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The effects of the centrally acting cholinesterase (ChE) inhibitors, tetrahydroaminoacridine (THA) and E2020 (1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl] methylpiperidine hydrochloride), potential drugs for the treatment of senile dementia, on the basal extracellular acetylcholine (ACh) concentration in the hippocampus of freely moving rats, were determined using a microdialysis technique without the use of a ChE inhibitor in the perfusion fluid and a sensitive RIA. The mean (+/- SEM) basal ACh content in the perfusate was 103.1 +/- 3.6 fmol/sample collected over 30 min when microdialysis probes with a length of 3 mm dialysis membrane were used. The content of ACh decreased to an almost undetectable level upon perfusion of magnesium, suggesting that, in the present study, most of the ACh detected in the perfusates was due to cholinergic neuronal activity. THA (1.65 mg/kg, i.p.) produced an insignificant increase in the extracellular ACh concentration, but a dose of 5 mg/kg, i.p. caused a prolonged and significant 5.5-fold increase from the control value. E2020 (0.65 and 2 mg/kg, i.p.) produced significant, prolonged and dose-dependent increases (4 and 12 times the control value, respectively), the peak effect occurring within 1 h. Perfusion with 10 mumol/l physostigmine produced an about 30-fold increase of ACh output, suggesting that the basal extracellular ACh concentration is highly dependent on ChE activity. When ChE was inhibited locally by perfusion with physostigmine, THA (5 mg/kg) produced a transient and, at its maximum, a 1.42-fold increase in extracellular ACh concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Danazol and gonadotropin-releasing hormone agonists which are used as therapeutic drugs for endometriosis, develop adverse reactions in association with their long-term use. The efficacy of anti-estrogens for endometriosis, an estrogen-dependent disorder, has not been demonstrated. A novel, orally active anti-estrogen, TZE-5323 ((2-cyclohexy-6-hydroxybenzo[b]thien-3-yl)[4-[2-(1- piperidinyl)ethoxy]phenyl] methanone hydrochloride, CAS 150797-71-0; free salt formula) was developed. TZE-5323 showed strong affinity for human estrogen receptor alpha (hER alpha) and beta (hER beta), and dose-dependently inhibited estradiol-stimulated transcriptional activation via hER alpha and hER beta. Furthermore, TZE-5323 dose-dependently reduced estrogen-increased uterine weight in ovariectomized rats. Tamoxifen showed agonistic activity on hER alpha, while TZE-5323 did not show such activity. In the experimental endometriosis model in rats in which endometrial tissue is autotransplanted into the renal subcapsular space, TZE-5323 dose-dependently reduced the volume of the endometrial implant as did danazol and leuprorelin acetate. Furthermore, the long-term administration of TZE-5323 neither showed a decrease in bone mineral density nor did it affect serum estradiol concentrations in intact rats. Therefore, TZE-5323 suggested its potential as a novel therapeutic drug for endometriosis which is effective also in long-term use.

Carboxypeptidase A, from porcine pancreas was purified to homogeneity by ammonium sulfate fractionation, autolysis, batch absorption and elution from DEAE-Sephadex, and crystallization. The overall purification was about 32-fold with a yield of 31 and the specific activity of the purified protein was 208 units/mg protein.The apparent relative molecular mass determined by gel filtration on a Sephadex G-200 column was 38900.The amino-terminal sequence of the porcine carboxypeptidase A, was Asn-Tyr-Ala-Thr-Tyr-His-Thr-Leu-GluGlu-Ile-Tyr-Asp-Phe-Met-Asp-Ile-Leu-Val-Ala-Glu-His-Pro-Gln-Leu-which was highly homologous to that of bovine carboxypeptidase A,. The purified enzyme was characterized with respect to isoelectric point (4.3). K,,, for N"-carbobenzoxyglycyl-L-phenylalanine (Cbz-Gly-LPhe) (20 mM), amino acid composition, pH optimum, pH stability, stability at different temperatures and effect of drying. The enzyme contained 1.01 mol zinc/mol and was inhibited by chelating agents such as EDTA and o-phenanthroline. Among substrates such as Cbz-Gly-LPhe, N"-benzoylglycyl-L-arginine, various kinds of amino acid esters, casein and elastin, porcine carboxypeptidase A, showed an enzymatic activity only towards Cbz-Gly-r.Phe and casein. These data are in good agreement with the substrate specificity of bovine carboxypeptidase A.As reported by Folk and Schirmer [I] in 1963, porcine pancreatic carboxypeptidase A exists in three electrophoretically distinct forms, carboxypeptidase A1, A2 and A3. These forms are generated by limited proteolysis on the aminoterminal region of procarboxypeptidase A 121. Carboxypeptidase A1 and Az were well characterized [I]. However, carboxypeptidase A3 was often contaminated with A2 and has not yet been fully characterized [l].Narayanan and Anwar [3] in 1970 reported a porcine carboxypeptidase A, designated as Ae, which was isolated from a twice-crystallized porcine elastase preparation. This species of carboxypeptidase A was different from carboxypeptidase A,, A2 and A3 in terms of solubility and amino acid composition, and was reported as a possible new genetic variant of carboxypeptidase A.More recently, Kobayashi et al. [4] have reported an isolation of carboxypeptidase Ap from a binary complex preparation of procarboxypeptidase A and a precursor of protease E in porcine secretion. They found that the binary complex consisted of one molecule of carboxypeptidase Ap and one molecule of protease E. The latter enzyme was inhibited by diisopropylfluorophosphate.During our studies on the development of a method for a large-scale production of carboxypeptidase in high yield from porcine pancreas, we isolated and crystallized porcine carboxypeptidase A,. Furthermore, it was found that carboxypeptidase A, thus obtained was associated with porcine elastase I in crystalline state.In this paper, we describe details of the purification method of carboxypeptidase A, from porcine pancreas and some properties of the enzyme. EXPERIMENTAL PROCEDURE MaterialsPancreatic glands were obtained from freshly s...

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