Neurogenic dural protein extravasation induced by meta-chlorophenylpiperazine (mCPP) involves nitric oxide and 5-HT2B receptor activation.

The compound m-chlorophenylpiperazine (mCPP), which is known to trigger migraine-like head pain in some subjects, was evaluated for its ability to induce dural plasma protein extravasation (PPE) in guinea pigs. Intravenous mCPP dose-dependently increased PPE. This effect was inhibited by non-selective 5-HT2 receptor antagonists (methysergide, LY53857, LY215840), by a peripherally restricted 5-HT2 receptor antagonist (xylamidine) and by a 5-HT2B selective receptor antagonist (LY202146). These data suggests that peripheral 5-HT2B receptors mediate mCPP-induced PPE. The nitric oxide synthase inhibitor L-NAME and 5-HT1 agonist sumatriptan also blocked mCPP-induced PPE, suggesting a role for nitric oxide (NO) and the trigeminal system, respectively. NO release has been linked to activation of the 5-HT2B receptor on the vascular endothelium. However, LY202146 did not block PPE induced by electrical stimulation of the trigeminal ganglion. These data are consistent with activation of peripheral 5-HT2B receptors initiating PPE and the theory that selective 5-HT2B antagonists might be effective prophylactic therapies for migraine.

Muscarinic analgesics with potent and selective effects on the gastrointestinal tract: potential application for the treatment of irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a pathopysiolocal condition characterized by abnormal bowel habits that are frequently accompanied by abdominal pain. Current therapy based on reducing high-amplitude GI contractions with nonselective muscarinic antagonists is limited in efficacy due to typical muscarinic side effects and provides no pain relief. We have previously found potent antinociceptive agents acting through muscarinic receptors. In the present work, new 1,2,5-thiadiazole-based structures with muscarinic activity have been evaluated both for activity as analgesics in the mouse withing assay and for activity in normalizing spontaneous cluster contractions in ferret jejunum as a model of IBS in humans. (5R,6R)-exo-6-[4-[(4,4,4-Trifluorobutyl)thio]-1,2,5-thiadiazol+ ++-3-yl] -1-azabicyclo[3.2.1]octane (35, LY316108/NNC11-2192) was found to offer an exceptional profile combining analgesic potency in mouse writhing (ED50 = 0.1 mg/kg) along with potency for normalization of GI motility (ED50 = 0.17 mg/kg). This combination of GI and analgesic potency suggests 35 as an excellent candidate for evaluation as a potential treatment of IBS.

Effects of LY267108, an erythromycin analogue derivative, on lower esophageal sphincter function in the cat.

BACKGROUND/AIMS: Erythromycin (EM-A) and some of its analogues stimulate gastrointestinal smooth muscle contractions. Because gastroesophageal reflux disease (GERD) in humans is in part caused by a reduction in lower esophageal sphincter (LES) pressure, the aim of this study was to investigate the effect of LY267108 (an EM-A analogue with no significant antimicrobial activity) on LES function. METHODS: In ketamine-anesthetized cats, LES pressure was recorded using a Dent sleeve. RESULTS: In cats, LY267108 increased LES pressure, as did motilin and EM-A. Neither LY267108, EM-A, nor motilin altered LES relaxation in response to a swallow. LY267108 increased LES pressure in cats in which the basal LES pressure was lowered experimentally by perfusing the distal esophagus with HCl (0.1 N for 3 days) or following isoproterenol (3.0 micrograms/kg intravenously). In summary, LY267108 increases LES pressure in normal cats, did not affect the relaxation of the LES in response to a swallow, and increases LES pressure in animals with an experimentally induced decrease in LES pressure. CONCLUSIONS: The results suggest that LY267108 may be useful in treating GERD because of its ability to increase LES pressure and thus present a barrier for gastroesophageal reflux.

Effect of basic fibroblast growth factor (bFGF) and insulin-like growth factors type I (IGF-I) and type II (IGF-II) on adult human keratinocyte growth and fibronectin secretion.

Effects of growth factors on keratinocyte migration and proliferation are of interest as an indication of their potential use in acceleration of wound re-epithelialization. Various growth factors were examined for effects on normal adult human keratinocyte growth and fibronectin (Fn) secretion for cells cultured in serum-free medium. Accumulation of Fn in the medium of cells growth with H + I + EGF + BPE paralleled growth during the exponential phase and declined as the cells approached confluence. Cells maintained in low Ca++ (0.15 mM) post-confluence and fed daily to prevent cornification continued to accumulate Fn in the medium, while those grown continuously in 1.2 mM Ca++ ceased Fn secretion at confluence. EGF, bFGF, and transforming growth factor-beta (TGF beta) stimulated keratinocyte Fn secretion in correlation with literature reports on the ability of these factors to stimulate the migration of these cells. In contrast, despite its marked effects on cell growth, BPE was found to consistently reduce the amount of Fn found in the medium when added to cultures containing either EGF or bFGF. Addition of BPE to cultures containing EGF or bFGF stimulated growth to the same extent, indicating that the effects of BPE on keratinocyte growth are not solely due to its content of bFGF.

Gamma glutamyl peptidase: a novel enzyme from hairless mouse epidermis.

Recent literature has suggested that pyroglutamate (PCA) formation in stratum corneum occurs by spontaneous cyclization of glutamine residues derived from filaggrin breakdown. This paper describes an enzymatic alternative. Epidermal homogenates from hairless mice were found to catalyze the formation of PCA from both glutamine and glutamic acid at pH 6.2. Enzyme activity responsible for the first step in this reaction, gamma-glutamyl peptide formation, was partially purified using ammonium sulfate precipitation followed by ion exchange, gel filtration, and hydroxylapatite chromatography. Enzyme preparations free of gamma-glutamyl cyclotransferase activity (which forms PCA from certain gamma-glutamyl peptides) catalyzed formation of gamma-glutamyl-glutamine from glutamine and gamma-glutamyl-glutamate from glutamic acid. Enzyme preparations catalyzed hydrolysis of a variety of gamma-glutamyl peptides but did not split non-gamma-glutamyl peptides or the transpeptidase substrate gamma-glutamyl-rho-nitroanilide. Ammonium sulfate fractions containing both gamma-glutamyl peptidase and gamma-glutamyl cyclotransferase activity catalyzed linear formation of PCA from glutamic acid for periods of up to 19 h. Using gamma-glutamyl-leucine as a substrate, gamma-glutamyl peptidase activity was found to be much higher in crude extracts from epidermis than in preparations from liver, kidney, spleen, intestine, lung, brain, or heart. This activity has not, to our knowledge, been previously described in mammalian tissues.

Biosynthesis of pyrrolidone carboxylic acid in hairless mouse epidermis.

[3H]Glutamic acid (PCA) was followed with time after a single subcutaneous injection. PCA specific activity increased slowly, reaching a peak at 3 to 4 days after injection of the labeled amino acid, after which it slowly decline. Incorporation of [3H]glutamic acid into epidermal PCA was markedly inhibited by a single topical application of cycloheximide. Topical application of cycloheximide 2 hr prior to [3H]glutamate injection caused a significantly greater reduction in PCA specific activity (determined 3 days after injection) than cycloheximide treatment 3 hr after administration of the labeled amino acid. Ninety-seven percent of the PCA content of hairless mouse epidermis was shown to reside in the stratum corneum. These observations indicate the involvement of protein synthesis in the formation of PCA from glutamic acid rather than a direct conversion of the amino acid. The high level of PCA in mammalian epidermis appears to be caused by its accumulation in the stratum corneum accompainied by a relatively slow rate of PCA turnover in comparison to other tissues.