Sensitive and selective liquid chromatography-electrospray ionization tandem mass spectrometry analysis of hydrochlorothiazide in rat plasma.

A sensitive and selective method for the determination of hydrochlorothiazide (HCTZ) concentrations in rat plasma was developed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). An aliquot of plasma (50 microl) was mixed with the solution of internal standard, hydrofluorothiazide (HFTZ), and extracted with tert-butyl methyl ether. The reconstituted extract was applied to the LC-MS/MS system with a reversed phase C8 column and eluted with distilled water/acetonitrile (85/15, v/v). To enhance negative ionization of HCTZ and HFTZ in the multiple reaction monitor (MRM), the solution consisting of acetonitlile/1% (v/v) ammonia solution (95/5, v/v) was delivered after column separation. This additional technique, so-called the post-column addition, increased sensitivity of HCTZ and HFTZ about 500- and 200-fold, respectively. The calibration curve showed good linearity (r = 0.999) over the range of 4-1000 ng/ml. Acceptable accuracy (100.8-113.1%) and precision (0.28-16.4%) were confirmed in the intra- and the inter-day analyses. It is indicated that this LC-MS/MS method is useful for pharmacokinetic studies of HCTZ in small animals, because it enabled the serial determination of plasma level of HCTZ in rats.

The defecation reflex in rats: fundamental properties and the reflex center.

While pharmacological and physiological studies in rats are now increasing, physiological properties of their defecation have been scarcely investigated. This study was performed to define the properties of defecation in decerebrate rats, with special reference to the pontine defecation reflex center, which has been postulated in dogs. Intraluminal pressure was recorded from the colon and rectum with balloon-pressure transducer method using balloons of 15-20 mm in length and 0.1-0.3 ml in volume. Distention of a balloon in the descending colon and rectum with an additional injection of 0.03-0.1 ml air induced propulsive contractions on the descending colon and rectum. The mean of threshold pressures to induce propulsive contraction was 17.0 +/- 5.8 mm Hg (mean+/-S.E.) in the proximal part and 18.3 +/- 3.3 mm Hg in the distal part of the descending colon, and 11.8 +/- 1.3 mm Hg in the rectum. The maximum amplitude of propulsive contractions was 55 mm Hg in the rectum, 47 mm Hg in the distal part of the descending colon and 38 mm Hg in the proximal part. Similar colorectal propulsive contractions were produced by gastric distention (5-10 ml, 20-30 mm Hg) and electrical stimulation of the anal canal. Contrarily, spontaneous contractions of the proximal colon were suppressed by rectal distention and anal-canal stimulation. These results suggest that the descending colon and rectum, but not the proximal colon, were innervated by the pelvic afferent and efferent fibers mediating the defecation reflex. Pontine transection at the cerebellar peduncle level abolished colorectal propulsive contractions induced by distention of the stomach, descending colon and rectum, and stimulation of the anal canal, although much smaller contractions were still induced after the pontine transection. These results suggest that the pontine defecation reflex center exists and works in rats, as in dogs.

Structure-activity relationships of a novel class of endothelin receptor selective antagonists; 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridines.

The synthesis and structure-activity relationships of 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridine class of ET(A) receptor selective antagonists were described. These derivatives were prepared from the optically active key intermediates (3, 4, 10, and 13). Optimization of the substituent at the 2-position of the bottom 4-methoxyphenyl ring of the lead compound 1 led to identification of 2-hydroxy-1-methylethoxy (2g and h), hydroxyalkyl (2i, m, and p), 3-methoxy-2-methylpropyl (2t and u), N-acetyl-N-methylaminomethyl (2v), and 2-(dimethylcarbamoyl)propyl (2w) derivatives that showed greater than 1000-fold selectivity for the ET(A) receptor over the ET(B) receptor with excellent binding affinity (IC(50)<0.10 nM). Further screening of these compounds by assessing the plasma exposures at 1 h, 4 h, and 8 h after oral administration (3 or 10 mg/kg) in rats led to identification of the hydroxymethyl (2i) and 3-methoxy-2-methylpropyl (2u) derivatives exhibiting good oral bioavailability in rats.

Identification of membrane-type receptor for bile acids (M-BAR).

Bile acids play an essential role in the solubilization and absorption of dietary fat and lipid-soluble vitamins. Bile acids also modulate the transcription of various genes for enzymes and transport proteins for their own and cholesterol homeostasis through binding to nuclear receptors. Here we report a novel category of bile acid receptor, a membrane-type G protein-coupled receptor (GPCR), BG37. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in BG37-expressing cells, but not in mock-transfected cells, independently of nuclear receptor expression. The rank order of potency of various bile acids for BG37-expressing cells was different from that for the nuclear receptor-mediated response. These observations demonstrate the presence of two independent signaling pathways for bile acids; membrane-type GPCR for rapid signaling and nuclear receptors for delayed signaling. Expression of BG37 was detected in various specific tissues, suggesting its physiological role, although it remains to be further characterized.