SOCS2 is dispensable for BCR/ABL1-induced chronic myeloid leukemia-like disease and for normal hematopoietic stem cell function.

Suppressor of cytokine signaling 2 (SOCS2) is known as a feedback inhibitor of cytokine signaling and is highly expressed in primary bone marrow (BM) cells from patients with chronic myeloid leukemia (CML). However, it has not been established whether SOCS2 is involved in CML, caused by the BCR/ABL1 fusion gene, or important for normal hematopoietic stem cell (HSC) function. In this study, we demonstrate that although Socs2 was found to be preferentially expressed in long-term HSCs, Socs2-deficient HSCs were indistinguishable from wild-type HSCs when challenged in competitive BM transplantation experiments. Furthermore, by using a retroviral BCR/ABL1-induced mouse model of CML, we demonstrate that SOCS2 is dispensable for the induction and propagation of the disease, suggesting that the SOCS2-mediated feedback regulation of the JAK/STAT pathway is deficient in BCR/ABL1-induced CML.

Oncogenic receptor tyrosine kinase in leukemia.

Growth, survival and differentiation of hematopoietic cells are regulated by the interaction between hematopoietic growth factors and their receptors. While the defect in this interaction results in an insufficient hematopoiesis, the aberrantly elevated activation leads to the transformation of hematopoietic cells. The constitutive active mutations of receptor tyrosine kinase, such as c-Kit platelet-derived growth factor receptor (PDGFR) or fins-like tyrosine kinase 3 (Flt3), play a major role in the development of hematopoietic neoplasia. The constitutive activation is provoked by several mechanisms, such as making fusion genes by chromosomal translocations, or various mutations involving regulatory regions of the receptor. The chromosomal translocation brings the receptor intracytoplasmic domain juxtaposed to an unrelated molecule which has dimerization or multimerization motif, resulting in the constitutive dimerization of the receptor. The missense, insertion or deletion mutations in the regulatory regions, such as juxtamembrane domain, activation loop and extracellular domain, cause constitutive activation by releasing the respective auto-inhibitory functions of each regulatory region. Constitutive active receptors generate different signals quantitatively and qualitatively from wild type receptor, which mediate the oncogenic phenotype. Given the frequent involvement of constitutive active receptor tyrosine kinase in hematopoietic malignancies, targeted inhibitions of active tyrosine kinase and downstream aberrant signaling are rapidly developing novel therapeutic modality with much promise.

[Effect of amlodipine, a new calcium antagonist on isolated blood vessels].

Amlodipine (AML), a new 1,4-dihydropyridine derivative, similar to nifedipine (NIF), dose-dependently relaxed or inhibited the KCL-contraction (cont.) of dog coronary artery (DCA) and rat aorta (RA) and the spontaneous motility of rat portal vein (RPV). However, in contrast to the case of NIF, the maximum effect was obtained at 1-2 hr following an addition of AML, and the recovery of KCl-cont. after removal of AML was very slow. Similar effects of AML on KCl-cont. were observed in dog femoral artery (DFA), dog basilar artery (DBA) and rabbit aorta (RBA). The IC50 values of AML for producing the half-maximal effect were 6.5 x 10(-9) M, 6.5 x 10(-9) M, 1.1 x 10(-8) M, 1.7 x 10(-8) M and 4.8 x 10(-8) M in DCA, DFA, RPV, DBA and RBA, respectively. In these vessels, the potency of AML was slightly less than the potency of NIF. On the other hand, AML (10(-7), 10(-6) M) only slightly attenuated norepinephrine (NE)-induced cont. in DFA and RA. Based on the ratio of IC50 values in DFA, AML exhibited 846-fold higher selectivity toward KCl-cont. than NE-cont., and it was much higher than the selectivity of NIF. In calcium-free medium, AML failed to inhibit the NE-cont. in RA, but it markedly inhibited CaCl2-induced cont. These results indicate that AML significantly inhibits KCl-cont. in blood vessels at lower doses with slightly less potency than NIF, but with a higher selectivity than NIF in comparison with the potency against NE-cont., and they also confirmed that the effect of AML is of slow onset and long-lasting.

[Antihypertensive effects of amlodipine, a new calcium antagonist].

The antihypertensive effects of oral administration of amlodipine (AML), a new calcium antagonist, were investigated in hypertensive animals. AML (1-10 mg/kg) produced a dose-dependent reduction of blood pressure (BP) in spontaneously hypertensive rats (SHR). The effect of AML reached a maximum at 4-6 hr and was sustained even at 10 hr post-dose, in contrast to the case of nifedipine that produced a maximum effect at 1 hr with a rapid recovery. Heart rate (HR) was increased slowly and slightly by AML (10mg/kg), but increased rapidly and markedly by nifedipine (3, 10 mg/kg). The dose (ED 30) of AML required to decrease BP by 30 mmHg was 2.3 mg/kg and similar to the case of nifedipine. AML also produced long-lasting reduction of BP in renal and DOCA hypertensive rats. The ED30 values were 2.4 and 2.2 mg/kg and similar to the respective values of nifedipine (ED 30:2.4,2.1 mg/kg). In renal hypertensive dogs (RHD), the effect of AML (0.1,0.3,1.0 mg/kg) was maximum at 4-6 hr and long-lasting, producing similar reductions of both systolic and diastolic BP (ED30: 0.3-0.4 mg/kg respectively). In SHR (1 or 3 mg/kg/day, for 15 days) and RHD (0.2 mg/kg/day for 20 days) chronically receiving AML, there was an enhancement of the antihypertensive effect of AML within a few days after starting chronic dosing, and thereafter a significant reduction of BP at 24 hr after dosing and constant effects of AML during subsequent treatment. BP after cessation of the chronic dosing gradually recovered to the level before the start of the experiments. No significant changes in HR were observed throughout the experiments. These results indicate that AML produces the antihypertensive effect with a similar potency to nifedipine but with a profile of slow onset and long duration, and there was no development of tolerance to the antihypertensive effects and changes of HR during long-term treatment.

[Effects of 1-[2-[bis(fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl) piperazine dihydrochloride (I-893) on turnover of dopamine and norepinephrine in the brain].

Studies were performed to elucidate the effects of 1-[2-[bis (fluorophenyl) methoxy]ethyl]-4-(3-phenylpropyl) piperazine dihydrochloride (I-893), a newly synthesized aryl-1,4-dialkyl-piperazine derivative, on turnover of dopamine and norepinephrine in the rat brain. The contents of both monoamines were not affected by I-893 at an oral dose of 10 mg/kg. The oral administration of 50-250 mg/kg produced a transient increase in dopamine content of the caudate nucleus and hypothalamus, and thereafter, the content dose-dependently decreased. Norepinephrine levels in the hypothalamus and frontal cortex were slightly decreased by I-893. I-893 potentiated the rate of alpha-methyl-p-tyrosine-induced depletion of dopamine and norepinephrine. The 3-methoxytyramine content in animals treated with pargyline was increased by I-893 in the caudate nucleus and olfactory tubercle. NSD-1015-induced accumulation of DOPA was suppressed by larger doses of I-893. Oral administration of I-893 (10-50 mg/kg/day) for 14 days slightly attenuated the inhibitory effects of the drug on the norepinephrine level, while it did not affect the inhibitory effect on the dopamine level. These results suggest that I-893 facilitates the release of dopamine and norepinephrine and/or inhibits the uptake of the monoamines in the presynaptic nerve terminals.

Effects of avermectin B1a and picrotoxin on striatal release of dopamine with reference to replacement of extracellular chloride with nitrate.

The present study was an attempt to elucidate the effects of avermectin B1a (AVM) and picrotoxin, an anion channel opener and blocker, respectively, on the release of endogenous dopamine from the slices of caudate nucleus of the rat, using a superfusion method in order to determine the interaction between these agents with the gamma-aminobutyric acid (GABA) receptor-anion channel complex. Avermectin (1.14-11.4 microM) reduced the Ca2+-dependent release of dopamine stimulated by 40 mM KCl without affecting the basal release of dopamine. In contrast, picrotoxin in doses larger than 20 microM facilitated the K+-stimulated release of dopamine. The inhibitory effect of avermectin was completely antagonized by 10 microM picrotoxin and 0.1 mM bicuculline; these doses of both agents did not change the K+-stimulated release of dopamine. Replacement of chloride (Cl-) in the superfusion medium with nitrate (NO3-) markedly facilitated the K+-stimulated release of dopamine and the increase was antagonized by verapamil (10 microM) and tetrodotoxin (1 microM). In the nitrate medium, avermectin reduced the K+-stimulated release of dopamine and the inhibitory effect was antagonized by bicuculline. However, picrotoxin up to 100 microM did not affect the K+-stimulated release of dopamine either in the presence or absence of bicuculline. These results suggest that the dopaminergic nerve terminals in the caudate nucleus receive inhibitory regulation through the facilitation of anion channels. This regulation is apparently altered depending on the main anion in the extracellular fluid.

Novel microsomal anion-sensitive Mg2+-ATPase activity in rat brain.

Ethacrynic acid (EA) highly sensitive Mg2+-ATPase activity was demonstrated in rat brain microsomes. Marker enzyme studies suggested that the EA highly sensitive Mg2+-ATPase activity originated mainly from plasma membranes, and possibly from synaptic vesicles. Oligomycin did not affect the EA highly sensitive Mg2+-ATPase activity. Sulfhydryl reagents, such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid), and anion transport inhibitors, such as 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid and 2,4-dinitro-1-fluorobenzene, completely inhibited the EA highly sensitive Mg2+-ATPase activity with apparent Ki values at 5, 5, 8, 8 and 10 microM respectively. Treatment of microsomes with ethylenediaminetetraacetic acid and ammonium sulfate increased the EA highly sensitive Mg2+ and Na+,K+-ATPase activities, but not EA less sensitive Mg2+- or HCO3-ATPase activity, 2- to 3-fold that in crude microsomes. Relative substrate specificities of ATP much greater than GTP greater than ITP greater than UTP, CTP, a Km for ATP at 0.77 mM, and an optimal pH at pH 7.4 were observed. Among the anions tested (Cl-, Br-, F-, HCO3-, I-, SCN-, NO3-), EA highly sensitive Mg2+-ATPase activity was stimulated significantly by Cl- and reduced by NO3-. These data suggest that a novel, plasma membrane-located and anion-sensitive Mg2+-ATPase activity exists in the brain.

Rats with congenital tremor and curled whiskers and hair.

In a colony of Kyo: Wistar strain of rats, we found animals with curled whiskers and hair. These rats exhibited tremor when they moved. There were no sex differences in phenotype and behavior, and the affected animals of both sexes were sterile. Among pairs that produced at least one tremulous offspring, 21.8% of the females and 21.7% of the males were affected; these proportions suggest that the anomaly is caused by an autosomal recessive gene. When the presumed heterozygous males were crossed with WAG/Rij females, about half of their female F1 hybrids were heterozygous, and they produced 26.1% (43/165) of the affected offspring when backcrossed to the sires. Again these results suggested that the disorder is caused by an autosomal recessive gene. We tentatively designated the gene as tremor (tm). The main pathological changes were seen in the gonad and central nervous system. The gonads of both sexes were aplastic even in adult animals. Vacuole formation was seen widely in the central nervous system and sometimes exhibited a spongy appearance. After administration of alpha-methyl-p-tyrosine, the norepinephrine content of the cerebellum was high, indicating that some anomalies of catecholamine release were present. The mutation is being maintained by random mating of the littermates of affected animals. Detailed pathological, endocrinological, neuropharmacological, and genetical studies are proceeding.

Release of histamine and adrenaline in vivo following intravenous administration of neurotensin.

Plasma histamine levels of rats anesthetized with pentobarbital sodium were significantly increased by intravenous administration of neurotensin (NT, 1 nmole/kg) with the maximum effect at 3 min, and a return to the initial levels in 20 min. Treatment of animals with compound 48/80 or disodium cromoglycate completely inhibited the elevation of histamine level by NT, however, treatment with reserpine or diphenhydramine and adrenalectomy did not affect the elevation. Plasma adrenaline levels increased transiently 1 min after NT injection, and adrenalectomy and treatment with compound 48/80 or diphenhydramine markedly reduced the elevation of adrenaline levels after NT injection. Plasma levels of noradrenaline were unchanged upon NT injection. These results provide direct evidence of the release of endogenous histamine and adrenaline following NT administration, and suggest the contribution of these amines to the NT-induced triphasic blood pressure responses (the first depressor, second pressor and third depressor responses) reported previously.

Inhibitory effects of brotizolam, a new thienodiazepine, on limbic forebrain and neostriatal dopaminergic systems in vivo and in vitro.

Studies were performed to elucidate the effects of brotizolam, a newly synthesized thienodiazepine, chemically related to the benzodiazepines, on dopamine turnover in the limbic forebrain and neostriatum. Intraperitoneally administered brotizolam retarded the rate of alpha-methyl-p-tyrosine-induced depletion of dopamine in the olfactory tubercle (OT), nucleus accumbens (NA) and caudate nucleus (CN). Significant retardation was observed with brotizolam in doses ranging from 0.1-10 mg/kg in the olfactory tubercle, and from 1-10 mg/kg in the nucleus accumbens and caudate nucleus. These inhibitory effects of brotizolam were antagonized by bicuculline, a GABA antagonist, in all of the regions examined. Using slices of the olfactory tubercle, nucleus accumbens and caudate nucleus, the effects of brotizolam on dopaminergic nerve terminals were examined in vitro. Basal release of dopamine was not affected by brotizolam in concentrations up to 10(-6) M; however, K+-stimulated release of dopamine was significantly reduced by brotizolam at 10(-7) M or above. The reduction of K+-stimulated release of dopamine was antagonized by bicuculline, added in the superfusion medium. These data suggest that brotizolam inhibits the release of dopamine in the limbic forebrain and neostriatal systems probably through mechanisms including a facilitation of GABAergic action on dopaminergic nerve terminals.