Physiological regulation and NO-dependent inhibition of migrating myoelectric complex in the rat small bowel by OXA

Orexin A (OXA)-positive neurons are found in the lateral hypothalamic area and the enteric nervous system. The aim of this study was to investigate the mechanism of OXA action on small bowel motility. Electrodes were implanted in the serosa of the rat small intestine for recordings of myoelectric activity during infusion of saline or OXA in naive rats, vagotomized rats, rats pretreated with guanethidine (3 mg/kg) or N(omega)-nitro-L-arginine (L-NNA; 1 mg/kg). Naive rats were given a bolus of the orexin receptor-1 (OX1R) antagonist (SB-334867-A; 10 mg/kg), and the effect of both OXA and SB-334867-A on fasting motility was studied. Double-label immunocytochemistry with primary antibodies against OXA, neuronal nitric oxide synthase (nNOS), and OX1R was performed. OXA induced a dose-dependent prolongation of the cycle length of the migrating myoelectric complex (MMC) and, in the higher doses, replaced the activity fronts with an irregular spiking pattern. Vagotomy or pretreatment with guanethidine failed to prevent the response to OXA. The OXA-induced effect on the MMC cycle length was completely inhibited by pretreatment with L-NNA (P < 0.05), as did SB-334867-A. The OX1R antagonist shortened the MMC cycle length from 14.1 (12.0-23.5) to 11.0 (9.5-14.7) min (P < 0.05) during control and treatment periods, respectively. Colocalization of OXA and nNOS was observed in myenteric neurons of the duodenum and nerve fibers in the circular muscle. Our results indicate that OXA inhibition of the MMC involves the OX1R and that activation of a L-arginine/NO pathway possibly originating from OX1R/nNOS-containing neurons in the myenteric plexus may mediate this effect. Endogenous OXA may have a physiological role in regulating the MMC.     

Stimulatory effect of endogenous orexin A on gastric emptying and acid secretion independent of gastrin

Orexin A (OXA) increases food intake and inhibits fasting small bowel motility in rats. The aim of this study was to examine the effect of exogenous OXA and endogenous OXA on gastric emptying, acid secretion, glucose metabolism and distribution of orexin immunoreactivity in the stomach. Rats equipped with a gastric fistula were subjected to intravenous (IV) infusion of OXA or the selective orexin-1 receptor (OX1R) antagonist SB-334867-A during saline or pentagastrin infusion. Gastric emptying was studied with a liquid non-nutrient or nutrient, using 51Cr as radioactive marker. Gastric retention was measured after a 20-min infusion of OXA or SB-334867-A. Plasma concentrations of OXA, insulin, glucagon, glucose and gastrin were studied. Immunohistochemistry against OXA, OX1R and gastrin in gastric tissue was performed. OXA alone had no effect on either acid secretion or gastric emptying. SB-334867-A inhibited both basal and pentagastrin-induced gastric acid secretion and increased gastric retention of the liquid nutrient, but not PEG 4000. Plasma gastrin levels were unchanged by IV OXA or SB-334867-A. Plasma OXA levels decreased after intake of the nutrient meal and infusion of the OX1R antagonist. Only weak effects were seen on plasma glucose and insulin by OXA. Immunoreactivity to OXA and OX1R were found in the mucosa, myenteric cells bodies and varicose nerve fibers in ganglia and circular muscle of the stomach. In conclusion, endogenous OXA influences gastric emptying of a nutrient liquid and gastric acid secretion independent of gastrin. This indicates a role for endogenous OXA, not only in metabolic homeostasis, but also in the pre-absorptive processing of nutrients in the gut.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Orexins acting at native OX(1) receptor in colon cancer and neuroblastoma cells or at recombinant OX(1) receptor suppress cell growth by inducing apoptosis

Screening of 26 gut peptides for their ability to inhibit growth of human colon cancer HT29-D4 cells grown in 10% fetal calf serum identified orexin-A and orexin-B as anti-growth factors. Upon addition of either orexin (1 microM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC(50) of 5 nm peptide. Orexins did not alter proliferation but promoted apoptosis as demonstrated by morphological changes in cell shape, DNA fragmentation, chromatin condensation, cytochrome c release into cytosol, and activation of caspase-3 and caspase-7. The serpentine G protein-coupled orexin receptor OX(1)R but not OX(2)R was expressed in HT29-D4 cells and mediated orexin-induced Ca(2+) transients in HT29-D4 cells. The expression of OX(1)R and the pro-apoptotic effects of orexins were also indicated in other colon cancer cell lines including Caco-2, SW480, and LoVo but, most interestingly, not in normal colonic epithelial cells. The role of OX(1)R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX(1)R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX(1)R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX(1)R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX(1)R, are pro-apoptotic peptides. These findings add a new dimension to the biological activities of these neuropeptides, which may have important implications in health and disease, in particular colon cancer.     

The in vitro effect of progesterone on the orexin system in porcine uterine tissues during early pregnancy

Background

Orexin A (OXA) and orexin B (OXB) are hypothalamic-derived peptides that participate in the regulation of energy metabolism, food intake and reproductive function by influencing the hypothalamic-pituitary-ovarian axis. Orexins are also produced in the endometrium, myometrium and placenta, which suggests that they could act as a link between energy metabolism and the reproductive system. Changes in the expression of orexin and the orexin receptor genes and proteins during the oestrous cycle and early gestation in pigs imply that orexin activity may be regulated by local factors within the uterus. The aim of this study was to investigate the influence of progesterone (P₄) on the expression of orexin system genes, and proteins in the porcine uterus during early gestation. Gene expression was analyzed by real-time PCR. Adiponectin secretion was determined by ELISA, and the receptors proteins content was defined using western blot analysis.

Results

In the endometrium, P₄ enhanced OXA secretion on days 10 to 11 of gestation and OXB secretion on days 12 to 13. In the myometrium, P₄ inhibited the secretion of both orexins on days 15 to 16 and OXB secretion also on days 12 to 13. In the endometrium, P₄ inhibited the expression of orexin receptor 1 (OX1R) protein at nearly all times analyzed, whereas the expression of orexin receptor 2 (OX2R) protein was inhibited only on days 15 to 16 of gestation. In the myometrium, P₄ stimulated OX1R protein expression on days 12 to 13 and 15 to 16 of gestation and inhibited OX1R protein expression on days 27 to 28. The expression of OX2R protein in the myometrium increased on days 12 to 13 and decreased on days 10 to 11 and 15 to 16.

Conclusions

The results indicate that P₄ could regulate the expression of the orexin system in the porcine uterus during early pregnancy, which suggests the presence of a local feedback loop that could play an important role in the regulation of maternal metabolism during pregnancy. The findings may contribute to the existing knowledge of the mechanisms linking maternal energy metabolism with the regulation of the reproductive system during pregnancy.

     

Orexin-1 receptor expression after global ischemia in mice

Orexins are neuropeptides that have a range of physiological effects including the regulation of feeding behavior and the sleep-wakefulness cycle. Recently, we reported that level of orexin A in spinal fluid was decreased in the patients of some neurodegenerative diseases and it is considered that orexin A and the receptors might be related to central nervous system disorders. However, the expression and localization of orexin receptors is not elicited well. Therefore, the purpose of this study is to investigate the time-dependent changes and the cellular localization of orexin receptor focusing on orexin-1 receptor (OX1R) in the mouse brain after transient common carotid artery occlusion (tCCAO) model by using immunohistochemical techniques. OX1R immunoreactivity dramatically increased and peaked in the hippocampus and cortex 2 days after tCCAO, but remained unchanged in the hypothalamus. Using double-immunohistochemistry, the OX1R immunopositive cells at 2 days after tCCAO were co-localized not only with neuronal marker, NeuN-immunoreactivity but also with astroglial and oligodendroglial markers, GFAP- and CNPase-immunoreactivities, respectively. These results suggested that OX1R is induced other cells in addition to the neurons during stress such as ischemia and orexins and its receptor might play an important role for ischemic insult.     

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP – on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) – on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.     

OX2R activation induces PKC-mediated ERK and CREB phosphorylation

Deficiencies in brain orexins and components of mitogen activated protein kinase (MAPK) signaling pathway have been reported in either human depression or animal model of depression. Brain administration of orexins affects behaviors toward improvement of depressive symptoms. However, the documentation of endogenous linkage between orexin receptor activation and MAPK signaling pathway remains to be insufficient. In this study, we report the effects of orexin 2 receptor (OX2R) activation on cell signaling in CHO cells over-expressing OX2R and in mouse hypothalamus cell line CLU172. Short-term extracellular signal-regulated kinase (ERK) phosphorylation and long-term cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) phosphorylation were subsequently observed in CHO cells that over-express OX2R while 20 min of ERK phosphorylation was significantly detected in mouse adult hypothalamus neuron cell line CLU172. Orexin A, which can also activate OX2R, mediated ERK phosphorylation was as the same as orexin B in CHO cells. A MAPK inhibitor eliminated ERK phosphorylation but not CREB phosphorylation in CHO cells. Also, ERK and CREB phosphorylation was not mediated by protein kinase A (PKA) or calmodulin kinase (CaMK). However, inhibition of protein kinase C (PKC) by GF 109203X eliminated the phosphorylation of ERK and CREB in CHO cells. A significant decrease in ERK and CREB phosphorylation was observed with 1 μM GF 109203X pre-treatment indicating that the conventional and novel isoforms of PKC are responsible for CREB phosphorylation after OX2R activation. In contrast, ERK phosphorylation induced by orexin B in CLU172 cells cannot be inhibited by 1 μM of protein kinase C inhibitor. From above observation we conclude that OX2R activation by orexin B induces ERK and CREB phosphorylation and orexin A played the same role as orexin B. Several isoforms of PKC may be involved in prolonged CREB phosphorylation. Orexin B induced ERK phosphorylation in mouse hypothalamus neuron cells differs from CHO cell line and cannot be inhibited by PKC inhibitor GF 109203X. And hypothalamus neuron cells may use different downsteam pathway for orexin B induced ERK phosphorylation. This result supports findings that orexins might have anti-depressive roles.     

Gastrin releasing peptides increase Fos-like immunoreactivity in the enteric nervous system and the dorsal vagal complex

We and others have shown that gastrin-releasing peptide (GRP) reduces food intake. In this study, we determined the activation of the gastrointestinal and dorsal vagal complex (DVC) neurons by various forms of GRP to determine the pathway involved in this reduction. We found the following: (1) GRP-10, -27 and -29 (2.1 nmol/kg, i.p.) increased the Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of the stomach and the area postrema (AP) of the DVC; (2) GRP-27 and GRP-29 increased the Fos-LI in the myenteric plexus of the duodenum; and (3) only GRP-29 increased the Fos-LI in the submucosal plexus of the duodenum. In conclusion, GRP may reduce food intake by activating the area postrema. The enteric neurons may have a potential role in this reduction through the direct activation of the AP or exerting local gut actions, such as the stimulation of gut motility or secretions.     

Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Orexin-A does not stimulate food intake in old rats

Aging is associated with a progressive decrease in appetite and food intake. Both A and B orexins, expressed in specific neurons of the lateral hypothalamic area, have been implicated in the regulation of sleep and feeding. In this study, the stimulatory effect of intracerebroventricular administration of the orexins on food intake was compared between young (4-mo-old) and old (25- to 27-mo-old) male Wistar rats. A stainless steel cannula was implanted stereotactically into the left lateral ventricle. After a 7-day recovery period, different doses (0-30 nmol) of orexins were injected into the left lateral ventricle without anesthesia. Food and water consumptions were measured at 1, 2, and 4 h after injection. The protein levels of orexin receptors, a specific receptor for orexin-A (OX1R) and a receptor for both orexin-A and -B (OX2R), in the hypothalamus were determined by Western blot analysis and compared between young and old rats. Intracerebroventricular administration of orexin-A stimulated food intake in a dose-dependent manner in young rats. However, no effects were observed at any dose in old rats. The protein level of OX1R in the hypothalamus was significantly lower in old rats than in young rats, although the protein level of OX2R was comparable between groups. Results of the present study indicate that the function of the orexin system is diminished in old rats. The decrease in the OX1R protein level in the hypothalamus could be responsible for orexin-A's lack of stimulation of food intake in old rats.     

Distribution of neurokinin A-like and neurokinin B-like immunoreactivity in human peripheral tissues.

Using specific radioimmunoassay and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of the two peptides in human peripheral tissues were studied. Both NKA-like immunoreactivity (NKA-LI) and NKB-like immunoreactivity (NKB-LI) were present in the walls of the gut and gall bladder and in the pancreas. In the gut, the values for NKA-LI were 0.56-35.73 pmol/g wet weight, while those in pancreas and gall bladder were 0.64-0.68 and 0.36 pmol/g wet weight, respectively. The values of NKB-LI were 0.45-2.66 pmol/g wet weight in the gut, 0.93-1.65 pmol/g wet weight in the pancreas, and 0.30 pmol/g wet weight in the gall bladder. The immunocytochemical reactivity to both peptides was localized to ganglia of the submucosal and myenteric nerve plexuses in the gut wall, and to neurons in the muscle layer and mucosa of the gut wall. Weak but positive NKA-LI appeared in nerve cells of the pancreas, while NKB-LI was not detectable in the pancreas. Conversely, in the gall bladder wall, NKA-LI was undetectable while a very faint NKB-LI was found in the muscle layer. The localization of NKA corresponded closely to that of NKB in the tissues although the relative concentrations of the peptides varied from organ to organ.     

Chemical coding of neurons expressing δ- and κ-opioid receptor and type I vanilloid receptor immunoreactivities in the porcine ileum

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.     

Expression of the orexin system in the porcine uterus,conceptus and trophoblast during early pregnancy

Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.     

Central vagal stimulation activates enteric cholinergic neurons in the stomach and VIP neurons in the duodenum in conscious rats

The influence of central vagal stimulation induced by 2h cold exposure or intracisternal injection of thyrotropin-releasing hormone (TRH) analog, RX-77368, on gastro-duodenal enteric cholinergic neuronal activity was assessed in conscious rats with Fos and peripheral choline acetyltransferase (pChAT) immunoreactivity (IR). pChAT-IR was detected in 68%, 70% and 73% of corpus, antrum and duodenum submucosal neurons, respectively, and in 65% of gastric and 46% of duodenal myenteric neurons. Cold and RX-77368 induced Fos-IR in over 90% of gastric submucosal and myenteric neurons, while in duodenum only 25-27% of submucosal and 50-51% myenteric duodenal neurons were Fos positive. In the stomach, cold induced Fos-IR in 93% of submucosal and 97% of myenteric pChAT-IR neurons, while in the duodenum only 7% submucosal and 5% myenteric pChAT-IR neurons were Fos positive. In the duodenum, cold induced Fos in 91% of submucosal and 99% of myenteric VIP-IR neurons. RX-77368 induces similar percentages of Fos/pChAT-IR and Fos/VIP-IR neurons. These results indicate that increased central vagal outflow activates cholinergic neurons in the stomach while in the duodenum, VIP neurons are preferentially stimulated.     

Changes in plasma orexin A and orexin B concentrations during the estrous cycle of the pig

Orexin A (OXA) and orexin B (OXB) are neuropeptides synthesized mainly in the lateral hypothalamus, which are involved in the control of various physiological functions such as energy homeostasis, sleep, wakefulness and feeding behavior. The present study analyzes orexins A and B levels in the porcine plasma during the estrous cycle. The highest plasma concentrations of orexin A were observed on days 2–3 of the estrous cycle (p < 0.05 relative to days 10–12 and 14–16) and the lowest (p < 0.05) on days 14–16. The highest orexin B levels in the blood plasma were noted on days 17–19 (p < 0.05 vs. days 14–16). We demonstrated the presence of OXA and OXB in porcine blood plasma and the impact of the phase of the estrous cycle on the observed changes in plasma orexin levels.