Leukotriene B4 mediates inflammation via TRPV1 in duct obstruction-induced pancreatitis in rats.

OBJECTIVES: We tested the hypothesis that leukotriene B4 (LTB4) mediates pancreatic inflammation in rats via activation of the transient receptor potential vanilloid 1 (TRPV1). METHODS: Leukotriene B4 or a vehicle was administered to adult rats via celiac axis injection after pretreatment with the TRPV1 antagonist, capsazepine, or vehicle, and the severity of subsequent pancreatitis was assessed by measuring pancreatic edema, myeloperoxidase (MPO) activity, and histological grading. In a second experiment, acute pancreatitis was induced by common pancreaticobiliary duct ligation. Six hours after surgery, pancreatic tissue levels of LTB4 were determined by enzyme-linked immunosorbent assay. Also, the effects of inhibition of LTB4 biosynthesis by pretreatment with the 5-lipoxygenase-activating peptide inhibitor, MK-886, were determined. RESULTS: Celiac axis administration of LTB4 significantly increased pancreatic edema and MPO activity, and produced histological evidence of pancreatic edema, neutrophil infiltration, and necrosis. Capsazepine pretreatment significantly reduced all inflammatory parameters in LTB4-induced pancreatitis. Pancreatic tissue levels of LTB4 were significantly elevated in rats that underwent common pancreaticobiliary duct ligation compared with control rats. MK-886 pretreatment significantly inhibited pancreatic edema, histological damage, and pancreatic MPO concentrations. CONCLUSIONS: Common pancreaticobiliary duct obstruction causes an increase in pancreatic LTB4 concentrations that in turn mediates activation of TRPV1 resulting in acute pancreatitis.

The role of leukotriene B4 in Clostridium difficile toxin A-induced ileitis in rats.

BACKGROUND & AIMS: Clostridium difficile toxin A is a potent intestinal inflammatory agent that has been shown to act at least partially by neurogenic mechanisms involving activation of the transient receptor potential vanilloid 1 (TRPV1) (capsaicin) receptor. We tested the hypothesis that leukotriene B4 (LTB4) mediates the effects of toxin A via activation of the TRPV1 receptor. METHODS: Isolated rat ileal segments were pretreated with pharmacologic agents before intraluminal injection of toxin A or LTB4. After 3 hours, the treated segments were removed and inflammation was assessed by luminal fluid accumulation, myeloperoxidase activity, and histology. RESULTS: LTB4 caused ileitis similar to that caused by toxin A and antagonism of TRPV1 receptors but not LTB4 receptors inhibited LTB4-induced inflammation. LTB4 also stimulated TRPV1-mediated substance P release and pretreatment with a specific substance P-receptor antagonist blocked LTB4-induced substance P action and ileitis. Inhibition of the LTB4 biosynthetic enzyme 5-lipoxygenase inhibited toxin A-induced increases in ileal LTB4 levels and toxin A- but not LTB4-induced ileitis. CONCLUSIONS: LTB4 mediates the inflammatory effects of toxin A via activation of TRPV1 receptors.

Inhibition of Clostridium difficile toxin A-induced colitis in rats by APAZA.

A new compound, APAZA, consisting of a molecule of 5-aminosalicylic acid linked to one molecule of 4-aminophenylacetic acid by an azo bond, was testedfor its ability to inhibit acute colitis in rats caused by Clostridium difficile toxin A. When administered chronically for 5 days in drinking water, APAZA significantly inhibited toxin A-induced myeloperoxidase activity, luminal fluid accumulation, and structural damage to the colon at doses of from 1 to 100 mg/kg x day. For comparison, sulfasalazine was administered in identical doses and was found to significantly inhibit toxin A-induced colitis only at the dose of 100 mg/kg x day. When 4-aminophenylacetic acid alone was administered chronically in drinking water, it also inhibited toxin A-induced colonic inflammation at a dose of 100 mg/kg x day. In order to determine if 4-aminophenylacetic acid has a direct anti-inflammatory effect on the colon rather than a systemic effect, 4-aminophenylacetic acid was administered acutely to surgically prepared isolated colonic segments by intraluminal injection in anesthetized rats 30 min before toxin A was injected. 4-Aminophenylacetic acid strongly and significantly inhibited toxin A-induced colitis in this experiment at doses as low as 10 microg/segment. It is concluded that APAZA is a potent inhibitor of toxin A-induced colonic inflammation in rats and that its constituent, 4-aminophenylacetic acid, is responsible for this increased protection against colitis compared to the 5-aminosalicylic acid component of sulfasalazine.

Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties.

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.

Endocannabinoids induce ileitis in rats via the capsaicin receptor (VR1).

Intraluminal administration of the endocannabinoids N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) causes inflammation similar to that caused by Clostridium difficile toxin A in the rat ileum. The effects of anandamide and 2-AG were significantly inhibited by pretreatment with the specific capsaicin receptor (vanilloid receptor subtype 1; VR1) antagonist capsazepine. Pretreatment with the CB1 and CB2 cannabinoid receptor antagonists N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716) and N-[1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) did not affect the responses to anandamide. It has previously been shown that intraluminal toxin A stimulates substance P (SP) release from primary sensory neurons and that pretreatment with SP receptor [neurokinin (NK)-1 receptor] antagonists inhibits the inflammatory effects of toxin A. Anandamide stimulated SP release and this was blocked by capsazepine pretreatment. Also, pretreatment with the specific NK-1 receptor antagonist (2S,3S)-3-([3,5-bis[trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine (L-733,060) significantly inhibited the inflammatory effects of both toxin A and anandamide. Toxin A increased tissue concentrations of anandamide and 2-AG in the ileum, and these effects were enhanced after pretreatment with inhibitors of fatty acid amide hydrolase, a major endocannabinoid-degrading enzyme. The toxin A-stimulated release of anandamide but not 2-AG was selective over their congeners. These results demonstrate that the endocannabinoids anandamide and 2-AG stimulate intestinal primary sensory neurons via the capsaicin VR1 receptor to release SP, resulting in enteritis, and that endocannabinoids may mediate the inflammatory effects of toxin A.

Differences in receptor binding and stability to enzymatic digestion between CCK-8 and CCK-58.

INTRODUCTION AND AIMS: It has been proposed that distinct tertiary structures of the C-terminus of CCK-8 and CCK-58 result in differences in stimulation of pancreatic amylase secretion. Binding of CCK-8 and CCK-58 to CCK-A and CCK-B receptors and stability to enzymatic digestion were used as independent probes for tertiary structure of the C-terminus. METHODOLOGY: Canine CCK-58 was purified from intestinal extracts and CCK-8 was purchased. Their amounts were determined by amino acid analysis. The effect of tertiary structure on receptor binding at CCK-A receptors and CCK-B receptors was evaluated using membrane preparations from mouse pancreas and brain. The influence of C-terminal tertiary structure on stability to enzymatic digestion was evaluated by reacting CCK-8 and CCK-58 with endopeptidase 24:11. RESULTS: CCK-58 was three times more potent than CCK-8 for binding mouse pancreatic membrane CCK-A receptors and equipotent to CCK-8 for binding mouse brain CCK-B receptors. CCK-8 was readily digested by endopeptidase 24:11, whereas CCK-58 was not. CONCLUSIONS: The results strongly support the hypothesis that differences in tertiary structure of the carboxyl terminus of CCK-8 and CCK-58 influence receptor binding and stability to enzymatic digestion.

Primary sensory neurons: a common final pathway for inflammation in experimental pancreatitis in rats.

We hypothesized that neurogenic inflammation is a common final pathway for parenchymal inflammation in pancreatitis and evaluated the role of primary sensory neurons in secretagogue-induced and obstructive pancreatitis. Neonatal rats received either the primary sensory neuron-denervating agent capsaicin (50 mg/kg s.c.) or vehicle. At 8 wk of age, pancreatitis was produced by six hourly injections of caerulein (50 microg/kg i.p.) or by common pancreaticobiliary duct ligation (CPBDL). The severity of pancreatitis was assessed by serum amylase, pancreatic myeloperoxidase (MPO) activity, histological grading, pancreatic plasma extravasation, and wet-to-dry weight ratio. Caerulein significantly increased MPO activity and wet-to-dry weight ratio, produced histological evidence of edematous pancreatitis, induced plasma extravasation, and caused hyperamylasemia. CPBDL increased MPO activity and produced histological evidence of pancreatitis. Neonatal capsaicin administration significantly reduced tissue MPO levels, histological severity scores, and wet-to-dry weight ratio and abolished plasma extravasation. These results demonstrate that primary sensory neurons play a significant role in the inflammatory cascade in experimental pancreatitis and appear to constitute a common final pathway for pancreatic parenchymal inflammation.

Characterization of pituitary IGF-I receptors: modulation of prolactin and growth hormone.

There have been no studies in any vertebrate that have localized insulin-like growth factor (IGF)-I receptors in prolactin (PRL) cells or that have correlated pituitary binding to the potency of IGF-I in regulating both PRL and growth hormone (GH) secretion. We show that IGF-I binds with high affinity and specificity to the pituitary gland of hybrid striped bass (Morone saxatilis x M. chrysops). IGF-I and IGF-II were equipotent in inhibiting saturable (125)I-IGF-I binding, whereas insulin was ineffective. IGF-I binds with similar affinity to the rostral pars distalis (>95% PRL cells) as the whole pituitary gland and immunohistochemistry colocalizes IGF-I receptors and PRL in this same region. Des(1-3)IGF-I, a truncated analog of IGF-I that binds with high affinity to IGF-I receptors but weakly to IGF-I binding proteins (IGFBPs), showed a similar inhibition of saturable (125)I-IGF-I binding, but it was more potent than IGF-I in stimulating PRL and inhibiting GH release. These results are the first to localize IGF-I receptors to PRL cells, correlate IGF-I binding to its efficacy in regulating GH and PRL secretion, as well as demonstrate that IGFBPs may play a significant role in modulating the disparate actions of IGF-I on PRL and GH secretion.