Enantioselective pharmacokinetics of homochlorcyclizine: III. Simultaneous determination of (+)- and (−)- homochlorcyclizine in human urine by high-performance liquid chromatography

A method is described for the simultaneous determination of (+)- and (−)-homochlorcyclizine (HCZ) in human urine by high-performance liquid chromatography on a chiral stationary phase of ovomucoid-bonded silica. The pH of the buffer and organic modifier in the mobile phase markedly affected the chromatographic separation. A mobile phase of methanol—0.02 M acetate buffer (pH 4.7) (25:75, v/v) at a flow-rate of 1.0 ml/min was used for the urine assays. The ultraviolet absorption was monitored at 240 nm, and diphenhydramine was employed as the internal standard for the quantitation. (+)-HCZ, (−)-HCZ and the internal standard were eluted at retention times of 15, 25 and 8 min, respectively. The limit of determination for HCZ enantiomers was ca. 50 ng/ml of urine. One of the metabolites in human urine, which was a quaternary ammonium-linked glucuronide, could also be determined in a manner similar to unchanged HCZ after β-glucuronidase hydrolysis. A pharmacokinetic study was conducted with three healthy volunteers, who each received a single oral dose of racemic HCZ (20 mg). Distinct differences were found between the two enantiomers, particularly in the metabolic process, that is, the urinary excretion as (−)-HCZ-glucuronide within 48 h was ca. four times higher than that of the (+)-isomer. This method should be very useful for enantioselective pharmacokinetic studies of HCZ.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus

Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.     

Anomalous permeabilities of the egg cell membrane of a starfish in K(+)-Tl(+) mixtures

The electrical properties of “inward” rectifying egg cell membranes of the starfish mediastera aequalis have been studied in the presence of K(+)-Tl(+) mixtures. When the ratio of the external concentrations of these ions is changed while their sum is kept constant, both the conductance and the zero-current membrane potential go through a minimum, showing clear discrepancies from theoretical results based on conventional electrodiffusion models (E.g., Goldman’s equation). By contrast, when the ration of the two concentrations is fixed and their sum varied, the potential follows an ideal Nernst slope, consistent with Goldman’s equation. The membrane conductance which, according to previous studies on similar membranes, is to be viewed as a function of the displacement of the membrane potential from its resting value δV, shows marked differences between the cases in which K(+) or Tl(+) are the predominant ions: when K(+) is the predominant permeant ion in solution, the addition of small amounts of Tl(+) inhibits the current, while corresponding blocking effects of K(+) on the current are not observed when Tl(+) is the predominant permeant ion. Also, the time course of the conductance during voltage clamp is different in the two cases, being much faster in Tl(+) than in K(+) solution for comparable values of δV. Most of the above features are accounted for by a model in which it is assumed that the ionic channels have external binding sites for cations and that their permeability properties depend on the species of the cation bound (K(+)or Tl(+) in the present experiments).     

Loperamide in rat intestines: A unique disposition

When everted sacs of rat duodenum, jejunum and ileum were incubated with [¹⁴C]loperamide in vitro, unchanged drug and its metabolites were found not only in tissues but also in media of the mucosal side with virtually no radioactivity in media of the serosal side. The amounts of metabolites found in media of the mucosal side were comparable to or larger than those in tissues. Di-desmethyl loperamide was more predominant in the media as compared with mono-demethylated one than in the tissues. Therefore, a portion of loperamide absorbed in intestines can be metabolized there and directly secreted back into lumen. Oral loperamide thus undergoes a unique disposition, likely constituting one of mechanisms for its distinct dissociation of central and antidiarrheal activities.     

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.     

The anti-inflammatory mechanism of MK-447 in rat carrageenin-induced pleurisy

Intrapleural injection of 2% λ-carrageenin caused the accumulation of exudate up to 19 hr. The rate of plasma exudation, measured by the exuded dye amounts for 20 min in the pleural cavity after intravenous injection of pontamine sky blue, showed a peak at 5 hr. Aspirin (100 mg/kg, i. p.) suppressed the dye exudation up to 5 hr, but did not at 7 hr. This inhibition coincided with the decrease of the PG and TXB₂ levels, which were measured by gas chromatography-mass spectrometry, in the pleural exudate. In _vitro experiments, MK-447, a phenolic compound, stimulates PG endoperoxide biosynthesis at lower doses and inhibits it at higher doses, acting as a tryptophan-like cofactor required by PG endoperoxide synthetase. This drug (0.3, 1.0 and 3.0 mg/kg, i. p.) suppressed the dye exudation dose-dependently up to 5 hr, but did not at 7 hr even at a higher dose, in combination with the dose-dependent decrease of the pleural level of PGE₂, which was reported to be a major PG among PGs and TXB₂ in the exudate in inducing the plasma exudation (Harada ; Prostaglandins, : 881, 1982). Thus, the anti-inflammatory action of MK-447 can be explained by inhibition of PGE₂ generation, giving no consideration to the role of oxygen-derived free radicals as a prime mediator in inflammation.     

Desensitization of the D-2 dopamine receptor and the β2-adrenoceptor in the intermediate lobe of the rat pituitary gland

A D-2 dopamine receptor and a β₂-adrenoceptor occur in the intermediate lobe of the rat pituitary gland (IL). Exposure of intact IL tissue to a D-2 agonist diminished the ability of dopaminergic agonists [but not 5′-guanylyl imidodiphosphate (Gpp(NH)p)] to inhibit adenylate cyclase activity. Conversely, exposure of intact IL tissue to a β-adrenergic agonist diminished the ability of a β-adrenergic agonist (but not forskolin) to stimulate adenylate cyclase activity. Treatment of ovariectomized rats with 17β-estradiol desensitizes the β₂-adrenoceptor but not the D-2 receptor. Desensitization of the IL catecholamine receptors is discussed within the framework of a previously published “working model” of these receptors.