Hiromichi Nakamuta

Glutathione-induced inhibition of Na+-independent and -dependent bindings of L-[3H] glutamate in rat brain

Reduced glutathione (GSH, 10(-7)-10(-3) M) was found to exert a profound suppressive action on the Na+-independent and -dependent bindings of L-[3H]glutamic acid (Glu) in a temperature-independent manner. Similarly significant reduction of the bindings resulted from the addition of oxidized glutathione (GSSG). Scatchard analysis revealed that GSH as well as GSSG invariably decreased the affinity of the binding sites for [3H]Glu without significantly affecting the number of the binding sites. These results suggest that GSH (GSSG) may in part participate in the synaptic transmission at central Glu neurons through interaction with the receptors and/or the uptake sites for Glu.

Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix.

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9–19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161–174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu23,Ala24-sCT in which Pro23 and Arg24 were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic α-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9–19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.

Possible interaction of [3H]glutamate binding sites with anion channels in rat neural tissues

The effect of various ions on [(3)H]l-glutamic acid (Glu) binding was examined using crude synaptic membrane preparations from the rat brain. In vitro addition of sodium acetate (1-100 mM) exhibited a significant enhancement of the binding in a concentration dependent manner. Ammonium chloride (20 mM) prevented the potentiation by sodium acetate at 2 degrees C, whereas sodium acetate exerted an inhibitory action on the ammonium chloride-induced augmentation of the binding at 30 degrees C. Ammonium chloride (1-100 mM) itself elicited a temperature dependent stimulation of the binding, which was invariably attenuated by an antagonist for the anion channels such as picrotoxinin (10(?3) M) as well as by inhibitors of anion transport including ethacrynic acid (10(?3) M) and 4,4?-diisothiocyanatostilbene-2,2?-disulfonic acid (10(?4)?10(?3) M), respectively. The later two inhibitors also caused a significant additional raise of the sodium acetate-induced enhancement of the binding. A significant augmentation of the binding resulted from the addition (20 mM) of various anions known to penetrate the anion channels such as bromide, iodide, nitrate, bicarbonate and thiocyanate in a permeability related manner, while that of non-permeable anions including fluoride, sulfate, acetate, formate, phosphate, oxalate, lactate, succinate and tartarate had no such a profound effect on the binding. Addition of d-aspartic acid resulted in the complete abolition of the Na(+)-dependent binding while sparing the Cl(?)-dependent binding. Scatchard analysis revealed that Cl(?) ions induced a two-fold increase in the number of the binding sites without affecting their affinity, whereas Na(+) ions reduced the affinity with a concomitant increase of the number of the binding sites. Addition of quisqualic acid (10(?5)?10(?3) M) inhibited the Cl(?)-dependent binding of [(3)H]Glu to a significantly greater extent than the inhibition on Na(+)-dependent binding. N- Methyl- d -aspartic acid and kainic acid exerted no preventive action on the basal, Cl(?)-dependent and Na(+)-dependent binding. respectively. The highest basal binding activity was found in the retina among various central structures examined. A significant basal binding activity of [(3)H]Glu was also detected in the pituitary and adrenal but not in the kidney. Chloride ions exhibited a significant facilitation of [(3)H]Glu binding to central regions without altering that to peripheral tissues such as pituitary and adrenal. In contrast, Na(+) ions induced significant attenuation of the binding to the pituitary, adrenal and retina despite the occurrence of augmentation of the binding to other central structures. These results suggest the Glu binding sites may be linked to the anion channels in the rat central nervous system and that this linkage may be absent from the pituitary, adrenal and retina.

Development of a highly sensitive and specific two-site enzyme immunoassay for parathyroid hormone (1-34): Application to pharmacokinetic study on intranasal parathyroid hormone (1-34) in human

A highly sensitive and specific two‐site enzyme immunoassay for parathyroid hormone (1‐34) (PTH(1‐34)) and its usability for the pharmacokinetic study are described. Plasma samples were incubated simultaneously with 2,4‐dinitrophenylated anti‐PTH(1‐34) IgG and anti‐PTH(1‐34) Fab′‐ β‐D‐galactosidase conjugate. The immune complex formed of the three components was trapped onto (anti‐2,4‐dinitrophenyl group) IgG‐coated polystyrene balls. β‐D‐ Galactosidase activity bound to the polystyrene balls was assayed by fluorometry. The practical detection limit of PTH(1‐34) was 50 fg (12 amol)/0.05 ml of sample and 1 pg/ml as the concentration and practically no interference occurred by PTH(1‐84) and PTH‐related protein (1‐34) up to 300 pg/ml and 10 ng/ml, respectively. The application of this method has enabled us to directly estimate the bioavailability of PTH(1‐34) dosed intranasaly at the prescribed level (0.090 mg). The pharmacokinetic parameters of the intranasal PTH(1‐34) (n = 4) thus estimated were as follows: the area under the plasma concentration‐time curve (AUC) = 20,500 ± 15,900(SD) pg·min/ml; the mean residence time (MRT) = 194 ± 16.3(SD) min; and the maximal concentration (Cmax) = 98 ± 51(SD) pg/ml with the maximal time (Tmax) = 35.0 ± 12.2(SD) min. J. Clin. Lab. Anal. 12:268–275, 1998.

Osteoporosis Requires Bone-Specific Statins

Remedies for primary osteoporosis are increasing in brands but not always with concomitant improvements in efficacy and safety. Clinical studies suggest that nitrogen-containing bisphosphonates alone display sufficient practical effectiveness to survive as effective therapy. However, their less effectiveness in highly osteopenic patients due to their lack of genuine bone anabolic effect waits improvements. Pinpointing statins as the inducer of BMP-2 provoked a rush of clinical and laboratory studies to identify bone anabolic properties. Clinical studies, even if only through observational, suggest that under conventional dosing conditions for hyperlipemia, the liver-targeted statins now in use display insufficient bone anabolic effect, although laboratory studies seem to be clarifying the mechanisms underlying intrinsic bone anabolic properties. While incomplete, these studies indicate the possibility that, if bioavailability to bone could be improved by simply changing dosing methods and/or deliberate derivatization, the genuine anabolic properties of statins on bone could be extracted and put into therapeutic use.

A sandwich transfer enzyme immunoassay for salmon calcitonin: determination of the bioavailability of intranasal salmon calcitonin in human.

A sandwich transfer enzyme immunoassay for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The assay procedure consisted of the reaction of SCT with 2,4‐dinitrophenyl biotinyl anti‐SCT IgG and anti‐SCT Fab′‐β‐D‐galactosidase conjugate, trapping onto (anti‐2,4‐dinitrophenyl bovine serum albumin) IgG‐coated polystyrene balls, eluting with ϵN‐2,4‐dinitrophenyl‐L‐lysine and transferring to streptavidin‐coated polystyrene balls and fluorometric detection of β‐D‐galactosidase activity. The practical detection limit of SCT was 0.05 pg (15 amol)/50 μl of sample and 1 pg/ml as the concentration. The application of this method has enabled us to directly estimate the bioavailability of SCT dosed intranasally at the therapeutic level (160 IU, 31 μg) for its anti‐osteoporotic effect as compared to an intramuscular dose (10 IU, 1.9 μg). The pharmacokinetic parameters of the intranasal SCT (n = 6) thus estimated were as follows: the area under the blood concentration‐time curve (AUC) = 9400 ± 5400 (SD) pg·h/ml, and the mean residence time (MRT) = 42 ± 14 (SD) min, when the AUC for the intramuscular SCT (n = 3) = 5600 ± 2000 (SD) pg·h/ml and the MRT = 39 ± 19 (SD) min. J. Clin. Lab. Anal. 11:380–387, 1997.

PLASMA LEVEL MONITORING OF NASAL SALMON CALCITONIN IN THE RAT BY A HETEROGENEOUS TWO-SITE ENZYME IMMUNOASSAY

The experimental and clinical effectiveness of nasal salmon calcitonin (SCT) for treatment of osteoporosis in humans has been well established, but none is known yet about the pharmacokinetic property in relation to therapeutic efficacy, especially when used in a therapeutic dose range. This preclinical study was designed to evaluate such a property, first of all in rats, using a novel heterogeneous two‐site enzyme immunoassay that has allowed us to evaluate the pharmacokinetic property of parenteral SCT in rats due to the high sensitivity (the detection limit=2 pg of SCT/ml of plasma). It was found that as early as 10 min after the nasal dosing of 1.25, 5, or 20 U/rat, the SCT immunoactivity became detectable in plasma and thereafter it waned rapidly with time. Hypocalcemia developed in a dose‐dependent manner, but with a delay of ∼20 min from the peak of the immunoactivity and lasted hours. The pharmacokinetic parameters measured for the doses (1.25, 5, and 20 U/rat) were as follows; the AUCs (pg·hr/ml)=20.8, 89.0, and 189, and the MRTs (min) = 52, 54, and 45, respectively. The results appear to suggest: (1) the unexpected quick transfer of nasal SCT into and from the circulation, (2) a delayed onset of hypocalcemia and possibly its anti‐osteopenic action, both of which may last longer, (3) that keeping the plasma SCT above the in vitro anti‐osteoclastic level (∼1 pM) only for a few hours per days would be enough for including the distinct anti‐osteopenic effect in rats, and (4) the feasibility of designing the clinical study as to the pharmacokinetics and pharmacodynamics of nasal SCT on humans. J. Clin. Lab. Anal. 11:129–131, 1997.

A noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for salmon calcitonin: Determination of the bioavailability of subcutaneous salmon calcitonin and its correlation with the hypocalcemic activity in rats

A noncompetitive enzyme immunoassay method (hetero‐two‐site enzyme immunoassay) for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The method in brief proceeds as follows: centrifugal filtration through a polysaccharide membrane to remove plasma proteins, biotinylation, trapping onto an anti‐SCT IgG‐coated polystyrene ball, acid elution, coupling with affinity‐purified anti‐SCT Fab1‐peroxidase conjugate, final trapping onto streptavidin‐coated polystyrene balls, and measurement of peroxidase activity bound to the balls by fluorometry. The practical detection limit of SCT was 0.1 pg (30 amol)/assay and 2 pg/ml as the assay samples concentration, which was at least fivefold lower than those previously reported by competitive radioimmunoassays. The application of this method has enabled us to 1) directly estimate the bioavailability of SCT dosed subcutaneously at the therapeutic levels (1.2 and 4.7 μg/kg) for its antiosteoporotic effect as compared to an intravenous dose (1.2 μg/kg) and 2) search for the relationship between blood level and the hypocalcemic activity of SCT. The pharmacokinetic parameters of subcutaneous SCT (1.2 and 4.7 μg/kg) thus estimated were as follows: the area under the blood concentration‐time curve (AUC) = 89 and 550 pg hr/ml, and mean residence time (MRT) = 44 and 65 minutes, respectively, when the AUC for an intravenous SCT (1.2 μg/kg) = 160 pg hr/ml and the MRT = 10 minutes.

Characterization of [3H]glutamate binding sites on frozen sections from rat adrenal

Some biochemical characteristics of [(3)H]glutamate (Glu) binding sites on frozen sections from the rat adrenal glands were studied. Adrenal frozen sections exhibited stereo-selective, saturable and temperature-dependent binding of [(3)H]Glu. An agonist for one of the subclasses of central Glu receptors, quisqualic acid (QA), elicited a significant inhibition of the binding, whereas neither N-methyl-d-aspartic acid nor kainic acid, agonists for other subclasses of the receptors, had such a significant effect on the binding at the concentration range similar to QA. In vitro addition of sodium acetate (100 mM) resulted in a significant inhibition of [(3)H]Glu binding to frozen sections of the rat adrenal glands. It thus appears that there exist QA-sensitive binding sites of [(3)H]Glu in the rat adrenal glands which exhibit pharmacological characteristics distinctly different from those in the brain.

サケカルシトニン(sCT)のラットにおける中枢性と末梢性摂食抑制作用 新しい誘導体 [Gly8]-sCTにおける分離

It has been reported based on animal studies that salmon calcitonin (sCT), besides hypocalcemic action, exhibits a variety of pharmacological actions. The anorectic action has been observed to ensue not only by central administration but also by peripheral injection, indicating that in clinical use to induce hypocalcemia or for other therapeutic purposes, the anorectic action may develop as a side effect. While studying the anorectic effect of [Gly8]-sCT in rats, a derivative of sCT having rather stronger hypocalcemic potency than the mother molecule, it was found that on peripheral injection, the derivative practically lacks the anorectic effect. Thus, a pharmacological evaluation of the novel peptide was made. When injected subcutaneously in rats at a dose level of 1 U/kg, sCT and the derivative induced similar patterns of hypocalcemia in either of which hypocalcemia reached a peak between 1 and 3 hr after injection. No notable difference existed between the action of the two peptides. In rats which were trained to take the daily food need within 2 hr (17:00 approximately 19:00), an intracerebroventricular injection of 1 U/rat of either peptide 30 min before food presentation significantly reduced both food and water intake, causing a loss of body weight as compared with the control which received the injection of saline alone. By subcutaneous injection of 100 U/kg, sCT was also active to decrease food and water intake. The effect was found to last longer than 24 hr and to cause a marked loss in body weight. In contrast, such effects did not develop in rats treated with the derivative. Both peptides were able to suppress the specific binding of 125I-sCT to the membrane fraction of rat brain and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)