (Kd, 283 +/- 16 microM). However, these quantitative differences could not fully explain the blunted [Ca2+]c and phosphorylase responses to methoxamine in rat hepatocytes. Treatment with chlorethylclonidine dose dependently suppressed 125I-BE binding sites and norepinephrine-induced phosphorylase activation in rat hepatocytes, whereas in rabbit aorta it resulted in only a 31% decrease in 125I-BE binding sites, with little effect on phosphorylase activation. Furthermore, alpha 1-adrenergic receptor-mediated cellular events of phosphatidylinositol (PI) hydrolysis and phosphorylase activation were unaffected by the removal of extracellular Ca2+ in rat hepatocytes, whereas both responses were markedly attenuated in rabbit aorta. The results indicate that two different alpha 1-adrenergic receptor subtypes activate glycogen phosphorylase, through different mechanisms for increasing [Ca2+]c in the two systems. In rat hepatocytes, alpha 1 receptors are closely linked to PI hydrolysis and Ca2+ release from intracellular stores and cause phosphorylase activation. In rabbit aorta, on the other hand, activation of alpha 1 receptors increases [Ca2+]c by Ca2+ influx from the extracellular fluid as well as by Ca2+ release, and both PI hydrolysis and phosphorylase activation are caused mainly by the Ca2+ entry. Methoxamine interacts with both chlorethylclonidine-sensitive and -insensitive alpha 1 receptor subtypes but selectively stimulates the alpha 1 receptor subtype that closely couples with the Ca2+ influx.

Animals↗