Isolation of PSD-Zip45, a novel Homer/vesl family protein containing leucine zipper motifs, from rat brain.

Using monoclonal antibody against the 45 kDa postsynaptic density protein, we isolated a novel isoform of Homer/vesl. The NH2-terminal region containing a PDZ domain of this protein is identical to that of Homer/vesl, and the COOH-terminal region containing unique leucine zippers shows self-multimerization. We named this protein PSD-Zip45. In addition to specific binding of PSD-Zip45 mediated by a PDZ domain to the metabotropic glutamate receptors 1alpha or 5, the distribution of PSD-Zip45 transcripts is highly consistent with that of metabotropic glutamate receptor transcripts. The PSD-Zip45 is, therefore, the first candidate as receptor anchoring proteins containing leucine zipper motifs in the central nervous system.

Decreased absorption as a possible cause for the lower bioavailability of a sustained-release propranolol.

The influence of sustained absorption on the oral availability of propranolol (P) and the metabolic disposition of P were investigated by obtaining the partial metabolic clearances (CLm) following long-acting P (LA) dosing in comparison with the conventional propranolol tablet (CP). Ten healthy volunteers were given a single oral dose of an LA capsule (60 mg) and CP (20 mg x 3) using a crossover design. Blood and urine samples were collected over 24- and 48-h postdose periods, respectively. Concentrations of P, propranolol glucuronide (PG), 4-hydroxypropranolol (4P), 4-hydroxypropranolol glucuronide (4PG), 4-hydroxypropranolol sulfate (4PS), and naphthoxylactic acid (NLA) were determined by HPLC with fluorescence and UV detection. Significant differences were observed between LA and CP in the area under the plasma concentration-time curves (AUCs) for P, PG, and NLA and in the amounts excreted into urine (Ae) for all measured metabolites (i.e., PG, 4P, 4PG, 4PS, and NLA). The parallel decrease of the AUC for P and the excreted amounts of all measured metabolites following LA dosing resulted in partial metabolic clearances (CLm) and renal clearances (CL) for P and its metabolites that were similar to those observed for CP. Therefore, the hepatic metabolism of P would not be affected by the slower absorption at a single oral dose of 60 mg. These results indicate that the poor absorption of P from the gastrointestinal tract might be one of the factors causing the low bioavailability of P observed after administration of the sustained-release formulation.

Relationship between the therapeutic effects or side-effects and the serum disopyramide or mono-N-dealkylated disopyramide concentration after repeated oral administration of disopyramide to arrhythmic patients.

After we had developed a method to determine simultaneously the blood concentrations of disopyramide (DP) and its metabolite mono-N-dealkylated disopyramide (MND) by high-performance liquid chromatography, DP was administered repeatedly to arrhythmic patients in order to examine the relationship between the serum DP or MND concentration and the therapeutic effects or side-effects. To 79 arrhythmic patients (57 patients with ventricular premature contraction, 13 with supraventricular arrhythmia and 9 with atrial fibrillation), DP was administered repeatedly at an initial oral dose of 200 to 400 mg/day. Of the 61 patients which were possible to evaluate after reaching a steady state, 32 were evaluated as effective and 29 as non-effective, the effective rate being 52.5%; the mean blood DP concentration (+/- S.D.) was 2.14 +/- 0.65 and 1.74 +/- 0.62 micrograms/ml, respectively, with a significant difference between the two groups (p +/- 0.05). At the final dose, 40 patients were evaluated as effective and 18 as non-effective, the effective rate being 69.0%; the mean blood DP concentration was 2.03 +/- 0.67 and 2.09 +/- 0.68 micrograms/ml, respectively, with no significant difference between the two groups. Among 42 patients with premature contraction, 26 were evaluated as effective and 16 as non-effective, the effective rate being 62%; the mean blood DP concentration was 2.01 +/- 0.62 and 2.20 +/- 0.70 micrograms/ml respectively, with no significant difference between the two groups. The incidence of side-effects was 17.7%, and there were no significant differences in blood DP or MND concentrations between the groups with and without side-effects. A blood DP concentration more than 2 micrograms/ml may be required to achieve the therapeutic effect of DP administered repeatedly.

Translocation of cortactin (p80/85) to the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Cortactin (p80/85) was discovered as a src kinase substrate and an actin filament binding protein. We investigated translocation of cortactin to the cytoskeleton during thrombin receptor-mediated platelet activation. Only a few percent of total cortactin (minor cortactin pool) translocates to the cytoskeleton as early as 5 s after platelet activation, while about 40% of total cortactin (major cortactin pool) is thereafter recovered in the cytoskeleton during platelet aggregation. Pretreatment of platelets with cytochalasin D suppresses completely this translocation, indicating that the translocation is dependent on actin polymerization. Inhibition of platelet aggregation by a tetrapeptide with the sequence RGDS, chelator of extracellular Ca2+, or a nonstirring condition results in marked suppression of translocation of the major cortactin pool. These results suggest that a minor cortactin pool translocates to the cytoskeleton independent of GPII-bIIIa (alpha IIb beta 3 integrin) engagement, and a major pool requires GPIIbIIIa-mediated signals into the cell for the translocation. Methyl 2,5-hydroxycinamate, a tyrosine kinase inhibitor, inhibits tyrosine phosphorylation of cortactin without affecting its translocation, indicating that tyrosine phosphorylation is not essential for the translocation. Morphological studies reveal that cortactin is colocalized with filamentous actin in aggregated platelets and that it is localized at the cell peripheries along actin filaments in spread platelets. Taking these together, we have demonstrated in this paper that the translocation of cortactin is associated with the reorganization of the actin-based cytoskeleton during platelet activation, particularly with platelet aggregation.

Relative bioavailability of two disopyramide capsules in humans based on total, unbound, and unbound enantiomer concentrations.

The relative bioavailability of two 100-mg disopyramide formulations which showed almost an 8- to 10-fold difference in their dissolution rates at pH 1.2 and 6.8 was determined in eight healthy subjects using a randomized block design. Although no significant differences in relative bioavailability were observed between the two formulations when based on the total disopyramide concentration, an almost 30 per cent difference in the extent of bioavailability was observed when assessed in terms of the unbound (+/-)- and (-)-disopyramide concentration, due probably to stereoselective nonlinear plasma protein binding. This suggests that unbound enantiomer parameters would be more sensitive to differences in bioavailability between two disopyramide formulations. Therefore, the type of concentration used would be an important factor for precise evaluation of the relative bioavailability of racemic drugs.

Dosing rate-dependent relationship between propranolol plasma concentration and beta-blockade.

The effect of propranolol dosing rate on beta-blockade was studied in human volunteers after administration of a conventional tablet and a sustained release capsule. The slope of the plot of the percentage of reduction in the heart rate against log plasma propranolol concentration was significantly greater after administration of a sustained release capsule than after administration of a conventional tablet. A marked leftward shift of the plasma concentration-response curve was also observed in rabbits as the infusion rate was decreased over the same infusion period. This shift was not altered by pretreatment with 6-hydroxydopamine and plasma catecholamine levels were not affected by the rate of infusion, indicating that the contribution of sympathetic activation to the effect was minimal. By contrast to the anticlockwise hysteresis of the temporal response after propranolol, no such hysteresis was found with the more hydrophilic beta-adrenoceptor antagonist atenolol, or was there any leftward shift in the plasma concentration- response relationship. Data from the isolated guinea pig atrial preparation also showed anticlockwise hysteresis and a leftward shift of the concentration-response curve at a low propranolol input rate, whereas no shift was observed with more hydrophilic beta-blockers such as atenolol, pindolol and metoprolol.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate expression of alpha-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells.

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the alpha-tropomyosin (alpha-TM) gene, exon 2a rather than exon 2b is specifically spliced in alpha-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of alpha-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the down-regulation of beta-TM-SM, alpha-TM-SM converted to alpha-TM-F1 and alpha-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high Mr type to low Mr type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express alpha-TM-SM, beta-TM-SM, and high Mr caldesmon. In situ hybridization revealed specific coexpression of alpha-TM-SM and high Mr caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.

[Evaluation of stat assay of serum digoxin concentration by radioimmunoassay and its application for digoxin regimen based in pharmacokinetics (author's transl)].

Serum digoxin concentration measured by stat RIA (phadebas digoxin RIA kit) correlated well with results obtained by complete assays. The results of stat assay can be reported within 1 hour, measuring one or more samples together with 2 standard samples in duplicates. Precise measurement can be expected with serum digoxin concentration over 0.5 ng/ml. The stat assay allows to apply the theory of pharmacokinetics for the estimation of digoxin concentration at steady states measuring minimum digoxin concentration (Cn(min)) on the 3rd to 6th day after the start of digoxin therapy. The estimated serum digoxin levels were well agreed with measured values with the difference ranging from 1.6 to 8.6% in CV. The method is useful for the planning and assessment of appropriate digoxin regimen.