Does ghrelin regulate intestinal motility in rabbits? An in vitro study using isolated duodenal strips.

Rabbit duodenum has been used for examining the ability of motilin to cause muscle contraction in vitro. A motilin-related peptide, ghrelin, is known to be involved in the regulation of gastrointestinal (GI) motility in various animals, but its ability to cause rabbit GI contraction have not been well examined. The aim of this study is to clarify the action of rat ghrelin and its interaction with motilin in the rabbit duodenum. The mRNA expression of ghrelin and motilin receptors was also examined using RT-PCR. Rat ghrelin (10-9-10-6 M) did not change the contractile activity of the duodenum measured by the mean muscle tonus and area under the curve of contraction waves. In agreement with this result, the distribution of ghrelin receptor mRNA in the rabbit GI tract varied depending on the GI region from which the samples were taken; the expression level in the duodenum was negligible, but that in the esophagus or stomach was significant. On the other hand, motilin (10-10-10-6 M) caused a concentration-dependent contraction by means of increased mean muscle tonus, and consistently, motilin receptor mRNA was expressed heterogeneously depending on the GI region (esophagus = stomach = colon = rectum < duodenum = jejunum = ileum < cecum). Expression level of motilin receptor was comparable to that of ghrelin receptor in the esophagus and stomach. Pretreatment with ghrelin (10-6 M) prior to motilin did not affect the contractile activity of motilin in the duodenum. In conclusion, ghrelin does not affect muscle contractility or motilin-induced contraction in the rabbit duodenum, which is due to the lack of ghrelin receptors. The present in vitro results suggest that ghrelin might not be a regulator of intestinal motility in rabbits.

Kinematic characteristics of canine hindlimb movement during sit-to-stand and stand-to-sit motions.

Sit-to-stand and stand-to-sit motions are basic motions for daily animal life, and these motions are used as therapeutic exercises for dogs with functional impairments. The sit-to-stand motion is divided into several phases for kinesiological assessment in human rehabilitation and physical therapy. However, these motions in dogs have not been characterized in detail. We examined canine hindlimb kinematic characteristics during sit-to-stand/stand-to-sit motions and compared the characteristics with those during walking. In addition, we tried to classify phases of the movements based on kinematic characteristics of the transition of the range of motion of the hindlimb. We used a three-dimensional motion analysis system to evaluate the motions of eight clinically healthy beagles. During the sit-to-stand motion, the total range of motion (ROM) in the hip joint flexion/extension was half of that of during walking, but the total ROM of the hindlimb external/internal rotation relative to the pelvis and flexion/extension of the stifle and the tarsal joints were significantly larger than those of walking, suggesting that sit-to-stand exercise causes movements of hindlimb joints without marked changes in hip joint flexion/extension movement. Both sit-to-stand and stand-to-sit motions could not be divided into multiple phases only by the transition of the range of motion of the hindlimb.

Motilin is a regulator of gastric contraction in Japanese fire belly newts (Cynops pyrrhogaster), in vitro studies using isolated gastrointestinal strips of newts, rabbits, and chickens.

The effects of newt motilin on the contractility of the isolated gastrointestinal (GI) tract from Japanese fire belly newts (newt) were examined to clarify whether motilin regulates GI motility in urodele amphibians. In addition, contractile responsiveness to motilins from seven species of vertebrates (human, chicken, turtle, alligator, axolotol, newt and zebrafish) were compared in GI preparations from three different animals (rabbit duodenum, chicken ileum and newt stomach) to determine the species-specific action of motilin. Newt motilin (10-10 M - 10-6 M) caused a contraction of cognate gastric strips, while the upper, middle, and lower intestinal strips were insensitive. The rank order of motilins for contractile activity in newt gastric strips was newt > alligator > axolotol > chicken > turtle > human ≫ zebrafish. On the other hand, newt motilin caused a weak contraction in the rabbit duodenum (human > alligator = chicken > turtle > newt ≧ axolotol > zebrafish), and it was ineffective in the chicken ileum (chicken > turtle > alligator > human ≫ newt, axolotol and zebrafish). This study demonstrates that motilin induces contraction in the GI tract of a urodele amphibian, the newt, in a region (stomach)-specific manner and further indicates that a ligand-receptor interaction of the motilin system is a species-specific manner probably due to differences in the amino acid sequence of motilin.

Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido, Japan.

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006-2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.

Pheasant motilin, its distribution and gastrointestinal contractility-stimulating action in the pheasant.

Previously, pheasant motilin was identified as a 22-amino acid peptide with a sequence of FVPFFTQSDI QKMQEKERIK GQ. In the present study, the distribution of pheasant motilin mRNA was determined and compared with that of ghrelin, a motilin-related peptide. The effects of pheasant motilin on the cognate gastrointestinal (GI) muscle strips were also examined in an in vitro contraction study. The expression of pheasant motilin mRNA was highest in the small intestine (duodenum, jejunum and ileum), moderate in the colon and very low in the brain, lung, heart, pancreas, esophagus, proventriculus, gizzard and caecum, and this distribution was in contrast with that of ghrelin mRNA. Pheasant motilin caused contraction of the cognate GI tract in a region-dependent manner, similar to chicken motilin. The contraction in the small intestine was large and was not affected by atropine. In contrast, contraction in the proventriculus was small and was decreased by atropine. The crop and colon were insensitive to pheasant motilin. Neither GM109 nor MA2029, mammalian motilin receptor antagonists inhibited the contractions of pheasant motilin. Erythromycin was ineffective in the pheasant ileum, although it caused contraction of the rabbit duodenum. These results indicate that pheasant motilin caused contraction through an action on smooth muscles in the small intestine and an action on enteric cholinergic nerves in the proventriculus. This high responsiveness of the small intestine suggests that motilin is a regulator of small intestinal motility in avians, and the characteristic of the motilin receptor in the pheasant might be different from that in mammals, as is that in chickens.

Motilin Comparative Study: Structure, Distribution, Receptors, and Gastrointestinal Motility.

Motilin, produced in endocrine cells in the mucosa of the upper intestine, is an important regulator of gastrointestinal (GI) motility and mediates the phase III of interdigestive migrating motor complex (MMC) in the stomach of humans, dogs and house musk shrews through the specific motilin receptor (MLN-R). Motilin-induced MMC contributes to the maintenance of normal GI functions and transmits a hunger signal from the stomach to the brain. Motilin has been identified in various mammals, but the physiological roles of motilin in regulating GI motility in these mammals are well not understood due to inconsistencies between studies conducted on different species using a range of experimental conditions. Motilin orthologs have been identified in non-mammalian vertebrates, and the sequence of avian motilin is relatively close to that of mammals, but reptile, amphibian and fish motilins show distinctive different sequences. The MLN-R has also been identified in mammals and non-mammalian vertebrates, and can be divided into two main groups: mammal/bird/reptile/amphibian clade and fish clade. Almost 50 years have passed since discovery of motilin, here we reviewed the structure, distribution, receptor and the GI motility regulatory function of motilin in vertebrates from fish to mammals.

Oxidative stress inducers potentiate 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated pre-cardiac edema in larval zebrafish.

We reported the involvement of oxidative stress and prostaglandins including thromboxane and prostacyclin in pre-cardiac edema (early edema) caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While the involvement of oxidative stress in TCDD-induced toxicity has been frequently reported, the mechanism of its action is still unclear. In the present study, oxidative stress inducers including paraquat, hydrogen peroxide (H2O2) and rotenone augmented early edema (edema) induced by a low concentration of TCDD (0.1 ppb) at 55 hr post fertilization (hpf), while each of them alone did not cause edema. Edema caused by TCDD plus oxidative stress inducers was almost abolished by antioxidants, an antagonist for thromboxane receptor (ICI-192,605) and an agonist for prostacyclin receptor (beraprost), suggesting that the site of action of these inducers was in the regular signaling pathway after activation of aryl hydrocarbon receptor type 2 (AHR2) by TCDD. Oxidative stress inducers also enhanced edema caused by an agonist for the thromboxane receptor (U46619), and the enhancement was also inhibited by antioxidants. Sulforaphane and auranofin, activators of Nrf2 that is a master regulator of anti-oxidative response, did not affect U46619-evoked edema but almost abolished TCDD-induced edema and potentiation by paraquat in both TCDD- and U46619-induced edema. Taken together, the results suggest that oxidative stress augments pre-cardiac edema caused by TCDD via activation of thromboxane receptor-mediated signaling in developing zebrafish. As paraquat and other oxidative stress inducers used also are environmental pollutants, interaction between dioxin-like compounds and exogenous source of oxidative stress should also be considered.

Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice.

Parasympathetic signalling via muscarinic acetylcholine receptors (mAChRs) regulates gastrointestinal smooth muscle function. In most instances, the mAChR population in smooth muscle consists mainly of M2 and M3 subtypes in a roughly 80% to 20% mixture. Stimulation of these mAChRs triggers a complex array of biochemical and electrical events in the cell via associated G proteins, leading to smooth muscle contraction and facilitating gastrointestinal motility. Major signalling events induced by mAChRs include adenylyl cyclase inhibition, phosphoinositide hydrolysis, intracellular Ca2+ mobilisation, myofilament Ca2+ sensitisation, generation of non-selective cationic and chloride currents, K+ current modulation, inhibition or potentiation of voltage-dependent Ca2+ currents and membrane depolarisation. A lack of ligands with a high degree of receptor subtype selectivity and the frequent contribution of multiple receptor subtypes to responses in the same cell type have hampered studies on the signal transduction mechanisms and functions of individual mAChR subtypes. Therefore, novel strategies such as genetic manipulation are required to elucidate both the contributions of specific AChR subtypes to smooth muscle function and the underlying molecular mechanisms. In this article, we review recent studies on muscarinic function in gastrointestinal smooth muscle using mAChR subtype-knockout mice.

Functional assessment of the gluteus medius, cranial part of the biceps femoris, and vastus lateralis in Beagle dogs based on a novel gait phase classification.

In humans, walking analysis based on the gait phase classification has been used for interpretation of functional roles of different movements occurring at individual joints, and it is useful for establishing a rehabilitation plan. However, there have been few reports on canine gait phase classification, and this is one of the reasons for preventing progress in canine rehabilitation. In this study, we determined phases of the canine gait cycle (GC) on the basis of the phase classification for human gait. The canine GC was able to be divided into initial contact (IC) and the following 5 phases: loading response (LR), middle stance (MidSt), pre-swing (PSw), early swing (ESw), and late swing (LSw). Next, the hind limb joint angles of the hip, stifle and tarsal joints and results of surface electromyography of the gluteus medius (GM), cranial part of the biceps femoris (CBF) and vastus lateralis (VL) muscles in relation to the gait phases were analyzed. The activities of three muscles showed similar changes during walking. The muscle activities were high in the LR phase and then declined and reached a minimum in the PSw phase, but they increased and reached a peak in the LSw phase, which was followed by the LR phase. In conclusion, the multiphasic canine GC was developed by modification of the human model, and the GC phase-related changes in the muscle activity and joint angles suggested the functions of GM, CBF and VL muscles in walking.

Motilin- and ghrelin-induced contractions in isolated gastrointestinal strips from three species of frogs.

Ghrelin (GHRL) and motilin (MLN), gut peptides isolated from the mucosa of the stomach and duodenum, respectively, stimulate gastrointestinal (GI) motility in mammals and birds. However, the functions of MLN and GHRL in amphibian GI tracts have not been examined in detail. To clarify the regulation of GI motility by the two peptides, the effects of human MLN and rat GHRL on contractility of isolated GI strips from three species of frogs, the black-spotted pond frog (pond frog; Pelophylax nigromaculata), bullfrog (Lithobates catesbeiana) and Western clawed frog (Xenopus; Xenopus tropicalis), were examined in in vitro experiments. The GI tract of each frog was divided into the stomach, upper intestine, middle intestine and lower intestine. Human MLN caused contractions of the stomach in the pond frog and upper intestine in the bullfrog and Xenopus, but other GI regions were insensitive to human MLN. Erythromycin did not cause contraction of the upper intestine of the bullfrog and Xenopus. Rat GHRL did not cause contraction of the stomach and small intestines in the pond frog and bullfrog, but it caused a concentration-dependent contraction in the stomach and upper intestine of Xenopus, while des-acyl rat GHRL did not cause any contraction of them. In conclusion, human MLN caused the contraction of the stomach or upper intestine in the three species of frogs, but GHRL was effective only in the stomach and upper intestine of Xenopus. On the basis of these data, MLN but not GHRL causes the GI region-dependent contractions in the frogs.

Cytochrome P450 Expression and Chemical Metabolic Activity before Full Liver Development in Zebrafish.

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some cyp1s (1b1, 1c1, 1c2, 1d1) and cyp2r1 peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed cyp2y3, 2r1, and 3a65 transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-p-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4'-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4'-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

Blood vessels are primary targets for 2,3,7,8-tetrachlorodibenzo-p-dioxin in pre-cardiac edema formation in larval zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has adverse effects on the development and function of the heart in zebrafish eleutheroembryos (embryos and larvae). We previously reported that TCDD reduced blood flow in the mesencephalic vein of zebrafish eleutheroembryos long before inducing pericardial edema. In the present study, we compared early edema (pre-cardiac edema), reduction of deduced cardiac output and reduction of blood flow in the dorsal aorta and cardinal vein caused by TCDD. In the same group of eleutheroembryos, TCDD (1.0 ppb) caused pre-cardiac edema and circulation failure at the cardinal vein in the central trunk region with the similar time courses from 42 to 54 h post fertilization (hpf), while the same concentration of TCDD did not significantly affect aortic circulation in the central trunk region or cardiac output. The dependence of pre-cardiac edema on TCDD concentration (0-2.0 ppb) at 55 hpf correlated well with the dependence of blood flow through the cardinal vein on TCDD concentration. Several treatments that markedly inhibited TCDD-induced pre-cardiac edema such as knockdown of aryl hydrocarbon receptor nuclear translocator-1 (ARNT1) and treatment with ascorbic acid, an antioxidant, did not significantly prevent the reduction of cardiac output at 55 hpf caused by 2.0 ppb TCDD. TCDD caused hemorrhage and extravasation of Evans blue that was intravascularly injected with bovine serum albumin, suggesting an increase in endothelium permeability to serum protein induced by TCDD. The results suggest that the blood vessels are primary targets of TCDD in edema formation in larval zebrafish.

Identification of pheasant ghrelin and motilin and their actions on contractility of the isolated gastrointestinal tract.

Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.

Regulation of Gastrointestinal Motility by Motilin and Ghrelin in Vertebrates.

The energy balance of vertebrates is regulated by the difference in energy input and energy expenditure. Generally, most vertebrates obtain their energy from nutrients of foods through the gastrointestinal (GI) tract. Therefore, food intake and following food digestion, including motility of the GI tract, secretion and absorption, are crucial physiological events for energy homeostasis. GI motility changes depending on feeding, and GI motility is divided into fasting (interdigestive) and postprandial (digestive) contraction patterns. GI motility is controlled by contractility of smooth muscles of the GI tract, extrinsic and intrinsic neurons (motor and sensory) and some hormones. In mammals, ghrelin (GHRL) and motilin (MLN) stimulate appetite and GI motility and contribute to the regulation of energy homeostasis. GHRL and MLN are produced in the mucosal layer of the stomach and upper small intestine, respectively. GHRL is a multifunctional peptide and is involved in glucose metabolism, endocrine/exocrine functions and cardiovascular and reproductive functions, in addition to feeding and GI motility in mammals. On the other hand, the action of MLN is restricted and species such as rodentia, including mice and rats, lack MLN peptide and its receptor. From a phylogenetic point of view, GHRL and its receptor GHS-R1a have been identified in various vertebrates, and their structural features and various physiological functions have been revealed. On the other hand, MLN or MLN-like peptide (MLN-LP) and its receptors have been found only in some fish, birds and mammals. Here, we review the actions of GHRL and MLN with a focus on contractility of the GI tract of species from fish to mammals.

Transcriptional profiling of cytochrome P450 genes in the liver of adult zebrafish, Danio rerio.

Increasing use of zebrafish in biomedical, toxicological and developmental studies requires explicit knowledge of cytochrome P450 (CYP), given the central role of CYP in oxidative biotransformation of xenobiotics and many regulatory molecules. A full complement of CYP genes in zebrafish and their transcript expression during early development have already been examined. Here we established a comprehensive picture of CYP gene expression in the adult zebrafish liver using a RNA-seq technique. Transcriptional profiling of a full complement of CYP genes revealed that CYP2AD2, CYP3A65, CYP1A, CYP2P9 and CYP2Y3 are major CYP genes expressed in the adult zebrafish liver in both sexes. Quantitative real-time RT-PCR analysis for selected CYP genes further supported our RNA-seq data. There were significant sex differences in the transcript levels for CYP1A, CYP1B1, CYP1D1 and CYP2N13, with males having higher expression levels than those in females in all cases. A similar feature of gender-specific expression was observed for CYP2AD2 and CYP2P9, suggesting sex-specific regulation of constitutive expression of some CYP genes in the adult zebrafish liver. The present study revealed several "orphan" CYP genes as dominant isozymes at transcript levels in the adult zebrafish liver, implying crucial roles of these CYP genes in liver physiology and drug metabolism. The current results establish a foundation for studies with zebrafish in drug discovery and toxicology.

Genetic diversity of cytochrome P450 1A2 with different metabolic activities in domestic cats.

Knowledge of genetic polymorphisms of metabolizing enzymes of medical drugs and xenobiotics including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP1A2, one of the major CYP isoforms in the feline liver. Wild-type and three non-synonymous polymorphic variants, but no synonymous variant, were identified in feline CYP1A2 in 50 alleles of domestic cats in Japan. Metabolic parameters, Km and Vmax, of ethoxyresorufin hydroxylation by CYP1A2 were shown to range within two times for identified non-synonymous variants by using a heterologous coexpression system. The results confirmed the polymorphic nature of CYP1A2 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats as well as for hereditary disorders.

Genetic diversity of cytochrome P450 2A with different metabolic activities in domestic cats.

Knowledge of genetic polymorphisms of cytochrome P450 (CYP), the most important xenobiotic metabolizing enzyme, is very limited in cats. Preliminarily, we investigated genetic polymorphisms in CYP2A13, one of the major CYP isoforms in the liver and lung. Four synonymous and three non-synonymous polymorphic variants were identified in feline CYP2A13 in domestic cats in Japan, without an obvious major type. Metabolic parameters, Km and Vmax, of coumarin hydroxylation of CYP2A13 were shown to range within two times for the identified non-synonymous polymorphic variants by using heterologous coexpression system in Escherichia coli. The results confirmed the polymorphic nature of CYP2A13 as a basis for effective application of medicines and prevention of adverse reactions in treatment of domestic cats.

Genetic diversity of cytochrome P450 3A with different metabolic activity in domestic cats.

Knowledge on genetic polymorphisms of metabolising enzymes including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP3A131, one of the major CYP isoforms in the feline liver and small intestine. Eight non-synonymous variants and one synonymous variant of feline CYP3A131 were identified in 29 cats. A major non-synonymous type was not observed. Metabolic parameters (Km and Vmax) of dibenzylfluorescein hydroxylation were ranged within about 2 times for the identified non-synonymous variants by using a heterologous coexpression system of CYP3A131 and feline cytochrome P450 reductase in Escherichia coli. The results confirmed the polymorphic nature of CYP3A131 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats.

A verification study of gastrointestinal motility-stimulating action of guinea-pig motilin using isolated gastrointestinal strips from rabbits and guinea-pigs.

Motilin (MLN), a 22-amino-acid peptide hormone, is generally present in the mucosa of the upper gastrointestinal (GI) tract, mainly the duodenum of mammals, and it regulates GI motility, especially that related to interdigestive migrating contraction. However, MLN and its receptor are absent in mice and rats, and MLN does not cause any mechanical responses in the rat and mouse GI tracts. The guinea-pig is also a rodent, but expression of the MLN gene in the guinea-pig has been reported. In the present study, two guinea-pig MLNs, FIPIFTYSELRRTQEREQNKGL found in the Ensemble Genome Database (gpMLN-1) and FVPIFTYSELRRTQEREQNKRL reported by Xu et al. (2001) (gpMLN-2), were synthesized, and their biological activities were evaluated in the rabbit duodenum and guinea-pig GI tract in vitro. Both gpMLNs showed contractile activity in longitudinal muscle strips of the rabbit duodenum. The EC50 values of gpMLN-1 and gpMLN-2 were slightly higher than that of human MLN (hMLN), but the maximum contractions were as same as that of hMLN. Treatment with GM109 and hMLN-induced receptor desensitization decreased the contractile activity of both gpMLNs, indicating that the two gpMLN candidates are able to activate the MLN receptor (MLN-R) of the rabbit duodenum. In guinea-pig GI preparations, hMLN and gpMLNs did not show any mechanical responses in circular muscle strips from the gastric antrum or in longitudinal strips of the duodenum, ileum and colon although acetylcholine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) caused definite mechanical responses. The DMPP-induced neural responses in the gastric circular muscle and ileal longitudinal muscles were not modified by gpMLN-1. Even in the gastric and ileal strips with intact mucosa, no mechanical responses were seen with either of the gpMLNs. Furthermore, RT-PCR using various primer sets failed to amplify the gpMLN-2 mRNA. In conclusion, gpMLNs including one that was already reported and the other that was newly found in a database were effective to the rabbit MLN-R, whereas they did not cause any contractions or modification of neural responses in the guinea-pig GI tract, indicating that the MLN system is vestigial and not functional in regulation of GI motility in the guinea-pig as well as in other rodents such as rats and mice.

Limited expression of functional cytochrome p450 2c subtypes in the liver and small intestine of domestic cats.

1. Compared to information for herbivores and omnivores, knowledge on xenobiotic metabolism in carnivores is limited. The cytochrome P450 2C (CYP2C) subfamily is recognized as one of the most important CYP groups in human and dog. We identified and characterized CYP2C isoforms and variants in cat, which is an obligate carnivore. 2. Quantitative RT-PCR and immunoblot analyses were carried out to evaluate the expression of CYP2C in the liver and small intestine. A functional CYP2C isoform was heterologously expressed in yeast microsomes to determine the enzymatic activity. 3. Cat had two CYP2C genes, 21 and 41, in the genome; however, CYP2C21P was a pseudogene that had many stop codons. Three splicing variants of CYP2C41 were identified (v1-v3), but only one of them (v1) showed a complete deduced amino acid sequence as CYP2C protein. Transcripts of feline CYP2C41v1 were detected but the amounts were negligible or very small in the liver and small intestine. Immunoreactivity to an antihuman CYP2C antibody was confirmed in the recombinant feline CYP2C41v1 but not in the feline liver. 4. Recombinant feline CYP2C41v1 metabolized several substrates, including dibenzylfluorescein that is specific to human CYP2C. 5. The results suggest a limited role of functional CYP2C isoforms in xenobiotic metabolism in cat.