Transgenic Zebrafish Expressing Rat Cytochrome P450 2E1 (CYP2E1): Augmentation of Acetaminophen-Induced Toxicity in the Liver and Retina.

Metabolic activation is the primary cause of chemical toxicity including hepatotoxicity. Cytochrome P450 2E (CYP2E) is involved in this process for many hepatotoxicants, including acetaminophen (APAP), one of the most common analgesics and antipyretics. Although the zebrafish is now used as a model for toxicology and toxicity tests, the CYP2E homologue in zebrafish has not been identified yet. In this study, we prepared transgenic zebrafish embryos/larvae expressing rat CYP2E1 and enhanced green fluorescent protein (EGFP) using a ß-actin promoter. Rat CYP2E1 activity was confirmed by the fluorescence of 7-hydroxycoumarin (7-HC), a metabolite of 7-methoxycoumarin that was specific for CYP2 in transgenic larvae with EGFP fluorescence (EGFP [+]) but not in transgenic larvae without EGFP fluorescence (EGFP [-]). APAP (2.5 mM) caused reduction in the size of the retina in EGFP [+] larvae but not in EGFP [-] larvae, while APAP similarly reduced pigmentation in both larvae. APAP at even 1 mM reduced the liver size in EGFP [+] larvae but not in EGFP [-] larvae. APAP-induced reduction of liver size was inhibited by N-acetylcysteine. These results suggest that rat CYP2E1 is involved in some APAP-induced toxicological endpoints in the retina and liver but not in melanogenesis of the developing zebrafish.

Intravenous administration of xenin-25 accelerates cyclic ruminal contractions in healthy conscious sheep.

The present study aimed to determine the effect and mode of action of the intravenous injection of xenin-25 on cyclic contractions of the rumen in healthy conscious sheep and mode of its action. Clinically healthy male sheep were equipped with a rumen cannula by surgery under anesthesia, and ruminal contractions were recorded with manometry in conscious animals after the recovery period. Intravenous xenin-25 injection induced a cluster of premature ruminal phasic contractions in a dose-dependent manner between 0.03 and 1 nmol/kg, and the change at the highest dose was statistically significant. In contrast, intravenous neurotensin injection inhibited the amplitude of cyclic rumen contractions. The xenin-25 effect was not significantly altered by prior injection of the neurotensin receptor subtype-1 antagonist SR 48692 at 30 and 100 nmol/kg. After euthanasia the ruminal muscles were excised for in vitro experiments. A single xenin-25 application (0.3-10 µM) to the longitudinal and circular muscle strips of the rumen did not induce any change in tension or electric field stimulation-induced phasic contractions of the muscle strips. These results demonstrated that circulating xenin-25 stimulates rumen contractions by acting on sites except the intramural intrinsic nerve plexus or smooth muscles of the rumen, implying that xenin-25 acts on the gastric center and/or cholinergic efferent nerve innervated to the ovine rumen.

Neurotensin and xenin stimulates pancreatic exocrine secretion through the peripheral cholinergic nerves in conscious sheep.

The present study aimed to clarify the effects of neurotensin and xenin on pancreatic exocrine secretion in conscious sheep and their mechanism of actions. The animals were equipped with two silastic cannulae in the common bile duct to separately collect pancreatic fluid and bile, and a silastic cannula in the proximal duodenum to continuously return the mixed fluids. NT and xenin were intravenously injected at range of 0.01-3.0 nmol/kg during the phase I of duodenal migrating motor complex. A single intravenous NT injection significantly and dose-dependently increased pancreatic fluid, protein, and bicarbonate outputs. The effect of NT at 1 nmol/kg was completely inhibited by a background intravenous infusion of atropine methyl nitrate at a dose of 10 nmol/kg/min, however, the effect was not altered by a prior injection of the neurotensin receptor subtype (NTR)-1 antagonist SR 48692 at 60 nmol/kg. Moreover, a single intravenous xenin-25 injection significantly and dose-dependently increased pancreatic fluid and protein output, whereas the effect of xenin-25 did not clearly show dose-dependence. The prior SR 48692 injection at 30 nmol/kg did not significantly alter the effects of xenin-25 at 0.3 nmol/kg, while the atropine infusion significantly inhibited the increase in fluid secretion. Under the atropine infusion, xenin-25 at 0.3 nmol/kg did not increase protein and bicarbonate outputs, whereas the inhibitory effect of the atropine was not significant compared to that of the single injection of xenin-25. A single intravenous injection of NTR-2 agonist levocabastine at 0.1-3 nmol/kg did not alter pancreatic exocrine secretion. These results suggest that both NT and xenin-25 effectively stimulates pancreatic exocrine secretion through the peripheral cholinergic system in sheep and that NTR-2 is not involved in the regulation of pancreatic exocrine secretion, however, we did not precisely determine the role of NTR-1 in the actions of both the peptides on pancreatic exocrine secretion.

Effects of xenin-25 on insulin and glucagon secretions in healthy conscious sheep.

The aim of present study was to determine effect of an intravenous injection of xenin-25 on insulin and glucagon secretion in healthy conscious sheep. After feeding once at 17:00, the experiment was started from 9:00 on the next day. Xenin-25 was intravenously (i.v.) injected at a dose of 100 to 1000 pmol/kg with and without the simultaneous injection of glucose at a dose of 200 µmol/kg, and blood was withdrawn before and after the injections. A single xenin-25 injection at 100 and 300 pmol/kg significantly increased the plasma insulin concentration, whereas the 1000 pmol/kg dose did not elicit significantly enhanced insulin response. Plasma glucose and glucagon concentrations did not significantly change after a single xenin-25 injection. Xenin-25 injection significantly and dose-dependently augmented the glucose-induced insulin secretion. However, the changes in the plasma glucose and glucagon level after the glucose injection were not altered by xenin injection. A prior intravenous injection of the neurotensin receptor subtype-1 (NTR-1) antagonist SR 48692 at 100 nmol/kg did not modify the glucose-induced change in plasma insulin caused by xenin-25 at 300 pmol/kg, and intravenous injection of the NTR-2 agonist levocabastine at 1000 pmol/kg did not augment the insulin response to the glucose injection. On the other hand, no xenin-25 immunopositive cells were detected in the ovine pancreas. The mRNAs of the three NTR subtypes were highly expressed in the ovine pancreas in comparison with the expression in the abomasum. These results suggest that xenin-25 released from the upper gastrointestinal tract plays a role of an insulinotropic factor in sheep, possibly through NTRs in the pancreatic islets, but not via NTR-2.

Characterization of feline cytochrome P450 2B6.

1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

Assessment of testicular corticosterone biosynthesis in adult male rats.

Corticosterone is synthesized in the adrenal glands and is circulated throughout the body to perform regulatory functions in various tissues. The testis is known to synthesize and secrete testosterone and other androgens. We developed an accurate method to measure steroid content using liquid chromatography-mass spectrometry analysis. In the present study, significant levels of the precursor compounds of testosterone and corticosterone synthesis could be detected in rat testis using this method. After adrenalectomy, corticosterone remained in the blood and testicular tissue at approximately 1% of the amount present in the control testis. When the excised testicular tissue was washed and incubated with NADH, NADPH and progesterone, not only testosterone and its precursors but also 11-deoxycorticosterone and corticosterone were produced; the levels of 11-deoxycorticosterone and corticosterone increased with incubation time. The production rate of 11-deoxycorticosterone from progesterone was estimated to be approximately 1/20 that of 17-hydroxyprogesterone, and the corticosterone level was approximately 1/10 that of testosterone. These ratios coincided with those in the testicular tissue of the adrenalectomized rats, indicating that corticosterone was synthesized in the testis and not in the blood. A primary finding of this study was that corticosterone and testosterone were synthesized in a 1/10-20 ratio in the testis. It is concluded that corticosterone, which has various functions, such as the regulation of glycolysis and mediating spermatogenesis, is produced locally in the testis and that this the local production is convenient and functional to respond to local needs.

Identification and functional characterization of novel feline cytochrome P450 2A.

1. Cytochrome P450s are the major metabolizing enzymes for xenobiotics in humans and other mammals. Although the domestic cat Felis catus, an obligate carnivore, is the most common companion animal, the properties of cytochrome P450 subfamilies are largely unknown. 2. We newly identified the feline CYP2A13, which consists of 494 deduced amino acids, showing the highest identity to CYP2As of dogs, followed by those of pigs, cattle and humans. 3. The feline CYP2A13 transcript and protein were expressed almost exclusively in the liver without particular sex-dependent differences. 4. The feline CYP2A13 protein heterogeneously expressed in Escherichia coli showed metabolic activity similar to those of human and canine CYP2As for coumarin, 7-ethoxycoumarin and nicotine. 5. The results indicate the importance of CYP2A13 in systemic metabolism of xenobiotics in cats.

Tachykinin: recent developments and novel roles in health and disease.

Over 80 years has passed since the discovery of substance P (SP), and a variety of peptides of the tachykinin (TK) family have been found and investigated. SP, neurokinin A (NKA), and neurokinin B (NKB) are representative peptides in mammalian species. SP and NKA are major excitatory neurotransmitters in the peripheral nervous system, while NKB is primarily involved in the central nervous system (CNS). Moreover, TK peptides play roles not only as neurotransmitters but also as local factors and are involved in almost all aspects of the regulation of physiological functions and pathophysiological processes. The role of SP as a mediator of pain processing and inflammation in peripheral tissues in coordination with transient receptor potential channels is well established, while novel aspects of TKs in relation to hematopoiesis, venous thromboembolism, tendinopathy, and taste perception have been clarified. In the CNS, the NKB signaling system in the hypothalamus has been shown to play a crucial role in the regulation of gonadotropin hormone secretion and the onset of puberty, and molecular biological studies have elucidated novel prophylaxic activities of TKs against neurogenic movement disorders based on their molecular structure. This review provides an overview of the novel aspects of TKs reported around the world in the last 5 years, with particular focus on nociception, inflammation, hemopoiesis, gonadotropin secretion, and CNS diseases.

Fatty acid-binding protein expression in the gastrointestinal tract of calves and cows.

Fatty acid-binding protein (FABP) has high affinity for long-chain fatty acids and appears to participate in the metabolism and intracellular transport of lipids. Liver- and intestinal-type FABP (L-FABP and I-FABP, respectively) are expressed in the small intestine. However, in the gastrointestinal tract of ruminants, expression and localization of FABPs are unknown. In this study, we investigated the expression of I-FABP and L-FABP in the gastrointestinal tract of cattle. I- and L-FABP had higher messenger RNA (mRNA) and protein expression levels in the duodenum and jejunum relatively to other gastrointestinal regions in both calves and cows. Furthermore, L-FABP mRNA and protein expression were high in the colon. Both these protein types were confirmed to be in the cytosol of jejunal epithelial cells, where they were found in the villi rather than in the crypts. We concluded that duodenal and jejunal FABPs might be involved in the metabolism of fatty acids mainly in epithelial cells in cattle.

Role of tachykinins and neurokinin receptor subtypes in the regulation of motility of the forestomach and abomasum in conscious sheep.

The present study was planned to evaluate role of tachykinins (TKs) and neurokinin (NK) receptors in the regulation of gastric motility in sheep. We examined the effects of intravenous (i.v.) injection of neurokinin A (NKA) and substance P (SP) on motility of the rumen, omasum, and abomasum in conscious sheep and the effects of NK receptor blockade on the effect of TKs using NK-1 receptor antagonist L-732,138 and NK-2 receptor antagonist SR48968. Moreover, the effect of NK receptor blockade on omasal cyclic contractions was examined. Intravenous injection of NKA and SP induced tonic contraction of rumen, omasum, and abomasum, and the contractile effect of NKA was more potent than that of SP in all the gastric regions. Although the effect of SP was not inhibited by L-732,138, the effect of NKA was significantly inhibited by SR48968. However, single infusion of SR48968 and L-732,138 did not alter cyclic electromyographic activity and basal intraluminal pressure in the omasum. These results imply that NKA and NK-2 receptors play a primary role in non-cholinergic regulation of ovine gastric motility, though NK-2 and NK-1 receptors seem unlikely to be involved in the physiological regulation of omasal cyclic contractions.

Role of tachykinin and neurokinin receptors in the regulation of ovine omasal contractions.

The present study investigated a role of tachykinins (TK) and neurokinin (NK) receptors (NK-R) in the non-cholinergic regulation of omasal contractions in sheep. Semiquantitative reverse transcription (RT)-PCR revealed that both preprotachykinin (PPT)-A and PPT-B mRNA were distributed in the omasal muscle layers and that NK-R type-1 (NK-1R) and type-2 (NK-2R) mRNA were largely expressed in the same tissues. Cumulative application of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) at 0.03-10µM induced tonic contractions of omasal muscle strips, and the contractile amplitude increased in order of NKB

Role of nitrergic nerves in the regulation of motility of the omasum and abomasum in healthy sheep (Ovis aries).

We examined the physiological role of nitrergic nerves in the regulation of omasal and abomasal motility in conscious healthy sheep and omasal muscle specimens. Nitric oxide (NO)-donor, S-nitroso-acethyl-dl-penicillamine (SNAP, 3-30 nmol/kg per min, i.v.) significantly inhibited omasal electromyographic (EMG) activity, whereas it did not alter EMG activity in the abomasal antrum. However, NO synthase inhibitor, Nomega-nitro-l-arginine-methyl ester (L-NAME, 0.3-3.0 micromol/kg per min, i.v.) did not alter EMG activity of the omasum and abomasum. In the in vitro experiments, SNAP application (6-200 micromol/l) significantly inhibited bethanechol (10 micromol/l)-induced contraction of longitudinal and circular muscles of the omasum. L-NAME application (0.03-3.0 mmol/l) enhanced electric field stimulation-induced contractions of the circular muscles. The results suggest that the omasal muscles are responsive to exogenous NO and that nitrergic nerves innervate the circular muscle layer of the omasum, however, nitrergic nerves are not or scarcely involved the physiological regulation of omasal and possibly abomasal motility in healthy sheep.

Localization of CCK-1R in the omasum and role of CCK in the regulation of omasal contractions in sheep.

The present study examined localization of cholecystokinin receptor (CCK-R) mRNA in the muscle layer of the ovine omasum and role of CCK-R type 1 (CCK-1R) in the regulation of muscle contraction of the omasum. We demonstrated that not only CCK-R type 2 (CCK-2R) mRNA but also CCK-1R mRNA is highly expressed in the muscle layer of the ovine omasum. Application of CCK-8 to muscle strips of the greater curvature of the ovine omasum at 1-100 nM induced tonic contraction in a concentration-dependent manner, and the contractile effect of CCK-8 was inhibited by both CCK-1R antagonist lorglumide (IC(50) 2.7 and 7.9 microM in the longitudinal and circular muscle, respectively) and CCK-2R antagonist PD135,158 (IC(50) 51.4 microM in the longitudinal muscle), indicating that not only CCK-2R but also CCK-1R is functionally expressed in the plasma membrane of smooth muscles in the omasum and mediates action of exogenous CCK. Contractile effect of intravenous infusion of CCK-8 (1-30 pmol/kg/min) on omasal contraction was also confirmed in the in vivo experiments using conscious sheep in the absence and presence of atropine infusion (14.4 nmol/kg/min), and showed that circulating CCK increases omasal electromyographic (EMG) activity at lower plasma concentration than that it inhibits ruminal contractions. Taking account of our previous results in the in vivo study using other CCK-1R antagonist, it is suggested that circulating CCK, even at normal range of plasma concentration, plays a physiological role as a regulator of omasal contractions in sheep and CCK-1R mediates the action of CCK.

Localization and secretion of epidermal growth factor in the parotid gland and its intragastric kinetics in sheep.

Ruminants secrete a large quantity of saliva that is rich in electrolytes; however, it remains unclear whether their parotid saliva contains epidermal growth factor (EGF). The present study was set up to examine the distribution of EGF and transforming growth factor-alpha (TGF-alpha) in the ovine parotid and submandibular glands and the salivary secretion of EGF-like binding activity (EGF-LBA) as the sum of EGF and TGF-alpha in conscious sheep. We also measured changes in the intragastric concentration of EGF-LBA in the ovine rumen and abomasum, and examined the effect of bilateral diversion of parotid saliva on intragastric EGF-LBA concentration in sheep. Both the ovine parotid and, to a lesser extent, the submandibular glands contained EGF-LBA. Immunohistochemical study showed that EGF and TGF-alpha-immunoreactivities were localized in the ductal epithelium in both glands. Transcriptional expression of EGF and TGF-alpha mRNA was demonstrated in both glands by reverse transcription-polymerase chain reaction (RT-PCR). In conscious sheep, the parotid gland continuously secreted EGF-LBA in the saliva before feeding, and the secretion of parotid EGF-LBA was markedly increased during feeding. After diversion of the parotid saliva for 1 week, EGF-LBA concentration in the ruminal fluid, but not in the abomasal fluid, decreased in the postprandial period, indicating that parotid EGF-LBA is a primary source of EGF-LBA for the rumen fluid during the postprandial period in sheep. Moreover, RT-PCR detected the expression of TGF-alpha mRNA in the rumen and abomasum and that of EGF in the abomasum, implying that these stomachs possibly supply, in part, EGF-LBA to the luminal fluid.

Multiple regulation of peptide YY secretion in the digestive tract.

In the last two decades, multiple aspects of the peptide YY (PYY) secretion have been investigated. Besides fat and fatty acids, many luminal nutrients in the distal intestine appear to induce PYY release. Some studies have shown that bile acid, but not nutrients, plays a crucial role in the regulation of PYY secretion. Moreover, chyme in the proximal intestine also regulates the peptide release by indirect action through humoral and neuronal factors. Gastrin, cholecystokinin, and the vagus nerve are major candidates for mediators of these indirect actions. Several growth factors have been shown to regulate PYY synthesis in mucosa of the distal intestine. This review is aimed at presenting an overview of these recent studies on PYY secretion in the distal intestine.