We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.

Adrenergic alpha-Agonists↗

PubMed1999

Lymphocyte-sensitivity to glucocorticoid correlates with the sensitivity to cyclosporin A and tacrolimus in chronic renal failure patients.

AIMS: Association between lymphocyte-sensitivity to immunosuppressants in transplant recipients in vitro and clinical outcomes has been demonstrated. In general, renal transplant recipients are treated with a combination of immunosuppressants such as either glucocorticoid/cyclosporin A (CsA) or glucocorticoid/tacrolimus (FK506) but the pharmacological complementarity of these drugs is still controversial. We examined relationships between the lymphocyte-sensitivities to these immunosuppressants. METHODS: We examined lymphocyte-sensitivities to prednisolone (PSL), CsA, and FK506 in vitro in a total of 190 chronic renal failure (CRF) patients and 140 healthy subjects. The lymphocyte-sensitivity was evaluated from the IC50 value against mitogen-stimulated lymphocyte-blastogenesis in vitro. RESULTS: Statistically significant correlations of the IC50 values in CRF patients between the following pairs of drugs were observed: PSL and CsA (P<0.0001; n=129, r=0.419), PSL and FK506 (P<0.001; n=54, r=0. 441), and CsA and FK506 (P<0.0001; n=45, r=0.608). Similar correlations were also observed in lymphocytes from healthy subjects. The population of CRF patients who exhibited high IC50 values (low sensitivities) to PSL and FK506 was significantly larger than that of healthy subjects (P<0.05). CONCLUSIONS: Patients who showed low lymphocyte-sensitivity to either of the drugs also may exhibit low sensitivity to the others, and thus they may have a high risk of unsatisfactory outcome under combination therapy after renal transplantation. To overcome this risk, the selection of immunosuppressants is recommended to be restricted according to individual lymphocyte-sensitivities to these drugs in vitro, or alternatively, by addition of other drugs with different mechanisms for immunosuppression.

Adult↗

PubMed1999

Cystic partially differentiated nephroblastoma in an adult: an immunohistochemical, lectin histochemical and ultrastructural study.

AIMS: Cystic partially differentiated nephroblastoma (CPDN) is an uncommon renal multicystic tumour, usually affecting early infants. To our knowledge, this report describes the first case of CPDN occurring in an adult. METHODS AND RESULTS: A 45-year-old man was found incidentally to have a left renal cystic tumour, measuring 20 mm in diameter, at the lower pole far from the pelvis. The tumour was composed of multilocular cystic spaces of variable size and intervening septa without solid nodular areas. The cysts were lined by a single layer of flattened, hobnail, or columnar epithelium. The septa were made of mesenchymal cells, which were admixed with small numbers of loosely aggregated blastemal cells, occasional tubular structures in various stages of development, and a few glomeruloid structures. The tumour cells had no anaplasia, and mitoses were rare. Immunohistochemical and lectin histochemical studies revealed that the cyst lining epithelium and the tubular structures in the septa expressed predominantly the markers for distal tubules and collecting ducts. Ultrastructurally, the cyst lining cells closely resembled collecting duct cells while some tubular structures showed an immature nephrogenic morphology. The patient was alive and well without evidence of recurrence 11 months after surgery. CONCLUSIONS: CPDN does occur in adults, as experienced in Wilms' tumour, though its incidence is extremely low. This study suggests that CPDN may show maturation intermediate between cystic nephroma and Wilms' tumour, even in adult cases.

Biomarkers, Tumor↗

PubMed1999

Interstitial invasion of well-differentiated hepatocellular carcinoma and subsequent tumor growth.

Pathological processes of interstitial invasion of hepatocellular carcinoma (HCC) were investigated in 125 autopsy and seven surgical cases, where selection focused on those occurring in well-differentiated HCC. The invasive pattern of well-differentiated HCC was characterized by the formation of streaks of tumor cell cords running along connective tissue fibers, referred to as a streak pattern. These cell cords were unaccompanied by reticulin frameworks and type IV collagen, which are consistent components of HCC parenchyma. The typical streak pattern was observed only in HCC cases and could be useful in differential diagnosis. Following progressive accumulation or proliferation of the tumor cells within the interstitium, many capillaries appeared between the cell cords while collagen fibers tended to disappear. The tumor cell cords then showed branching and were accompanied by reticulin frameworks, type IV collagen and sinusoidal blood spaces. In the setting of interstitial invasion of well-differentiated HCC, it is suggested that interstitial tissue is converted into HCC parenchyma via the aforementioned steps.

Antigens, CD34↗

PubMed1999

Expression of protein tyrosine phosphatase PTP-RL10 and its isoform in the mouse testis.

BACKGROUND: The cytoplasmic-type protein tyrosine phosphatase PTP-RL10/PTPD1/PTP2E contains an ezrin-like domain and associates with the c-Src protein tyrosine kinase. Because tyrosine phosphorylation regulated by protein tyrosine kinases and phosphatases is involved in activation, migration, differentiation and proliferation of various cell types, the expression of PTP-RL10 and c-src in the mouse testis was investigated. METHODS: Testes of wild-type mice and W/W(v) mutant mice that lack germ cells were analyzed by northern blotting, in situ hybridization histochemistry and reverse transcriptase-polymerase chain reaction for the expression of PTP-RL10, its isoform PTP-RL10b and c-src. Expression in the Sertoli cell lines, TM4 and TAMA26 was also analyzed. RESULTS: PTP-RL10 transcripts of 5.7kb and 2.9kb in size were detected in the testis. In situ hybridization histochemistry demonstrated that the 5.7kb transcripts were expressed in pachytene spermatocytes and somatic cells including Sertoli cells, in which c-src transcripts were detected. The 2.9kb transcript encoded a novel isoform, PTP-RL10b, that has the catalytic domain but not the domains that associate with c-Src. PTP-RL10b was expressed mainly in the testicular somatic cells. TM4 and TAMA26 cells were found to co-express PTP-RL10, PTP-RL10b and c-src transcripts. CONCLUSION: PTP-RL10 and its isoform are expressed in the Sertoli cells and are suggested to play roles in spermatogenesis by interacting with c-Src and/or other protein(s).

Animals↗

PubMed1999

Binding pockets of the beta(1)- and beta(2)-adrenergic receptors for subtype-selective agonists.

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.

Adrenergic beta-1 Receptor Agonists↗

PubMed1999

Responsiveness of beta-escin-permeabilized rabbit gastric gland model: effects of functional peptide fragments.

We established a beta-escin-permeabilized gland model with the use of rabbit isolated gastric glands. The glands retained an ability to secrete acid, monitored by [14C]aminopyrine accumulation, in response to cAMP, forskolin, and histamine. These responses were all inhibited by cAMP-dependent protein kinase inhibitory peptide. Myosin light-chain kinase inhibitory peptide also suppressed aminopyrine accumulation, whereas the inhibitory peptide of protein kinase C or that of calmodulin kinase II was without effect. Guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) abolished cAMP-stimulated acid secretion concomitantly, interfering with the redistribution of H+-K+-ATPase from tubulovesicles to the apical membrane. To identify the targets of GTPgammaS, effects of peptide fragments of certain GTP-binding proteins were examined. Although none of the peptides related to Rab proteins showed any effect, the inhibitory peptide of Arf protein inhibited cAMP-stimulated secretion. These results demonstrate that our new model, the beta-escin-permeabilized gland, allows the introduction of relatively large molecules, e.g., peptides, into the cell, and will be quite useful for analyzing signal transduction of parietal cell function.

Aminopyrine↗

PubMed1999

[Pharmacological evaluation of the development candidate].

In recent years, drugs developed in Japan are used internationally in other countries. The pharmaceutical pre-clinical phase in critical in the drug developing cycle. Pharmacologists contribute to finding the leading compounds and evaluate the right compound for clinical trial. The selection of the right compound for development will save both time and money. In the pharmacological evaluation of a development candidate, in vivo animal studies that can assure the clinician that the drug is both effective and safe are usually required. Studies on the mechanism of action of the drug and how the drug works in the diseased state becomes important since a known target molecule usually serves as the basis for the development of a new drug.

Animals↗

PubMed1999

[beta gamma subunit of heterotrimeric G protein as a new target molecule for drug development].

Although ischemia-reperfusion produces reactive oxygen species and induces injury of the heart, the mechanism leading to injury is largely unknown. Hydrogen peroxide (H2O2) is widely used for a reagent to mimic the action of reactive oxygen species produced by ischemia-reperfusion. Treatment of the rat neonatal myocytes with H2O2 resulted in activation of mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38. To study the involvement of beta gamma subunit of heterotrimeric G protein in H2O2-induced activation of MAPKs, we expressed the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2-ct) which can bind beta gamma subunit and inhibit the interaction with various effector proteins. Expression of GRK2-ct inhibited the H2O2-induced activation of ERK by 70% and also inhibited the activation of Akt by 30%. In contrast with H2O2-induced activation of ERK, the activation of ERK induced by phorbol ester PMA and the activation of JNK and p38 induced by H2O2 were not affected by expression of GRK2-ct, indicating that the activation of ERK but not JNK and p38 is dependent on beta gamma subunit. Among several inhibitors for analyzing intracellular signaling pathways, wortmannin inhibited the activation of ERK by H2O2 treatment. These data suggest that treatment of the rat neonatal myocytes with H2O2 releases beta gamma subunit from heterotrimeric G protein, and leads to activation of ERK in part by phosphatidylinositol-3 kinase dependent pathway. Thus beta gamma subunit may be a novel target molecule to selectively modulate the intracellular signaling cascade.

Animals↗

PubMed1999

Treatment with l-cis diltiazem before reperfusion reduces infarct size in the ischemic rabbit heart in vivo.

l-cis Diltiazem, an optical isomer of diltiazem, protects against myocardial dysfunction in vitro, whereas its Ca2+ channel blocking activity is about 100 times less potent than that of diltiazem. However, there is no evidence that l-cis diltiazem actually protects against ischemia/reperfusion injury in vivo. To assess this, we employed an anesthetized rabbit model, where the left circumflex artery was occluded for 15 min and reperfused for 360 min. Treatment with diltiazem before and during ischemia (bolus 200 microg/kg and 15 microg/kg per minute for 25 min, i.v.; 575 microg/kg total) showed slightly depressed hemodynamic parameters, while l-cis diltiazem (1150 microg/kg) had no effect. Treatment with l-cis diltiazem produced a high recovery of the thickening fraction and limited the infarct size in a dose-dependent manner. Furthermore, the treatment with l-cis diltiazem (1150 microg/kg) or diltiazem (575 microg/kg) 5 min before reperfusion also limited the infarct size, but not after reperfusion. These results suggest that l-cis diltiazem affects some events in the onset of reperfusion, independently of Ca2+-channel-blocking action. Our observations are the first to show that l-cis diltiazem demonstrated its cardioprotective action in the ischemic rabbit heart in vivo.

Anesthesia↗

PubMed1999

Analysis of domain responsible for desensitization of beta1-adrenergic receptor.

When the wild type beta1-adrenergic receptor (WT-beta1AR) was expressed in Sf9 cells, the beta1AR-stimulated adenylyl cyclase activities were desensitized by prior treatment with isoproterenol. The extent of beta1AR desensitization was not modified, and the onset was not promoted by the overexpression of G protein-coupled receptor kinase 2 (GRK2), GRK5 or GRK6. However, overexpression of the dominant negative mutant of GRK2 appeared to inhibit desensitization of the beta1AR. The change of the potential protein kinase A phosphorylation site located at the intracellular third loop did not affect beta1AR desensitization. Desensitization of the truncated mutant, in which nearly all of the serine and threonine residues from the carboxyl terminus were eliminated, was the same as that of the WT-beta1AR. A deletion mutant that lacked serine and threonine residues of the intracellular third loop was also desensitized by isoproterenol stimulation. Furthermore, the deletion of serine and threonine residues from both the intracellular third loop and carboxyl terminus did not affect desensitization of the beta1AR. These results suggested that phosphorylation by endogenous GRKs in Sf9 cells contributed to desensitization of the beta1AR and that the regions other than third intracellular loop and carboxyl terminus may be responsible for beta1AR desensitization.

Adrenergic beta-1 Receptor Agonists↗

PubMed1999

Temporal differences in actions of calcium channel blockers on K+ accumulation, cardiac function, and high-energy phosphate levels in ischemic guinea pig hearts.

We investigated temporal differences in the protective action of three types of Ca2+ channel blockers in myocardial ischemia, focusing particularly on the blocking ability under depolarizing conditions. The effects of diltiazem, verapamil, and nifedipine on extracellular potassium concentration ([K+]e), acidosis, and level of metabolic markers were examined during 30-min global ischemia and postischemic left ventricular (LV) function in isolated guinea pig hearts. Diltiazem and verapamil, but not nifedipine, inhibited the late phase (15-30 min) of [K+]e elevation, whereas all three blockers delayed the onset of the early phase (0-8 min) of [K+]e elevation. Diltiazem and verapamil inhibited ischemic contracture and improved postischemic LV function to a greater extent. These differences appeared to be linked to preservation of ATP and creatine phosphate and delay of cessation of anaerobic glycolytic activity. Maneuvers to preserve energy sources during ischemia (decrease in external Ca2+ concentration or pacing at a lower frequency) attenuated the late phase of [K+]e elevation. Inhibition of LV pressure was potentiated 12- and 8.2-fold by diltiazem and verapamil, respectively, at 8.9 mM K+ as compared with 2.9 mM K+, whereas that by nifedipine was unchanged. These results indicate that the differential cardioprotection of Ca2+ channel blockers in the late period of ischemia correlates with preservation of high-energy phosphates as a result of different Ca2+ channel blocking abilities under high [K+]e conditions.

Acidosis↗

PubMed1999

[Effects of angiotensin II on the cerebral circulation and function].

The effect of angiotensin II (A II) on cerebral arteries is variable; A II would constrict or dilate cerebral arteries depending on species, the size of vessels examined, preexisting contractile levels, and the presence of functional endothelium. Hence exogenously applied A II may increase or decrease cerebral blood flow. The physiological role played by endogenous A II is even unclear in the cerebral circulation. Endogenous A II is unlikely to modulate cerebral blood flow. However, it may play some roles in the determination of the autoregulation of cerebral blood flow, at least judging from the effect of angiotensin converting enzyme inhibitors. Circulating A II elevates blood pressure, and promotes water and sodium intake. A II derived from the central renin-angiotensin system would play diverse roles as a neurotransmitter or a local hormone.

Angiotensin II↗

PubMed1999

[Actual state of involuntary admission by the prefectural governor--a research report on the involuntary admission in 1995. Committee on Psychiatric Services and Law. Sub-Committee on Measures of Security and Justice].

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Commitment of Persons with Psychiatric Disorders↗