Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes.

Tree shrews (Tupaia belangeri) are a non-rodent primate-like species sometimes used for biomedical research involving hepatitis virus infections and toxicology. Genome analysis has indicated similarities between tree shrews and humans in the numbers of cytochromes P450 (P450 or CYP), which constitute a family of important drug-metabolizing enzymes; however, P450s have not been fully investigated in tree shrews. In this study, we identified CYP1A1, CYP1A2, CYP1B1, and CYP1D1 cDNAs from tree shrew liver and compared their characteristics with dog, pig, and human CYP1As. The deduced amino acid sequences of tree shrew CYP1s were highly identical (82-87 %) to human CYP1s. In tree shrews, CYP1A1 and CYP1A2 mRNAs were preferentially expressed in liver, whereas CYP1D1 mRNA was preferentially expressed in kidney and lung. In contrast, CYP1B1 mRNA was expressed in various tissues, with the most abundant expression in spleen. Among the tree shrew CYP1 mRNAs, CYP1A2 mRNA was most abundant in liver, and CYP1B1 mRNA was most abundant in kidney, small intestine, and lung. All tree shrew CYP1 proteins heterologously expressed in Escherichia coli catalyzed caffeine and estradiol in a similar manner to tree shrew liver microsomes and human, dog, and pig CYP1 proteins. These results suggest that tree shrew CYP1A1, CYP1A2, CYP1B1, and CYP1D1 genes, different form human pseudogene CYP1D1P, are expressed in liver, small intestine, lung, and/or kidney and encode functional drug-metabolizing enzymes important in toxicology.

Evidence of micro-evolution in Crocidura russula from two abandoned heavy metal mines: potential use of Cytb, CYP1A1, and p53 as gene biomarkers.

Heavy metals accumulated in the environment due to the mining industry may impact on the health of exposed wild animals with consequences at the population level via survival and selection of the most resistant individuals. The detection and quantification of shifts in gene frequencies or in the genetic structure in populations inhabiting polluted sites may be used as early indicators of environmental stress and reveal potential 'candidate gene biomarkers' for environmental health assessment. We had previously observed that specimens of the Greater white-toothed shrew (Crocidura russula) from two heavy metal mines in Southern Portugal (the Aljustrel and the Preguiça mines) carried physiological alterations compared to shrews from an unpolluted site. Here, we further investigated whether these populations showed genetic differences in genes relevant for physiological homeostasis and/or that are associated with pathways altered in animals living under chronic exposure to pollution, and which could be used as biomarkers. We analysed the mitochondrial cytochrome b (Cytb) gene and intronic and/or exonic regions of four nuclear genes: CYP1A1, LCAT, PRPF31, and p53. We observed (1) population differences in allele frequencies, types of variation, and diversity parameters in the Cytb, CYP1A1, and p53 genes; (2) purifying selection of Cytb in the mine populations; (3) genetic differentiation of the two mine populations from the reference by the p53 gene. Adding to our previous observations with Mus spretus, we provide unequivocal evidence of a population effect exerted by the contaminated environment of the mines on the local species of small mammals.

Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew.

The function of the RIG-I-like receptors (RLRs; including RIG-I, MDA5, and LGP2) as key cytoplasmic sensors of viral pathogen-associated molecular patterns (PAMPs) has been subjected to numerous pathogenic challenges and has undergone a dynamic evolution. We found evolutionary evidence that RIG-I was lost in the Chinese tree shrew lineage. Along with the loss of RIG-I, both MDA5 (tMDA5) and LGP2 (tLGP2) have undergone strong positive selection in the tree shrew. tMDA5 or tMDA5/tLGP2 could sense Sendai virus (an RNA virus posed as a RIG-I agonist) for inducing type I IFN, although conventional RIG-I and MDA5 were thought to recognize distinct RNA structures and viruses. tMDA5 interacted with adaptor tMITA (STINGTMEM173/ERIS), which was reported to bind only with RIG-I. The positively selected sites in tMDA5 endowed the substitute function for the lost RIG-I. These findings provided insights into the adaptation and functional diversity of innate antiviral activity in vertebrates.

Solar radiation during rewarming from torpor in elephant shrews: supplementation or substitution of endogenous heat production?

Many small mammals bask in the sun during rewarming from heterothermy, but the implications of this behaviour for their energy balance remain little understood. Specifically, it remains unclear whether solar radiation supplements endogenous metabolic thermogenesis (i.e., rewarming occurs through the additive effects of internally-produced and external heat), or whether solar radiation reduces the energy required to rewarm by substituting (i.e, replacing) metabolic heat production. To address this question, we examined patterns of torpor and rewarming rates in eastern rock elephant shrews (Elephantulus myurus) housed in outdoor cages with access to either natural levels of solar radiation or levels that were experimentally reduced by means of shade cloth. We also tested whether acclimation to solar radiation availability was manifested via phenotypic flexibility in basal metabolic rate (BMR), non-shivering thermogenesis (NST) capacity and/or summit metabolism (Msum). Rewarming rates varied significantly among treatments, with elephant shrews experiencing natural solar radiation levels rewarming faster than conspecifics experiencing solar radiation levels equivalent to approximately 20% or 40% of natural levels. BMR differed significantly between individuals experiencing natural levels of solar radiation and conspecifics experiencing approximately 20% of natural levels, but no between-treatment difference was evident for NST capacity or Msum. The positive relationship between solar radiation availability and rewarming rate, together with the absence of acclimation in maximum non-shivering and total heat production capacities, suggests that under the conditions of this study solar radiation supplemented rather than substituted metabolic thermogenesis as a source of heat during rewarming from heterothermy.

Neuropeptide Y influences acute food intake and energy status affects NPY immunoreactivity in the female musk shrew (Suncus murinus).

Neuropeptide Y (NPY) stimulates feeding, depresses sexual behavior, and its expression in the brain is modulated by energetic status. We examined the role of NPY in female musk shrews, a species with high energetic and reproductive demands; they store little fat, and small changes in energy can rapidly diminish or enhance sexual receptivity. Intracerebroventricular infusion of NPY enhanced acute food intake in shrews; however, NPY had little affect on sexual receptivity. The distribution of NPY immunoreactivity in the female musk shrew brain was unremarkable, but energy status differentially affected NPY immunoreactivity in several regions. Similar to what has been noted in other species, NPY immunoreactivity was less dense in brains of ad libitum shrews and greater in shrews subjected to food restriction. In two midbrain regions, both of which contain high levels of gonadotropin releasing hormone II (GnRH II), which has anorexigenic actions in shrews, NPY immunoreactivity was more sensitive to changes in food intake. In these regions, acute re-feeding (90-180 min) after food restriction reduced NPY immunoreactivity to levels noted in ad libitum shrews. We hypothesize that interactions between NPY and GnRH II maintain energy homeostasis and reproduction in the musk shrew.

Bioaccumulation of lead, mercury, and cadmium in the greater white-toothed shrew, Crocidura russula, from the Ebro Delta (NE Spain): sex- and age-dependent variation.

We quantified bioaccumulation of lead, mercury, and cadmium in bones from 105 greater white-toothed shrews (Crocidura russula) collected at the Ebro Delta, a polluted area, and the Medas Islands, a control site. Lead and mercury levels varied with site, age, and sex, although statistical significances depended on each factor. Globally, shrews from the polluted area exhibited significantly higher concentrations of Pb and Hg. Increment of Pb with age was particularly remarkable in wetland animals and was interpreted in relation to human activities, namely hunting. Unlike males, females from the Ebro Delta maintained low Hg levels, which were associated with gestation and lactation. Cadmium levels did not differ between sites, sexes, or ages. This study provides the first data on heavy metals in mammals from this wetland and suggests that C. russula is a good bioindicator of metal pollution. We concluded that sex and age may represent an important source of variation in the bioaccumulation of these metals in wild populations.

Maximal enzyme activities, and myoglobin and glutathione concentrations in heart, liver and skeletal muscle of the Northern Short-tailed shrew (Blarina brevicauda; Insectivora: Soricidae).

We measured the enzymes of glycolysis, Krebs Cycle, beta-oxidation and electron transport in the heart, liver and skeletal muscle of the Northern Short-tailed Shrew, Blarina brevicauda. Additionally, we measured the amount of myoglobin in skeletal and heart muscle as well as the concentration of glutathione in heart. The picture that emerges is of an aerobically well-endowed animal with constrained anaerobic capacity as indicated by small activities of glycolytic enzymes and creatine kinase. Lipid metabolism and amino acid transamination, as well as gluconeogenesis, are predominant in processing carbon resources and probably reflect the large contribution lipid and protein make to the diet of this carnivore. The citrate synthase activity is the largest of any reported value for vertebrate heart (250 U/g). The additional, very active cytochrome c oxidase activity (220 U/g) and large myoglobin concentrations (8 mg/g) in heart are clearly the underpinnings of the rapid metabolic rates reported for small insectivores. The potential for generation of reactive oxygen species must be great since the total glutathione concentration (165 mumol/g) is 300-fold greater in shrew hearts than in hearts of rats.

Potential energetic implications of emesis in the house musk shrew (Suncus murinus).

During the course of studies investigating novel anti-emetic therapies we serendipitously observed a previously unreported behaviour related to emesis in the house musk shrew. This behaviour consisted of spontaneous ingestion of vomit in about half of the animals (males and females) in which emesis was induced by either nicotine (4 mg kg-1 sc.) or horizontal motion (1 Hz, 4 cm, 10 min). Analysis of vomit samples and gastric contents revealed that in a "typical" individual the gastric contents would be voided by as few as 3 vomits. Energetic calculations of the metabolisable energy of food, gastric contents, vomit and field metabolic rate (FMR) predict that a male weighing 60 g would lose 17.3% of its hourly energy requirement for FMR if it vomited once. A 40 g female, however, would experience an hourly energy loss of approximately 22.8%. The possible energetic consequences and resulting ecological implications of this unusual behaviour are discussed.

Immunohistochemical demonstration of c-fos protein in neurons of the medulla oblongata of the musk shrew (Suncus murinus) after veratrine administration.

We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting.

Immunolocalization of androgen receptors and aromatase enzyme in the adult musk shrew brain.

In the brain and other tissues, estrogens are produced by aromatization of androgens. Biochemical data suggest that aromatase enzyme is regulated by the androgen receptor (AR). Neurons that contain either AR or aromatase (AROM) enzyme reside in many of the same brain regions. In this report, we examined the codistribution of AR- and AROM-enzyme-immunoreactive (-ir) neurons in several regions of the adult male and female musk shrew brain. Data were collected from the intermediate nucleus of the lateral septum (LS), medial anterior (BNSTMA) and medial posterointerior (BNSTMP) divisions of the bed nucleus of the stria terminalis, medial preoptic area (mPOA), ventromedial nucleus of the hypothalamus (VMN), medial (MeA), cortical and central nuclei of the amygdala. Males had significantly more AR-ir neurons in the BNSTMP, mPOA, VMN and LS as compared to females. With the exception of the BNSTMA and LS, males had more AROM-ir neurons in each region than females. Furthermore, males had significantly more double-labeled neurons than females in the BNSTMP, mPOA, VMN, LS and MeA. The percentage of AROM-ir neurons that also contained AR immunoreactivity ranged from 13 to 82% depending on sex and region. The highest percentage of dual-labeled neurons (79% in females and 82% in males) was found in the VMN. Taken together, these data show that there is extensive cellular colocalization of AR and AROM enzyme in specific regions of the musk shrew brain. We propose that in both sexes, androgen receptors may act as transcription factors to regulate AROM enzyme.

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection.

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 microM and the calibration curve was linear at least up to 5 microM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 microM and from 3.5 to 6.7 at 5 microM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 microM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.

Sex differences in mammalian and chicken-II gonadotropin-releasing hormone immunoreactivity in musk shrew brain.

Many vertebrates have more than one molecular form of gonadotropin-releasing hormone (GnRH) present in brain. In all cases documented to date, GnRH neurons located in the forebrain are critical players in the brain-pituitary-gonadal feedback axis although the details of how steroids regulate GnRH remain elusive. The function of the second form, usually produced in cells in the midbrain, is not known. It has been hypothesized that this GnRH acts as a neurotransmitter. In the musk shrew (Suncus murinus), as in other mammals, the forebrain cells produce mammalian GnRH (mGnRH) and chicken-II GnRH (cGnRH-II) is present in midbrain neurons. Immunocytochemical analyses were performed to examine sex differences and determine whether the presence or absence of the gonads had any affect on cell number and/or fiber area in the major terminal fields of both forms of GnRH. We detected a significant sex difference in the numbers of immunoreactive (ir) neurons containing mGnRH and cGnRH-II. In both GnRH systems, males have significantly more GnRH-ir cells than females. Furthermore, ovariectomy significantly increased the number of mGnRH-ir and cGnRH-II-ir cell bodies in female brains. In females, changes in the size of the immunoreactive fiber area of the medial habenula were identical to those noted for cGnRH-II cells. In males, the major terminal field for the mGnRH fibers was significantly larger in gonad-intact than in castrated males. In sum, ovarian hormones regulate cGnRH-II production and release, as well as some aspects of mGnRH production in neurons. In males, mGnRH fiber area is sensitive to changes in testicular hormones. These data suggest that the phylogenetically conserved cGnRH-II form is regulated by ovarian hormones and, thus, may be involved in the brain-pituitary-gonadal feedback axis.

Induction of fos-like immunoreactivity in musk shrews after mating.

In many mammalian species the neuroendocrine regulation of male and female reproductive behavior is sexually dimorphic. By contrast, many features of female sexual behavior in the musk shrew (Suncus murinus) more closely resemble those of males than of females of other species. Female musk shrews require testosterone (T), which is neurally aromatized to estrogen, to induce sexual behavior. Aromatization occurs in the medial preoptic area (MPOA), and this region is critical for the expression of female receptivity. To compare neural responses to sexual behavior in females and males, we compared the number of Fos-like immunoreactive (Fos-ir) neurons after mating in musk shrews. In both males and females the number of Fos-ir neurons was increased by mating activity in the granule layer of the accessory olfactory bulb (gr-AOB), the bed nucleus of the stria terminalis (BNST), MPOA, the medial amygdala (MeA), and the region corresponding to the midbrain central tegmental field (CTF). Although Fos was induced by mating in several regions, this response was only dimorphic in the ventral medial nucleus of the hypothalamus (VMN), where mating significantly increased Fos-ir in females, but not in males. In both sexes, only the gr-AOB displayed an increase in Fos-ir after exposure to chemosensory cues alone. Thus, the pattern of Fos expression in the brain after mating is only sexually dimorphic in one region, the VMN. Further, in spite of past behavioral studies done in this species, which show a role for pheromones in induction of receptivity, these data show that exposure to pheromones does not induce Fos in structures caudal to the olfactory bulbs.

Behavioral interactions have rapid effects on immunoreactivity of prohormone and gonadotropin-releasing hormone peptide.

The nervous system responds to both internal and external cues, integrating these signals to coordinate behavior and physiology. Mating interactions can promote dramatic changes in neuroendocrine cells which trigger successful copulation, ovulation, fertilization, and pregnancy. The neurons that transduce behavioral cues into neuroendocrine signals are distributed in a loose continuum along the medial ventral forebrain where they produce and secrete gonadotropin-releasing hormone (GnRH). In the past we have reported changes in GnRH-immunoreactive (GnRH-ir) cell numbers in brains of female musk shrews sacrificed during, and after, brief mating interactions. The purpose of the current study was twofold: first to determine which aspect of intracellular GnRH production is stimulated by behavioral interactions; second, to characterize the specific aspects of the social exchange that trigger GnRH production. We report that 1 h after copulation the production of proGnRH protein is stimulated. Non-copulatory behavioral interactions resulted in a rapid decrease in the numbers of neurons containing GnRH-ir peptide. This change was accompanied by an increase in the GnRH-ir fibers in the median eminence, but no surge in luteinizing hormone. These data suggest that behavioral interactions stimulate release of mature GnRH peptide from cell bodies followed by accumulation of available GnRH in cell terminals. Copulation triggers increased production of proGnRH in cell bodies. The data highlight the usefulness of behavioral paradigms for the examination of the dynamics of neuropeptide production.

Presence and differential distribution of distinct forms of immunoreactive gonadotropin-releasing hormone in the musk shrew brain.

Gonadotropin-releasing hormone (GnRH) immunoreactive cells and fibers were revealed in olfactory regions, the ventral forebrain, and in the midbrain of the musk shrew (Suncus murinus). Immunoreactive neurons in olfactory and telencephalic areas were specific for the mammalian form of GnRH. Cell bodies in the midbrain, however, cross-reacted with an antibody specific for chicken-II GnRH. High-performance liquid chromatography and radioimmunoassay analyses confirmed these results; high levels of chicken II GnRH were present in the midbrain, and mammalian GnRH was detected in both forebrain and midbrain. In addition, a third, late-eluting form of GnRH was revealed using high-performance liquid chromatography in both forebrain and midbrain of the musk shrew. Midbrain neurons containing GnRH have not been reported previously in a mammal, although mesencephalic GnRH immunoreactivity within cell bodies is common among nonmammalian vertebrates. Likewise, while multiple forms of GnRH have been reported in nonmammalian vertebrates and several metatherian species of mammals, this is the first report on multiple forms of GnRH in the brain of a placental mammal. Taken together, the findings suggest that this primitive eutherian mammal has retained the ability to produce GnRH protein in the midbrain. This feature of the GnRH system has been conserved among nonmammalian vertebrates, but appears to have been lost in modern placental mammal species. The functional significance of this group of neurons has yet to be determined.

Co-existence of gonadotrophins (FSH, LH) and thyrotrophin (TSH) in single anterior pituitary cells of the musk shrew, Suncus murinus.

According to recent immunocytochemical studies of anterior pituitary cells, it is obvious that the "one cell-one hormone" theory must be modified. Many pituitary morphologists have demonstrated that there are some cells that contain two hormones. In this study, we demonstrate by means of immuno-electronmicroscopy the co-existence of gonadotrophins (FSH and LH) and thyrotrophin (TSH) in the same anterior pituitary cells of the musk shrew. These cells were remarkably altered in their ultrastructural features by either gonadectomy or thyroidectomy. Double labeling for gonadotrophins and thyrotrophin was present not only in the same cells but also in the same secretory granules. Our ability to demonstrate co-existence of gonadotrophins and thyrotrophin in the same cell may be due to our selection of fixative and embedding media for electron-microscopic immunocytochemistry. Our conclusion that gonadotrophins and thyrotrophin are produced in a single cell type of the anterior pituitary gland in the musk shrew, i.e., thyrogonadotrophs, suggests the need to consider a modification of the classic scheme for classification of anterior pituitary cells.

Immunohistochemistry of the pituitary pars distalis of the musk shrew, Suncus murinus.

Immunocharacteristics of the pituitary pars distalis cell types of the musk shrew, Suncus murinus, were studied by the unlabeled antibody enzyme technique, using peroxidase-antiperoxidase or avidin-biotin-peroxidase complex. The thyrotropin (TSH)-, gonadotropin (GTH)-, corticotropin (ACTH)-, prolactin (PRL)-, and growth hormone (GH)-secreting cells of the PD were identified on the basis of their immunoreactivity with different heterologous antisera. The TSH cells showed specific immunoreactivity with antisera against human (h) TSH beta and rat (r) TSH beta. Cells showing immunoreactivity with the antisera against hLH beta and ovine (o) LH beta were designated as GTH cells as no immunoreactivity was observed with antisera against hFSH beta and oFSH beta. The ACTH cells as well as the cells of the pars intermedia were revealed by anti-ACTH1-24 and anti-ACTH1-10 sera. Whereas the PRL cells were recognized by their immunoreactivity with antisera against hPRL and oPRL, the GH cells were identified with anti-hGH, anti-oGH, and anti-bovine (b) GH sera. TSH and GTH, TSH and ACTH, GTH and ACTH, ACTH and GH, ACTH and PRL, and GH and PRL cells were visualized in the same section using the dual immunoperoxidase technique. Comparison of the immunohistochemically identified cells with those described histochemically reveals several discrepancies, which expose the limitations of the latter techniques identifying adenohypophysial cells.

Hazardous exposure of ground-living small mammals to cadmium and lead in contaminated terrestrial ecosystems.

The dietary exposure to cadmium and lead of two ground-living species of small mammals, i.e., shrews Sorex araneus (Insectivora) and voles Microtus agrestis (Rodentia), was investigated and related to metal loads in target organs (kidneys and liver). The study was done in two natural areas polluted with cadmium and lead originating from urban and industrial metal sources. The average intake of cadmium by the herbivorous voles varied between 0.1 and 0.4 micrograms/g/day and of lead between 2 and 10 micrograms/g/day. The carnivorous shrews showed a considerably higher metal intake rates, i.e., cadmium 3 to 16 micrograms/g/day and lead 19 to 53 micrograms/g/day, which was largely due to the consumption of contaminated earthworms (Oligochaeta). An average cadmium intake of 15 micrograms/g/day or a lead intake of 20 micrograms/g/day corresponded with critical renal metal loads of 120 micrograms/g for cadmium and 25 micrograms/g for lead, which are indicative of adverse health effects. The renal metal loads in shrews reached the critical level, but they remained far below this level in voles. The results indicate a greater risk of toxic exposure to cadmium and lead in soricine shrews than in microtine rodents.

An immunohistochemical study on the distribution of endocrine cells in the gastrointestinal tract of the musk shrew, Suncus murinus.

The endocrine cells in the gastrointestinal tract of the musk shrew were studied immunohistochemically. Eleven kinds of endocrine cells, immunoreactive for serotonin, somatostatin, gastrin, cholecistokinin, gastric inhibitory polypeptide, motilin, secretin, neurotensin, pancreatic glucagon, enteroglucagon and bovine pancreatic polypeptide, were revealed. In the stomach, serotonin-, somatostatin-, gastrin-, pancreatic glucagon- and enteroglucagon-immunoreactive cells were detected. The first three types of cells predominated and were more abundant in the pyloric glands than in the other stomach regions. In the small intestine, all types of endocrine cells were found, each having different distributions and relative frequencies. In the large intestine, 10 types of endocrine cells except cholecystokinin-immunoreactive cells were detected. Serotonin- and bovine pancreatic polypeptide-immunoreactive cells were more numerous in the large intestine than in the small intestine.