Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite actions on rat nodose neuron calcium currents

1. The whole-cell variation of the patch-clamp technique was used to study the effect of neuropeptide Y (NPY) and preferential agonists for the NPY-1 and NPY-2 receptor subtypes on voltage-dependent calcium currents in acutely dissociated postnatal rat nodose ganglion neurons. 2. Both low- and high-threshold calcium current components were present. NPY altered voltage-dependent calcium currents in approximately 50% of neurons studied. NPY (0.1-100 nM, ED50 6 nM) decreased the peak amplitude of transient high-threshold calcium currents in approximately 45% of the neurons. NPY (100 nM) decreased the peak amplitude of these currents 31 +/- 5% (mean +/- SE). However, in approximately 5% of the neurons NPY (100 nM) caused a reversible and reproducible increase in transient high-threshold calcium currents of 21 +/- 4%. NPY did not affect either transient low-threshold or slowly inactivating high-threshold calcium current components. 3. Application of the C-terminal fragment NPY 13-36 (100 nM), a preferential agonist for NPY-2 receptors, reversibly decreased the peak amplitude of transient high-threshold calcium currents by 26 +/- 5% in 9 of 20 cells (45%). Application of [Pro34]-NPY (100 nM), a preferential agonist for NPY-1 receptors, reversibly increased the peak amplitude of transient high-threshold calcium currents 20 +/- 4% in 23 out of 48 neurons (48%). Six of 20 neurons (30%) responded to application of both agonists. Neither the NPY-1 nor NPY-2 agonists affected transient low-threshold or slowly inactivating high-threshold calcium current components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster

Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1-15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility.     

Localization of regulatory peptides in the gastrointestinal tract of the striped dolphin, Stenella coeruleoalba (Mammalia: Cetacea). An immunohistochemical study

Samples of oesophagus, first, second and third stomach, duodenal ampulla, proximal intestine and distal intestine including rectum were obtained from striped dolphins (Stenella coeruleoalba) stranded along Italian coasts, fixed in formalin and used for immunohistochemistry. The possible presence of neuropeptides and the biogenic amine serotonin was investigated by a labelled streptavidin-biotin method. Neuropeptide Y (NPY)-, substance P-, calcitonin gene-related peptide (CGRP)-, metenkephalin-, gastrin releasing peptide (GRP)/bombesin-, and somatostatin-like immunoreactivities were present in the submucosal as well as the myenteric plexuses, even with differences of distribution in the various organs. Vasoactive intestinal poly-peptide (VIP)-like immunoreactivity was detected in the submucosal plexus, whereas beta-endorphin- and leu-enkephalin-like immunoreactivities were shown in the myenteric plexus only. NPY-, substance P-, CGRP- and VIP-like-immunoreactivities were also observed in perivascular nerve fibres. In addition, VIP-, GRP- and somatostatin-like immunoreactivities were detected in myelinated nervous bundles. These were localized in the submucosal and muscular layers all along the gastrointestinal tract, and possibly sustain an exceptionally rapid response of the target structures. It is note-worthy that peptidergic axons in the wall of the gut of the majority of mammals are unmyelinated. A somatostatin-like peptide was identified in epithelial cells only in the second stomach, whereas in terrestrial mammals this endocrine cell type occurs widely. Immunoreactivity to serotonin was never detected, and this is a further difference in comparison with the majority of other mammals.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D28, and s-100. Cultured cells or tissues were incubated with 10 microM BODIPY forskolin for 30 min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D28 (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.     

Colocalization of NADPH-diaphorase staining and VIP immunoreactivity in neurons in opossum internal anal sphincter

Nitric oxide and vasoactive intestinal polypeptide (VIP) are important inhibitory neurotransmitters mediating relaxation of the internal anal sphincter. The location and coexistence of these two neurotransmitters in the internal anal sphincter has not been examined. We performed a double-labeling study to examine the coexistence of nitric oxide synthase and VIP in the opossum internal anal sphincter using the NADPH-diaphorase technique which is a histochemical stain for nitric oxide synthase. In perfusion-fixed, frozen-sectioned tissue, VIP-immunoreactive neurons were labeled using immunofluorescence histochemistry. After photographing the VIP-immunoreactive neurons, nitric oxide synthase was labeled using the NADPH-diaphorase technique. Ganglia containing neuronal cell bodies were present in the myenteric plexus for the entire extent of the internal anal sphincter. VIP-immunoreactive and NADPH-diaphorase-positive neurons were present in ganglia in the myenteric as well as the submucosal plexuses. Most of the VIP-immunoreactive neurons were also NADPH-diaphorase positive. VIP and nitric oxide synthase are present and frequently coexist in neurons in the internal anal sphincter of the opossum. These neurons may be an important source of inhibitory innervation mediating the rectoanal reflex-induced relaxation of the sphincter. The demonstration of the coexistence of these two neurotransmitters will be of fundamental importance in unraveling their relationship and interaction in the internal anal sphincter as well as other systems.     

Purinergic fast excitatory postsynaptic potentials in myenteric neurons of guinea pig: distribution and pharmacology

BACKGROUND & AIMS: Adenosine triphosphate (ATP) acting at P2 receptors mediates some fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons of guinea pig ileum. The present studies investigate the distribution of purinergic fEPSPs along the length of the gut and characterize the P2-receptor subtype mediating fEPSPs. METHODS: Conventional intracellular electrophysiological methods were used to record from myenteric neurons in vitro. RESULTS: At a membrane potential of -97 +/- 1 mV, the amplitude (25 +/- 1 mV; n = 307) of fEPSPs was similar along the gut. Hexamethonium (100 micromol/L) inhibited fEPSPs in the gastric corpus by 98% +/- 1% (n = 31) and in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 42%-55%. In the presence of hexamethonium, suramin (100 micromol/L) or the P2X antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 micromol/L) reduced the control fEPSP amplitude in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 71%-84%. The pharmacology of the purinergic fEPSPs was investigated in detail in the ileum. Noncholinergic fEPSPs were concentration-dependently (1-30 micromol/L) inhibited by PPADS (50%-inhibitory concentration, 3 micromol/L). In addition, alpha,beta-methylene 5'-adenosine triphosphate (1 micromol/L) also reduced purinergic fEPSPs. CONCLUSIONS: Fast EPSPs mediated in part through P2X receptors are prominent in myenteric neurons along the small and large intestines but are rare in the gastric corpus.     

Nitric oxide modulates neuropeptide Y regulation of ion transport in mouse ileum

The possible involvement of nitric oxide in the regulation of intestinal ion transport induced by neuropeptide Y (NPY) was investigated by evaluating the effects of NG-methyl-L-arginine (L-NMA), L-arginine and S-nitroso-N-acetylpenicillamine (SNAP) on NPY activity in mouse ileum mounted in Ussing chambers in vitro. Serosal NPY (10 nM) produced a sustained decrease in basal transmural short circuit current (Isc) and potential difference without altering the tissue conductance. Pretreatment of tissues with L-arginine (3 mM), but not D-arginine (10 mM), blocked the NPY-mediated changes in Isc. This L-arginine effect on NPY activity was reversed by L-NMA (3 mM), and not by NG-methyl-D-arginine (10 mM). The L-arginine effect on NPY activity was concentration-related with an A50 (95% CL) value of 1.6 (0.9-2.3) mM. In contrast to L-arginine, L-NMA (1 mM) pretreatment of tissues produced an enhancement of NPY activity, resulting in a 3.8-fold leftward displacement of the NPY concentration-response curve; NG-methyl-D-arginine was without effect. The effect of L-NMA on NPY activity was concentration-related with an A50 (95% CL) value of 45.3 (23.2-68.8) microM. Serosal application of SNAP, a nitric oxide donor, produced a concentration-related decrease in basal Isc and potential difference without altering tissue conductance with an A50 (95% CL) value of 22.5 (11.1-40.5) microM. Pretreatment of tissue with SNAP (100 microM) reduced the NPY activity with rightward displacement of NPY concentration-response curve. Pretreatment of tissues with L-arginine also blocked the reduction of Isc by [D-Pen2, D-Pen5]enkephalin (10-30 nM), H2N-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (10-30 nM) and somatostatin (0.3-1.0 microM), but had no effect on norepinephrine (0.1-0.3 microM)-induced decrease in mouse ileal Isc. These results show that [fgc]l-arginine and SNAP block NPY-mediated changes in ion transport, suggesting that nitric oxide may play a role in the regulation of NPY-mediated ion transport in the mouse ileum.     

Reduction of neuropeptide Y binding sites in the rat hippocampus after electroconvulsive stimulations

The role of vasoactive intestinal peptide (VIP) was investigated when mucosal stroking and 5-hydroxytryptamine (5-HT) were used to activate neural reflexes that stimulate chloride secretion in the guinea pig colon. Muscle-stripped segments of colon containing intact submucosal ganglia without myenteric ganglia were set up in modified flux chambers in order to record short-circuit current (Isc). Mucosal stroking with a brush for 1 s or a pulse of 5-HT (injection of 15 microliters of 100 microM 5-HT into 1.5 ml of mucosal solution) caused an increase in Isc that was reduced by the VIP antagonist, neurotensin6-11-VIP7-28, in a concentration-dependent manner. The Isc responses to mucosal stroking and a 5-HT pulse were reduced by 53% and 58%, respectively, by 2 microM neurotensin6-11-VIP7-28. The residual Isc response in the presence of neurotensin6-11-VIP7-28 was abolished by atropine. Blockade of 5-HT1P receptors on submucosal afferent neurons decreased Isc responses to stroking or a 5-HT pulse. The residual Isc response after 5-HT1P receptors were blocked was reduced by only 11-14% by neurotensin6-11-VIP7-28. In the presence of blockade of both 5-HT1P and VIP receptors, atropine abolished the Isc response to both stimuli. The observations suggest that the neural circuitry activated by stroking includes at least two independent pathways. One pathway contains VIP neurons which receive inputs directly or indirectly from 5-HT1P receptor-containing afferents. A second pathway involves muscarinic cholinergic transmission that is independent of 5-HT1P and VIP receptor activation.     

In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum

We investigated the effect of the cannabinoid agonist (+)WIN-55212-2 on human ileum longitudinal smooth muscle preparations, either electrically stimulated or contracted by carbachol. Electrical field stimulation mostly activated cholinergic neurons, since atropine and tetrodotoxin (TTX), alone or coincubated, reduced twitch responses to a similar degree (85%). (+)WIN-55212-2 concentration-dependently inhibited twitch responses (IC50 73 nM), but had no additive effect with atropine or TTX. The cannabinoid CB1 receptor antagonist SR 141716 (pA2 8.2), but not the CB2 receptor antagonist, SR 144528, competitively antagonized twitch inhibition by (+)WIN-55212-2. Atropine but not (+)WIN-55212-2 or TTX prevented carbachol-induced tonic contraction. These results provide functional evidence of the existence of prejunctional cannabinoid CB1-receptors in the human ileum longitudinal smooth muscle. Agonist activation of these receptors prevents responses to electrical field stimulation, presumably by inhibiting acetylcholine release. SR 141716 is a potent and competitive antagonist of cannabinoid CB1 receptors naturally expressed in the human gut.     

Use of D-myo inositol 1,2,6 trisphosphate to inhibit contractile activity in rat ventricular cardiomyocytes induced by neuropeptide Y and other cardioactive peptides through phospholipase C

1. D-Myo inositol 1,2,6 trisphosphate (alpha-trinositol, pp56), an isomer of the second messenger substance, inositol 1,4,5 trisphosphate, has an interesting pharmacological profile that includes antagonism of a number of neuropeptide Y (NPY)-mediated cellular processes. The ability of pp56 to inhibit selectively the myocardial contraction mediated by NPY in relation to the responses to other cardioactive peptides, including endothelin-1, calcitonin gene-related peptide (CGRP), secretin and vasoactive intestinal peptide (VIP), was assessed. In order to investigate the possible interaction of pp56 with mechanisms of inositol phosphate signalling generated in heart muscle cells by activation of the beta-isoenzyme of phospholipase C (PLC beta), noradrenaline was used as a positive control, and isoprenaline and forskolin were included as negative controls. 2. Ventricular cardiomyocytes, isolated from the hearts of adult rats, were stimulated to contract at 0.5 Hz in the presence of calcium ion (2 mM). The concentrations of agonists used were in the region of their maximally effective concentrations for myocyte contraction and the concentration of pp56 was in the range of 1-100 microM. Contractile activity was monitored by video microscopy and maximum shortening determined by image analysis. 3. In the absence of agonist, contractile amplitudes following 20 min preincubation with pp56 were not different from that observed in the absence of pp56. Pp56 (1-100 microM) inhibited significantly the positive contractile response to noradrenaline (5 microM) in the presence of propranolol (500 nM), such that the response was almost completely attenuated at the highest concentration of the inhibitor. Pp56 did not inhibit the positive contractile responses to forskolin (40 microM) or isoprenaline (100 nM). 4. NPY alone does not influence the basal level of contraction of cardiomyocytes, but can attenuate isoprenaline-stimulated contraction and can increase contractile amplitude from basal when the transient outward current is blocked with 4-aminopyridine. In the presence of isoprenaline (100 nM), the negative response to NPY (100 nM) was attenuated significantly by pp56 (1-100 microM). With 4-aminopyridine, the positive contractile response to NPY (200 nM) was decreased by pp56, although this was not statistically significant. 5. Pp56 inhibited the positive contractile responses to CGRP (1 nM) and endothelin-1 (20 nM) completely, but did not affect the responses to secretin (20 nM) or VIP (20 nM). 6. In conclusion, these data challenge the previously obtained selectivity of pp56 as an antagonist of NPY-mediated cellular processes, since responses to CGRP and endothelin-1 were at least equally sensitive. Furthermore, as pp56 discriminated clearly in its inhibition of responses to alpha-adrenoceptor by comparison with beta-adrenoceptor/adenylate cyclase stimulation, it appears that pp56 may be a useful pharmacological agent with which to distinguish between PLC beta-dependent and PLC beta-independent coupling mechanisms. On this basis, further evidence has been obtained that, in rat cardiomyocytes, the contractile responses to NPY, CGRP and endothelin-1 are attributable to the activation of PLC beta-dependent pathways, whereas the responses to secretin and VIP are mediated by PLC beta-independent pathways.     

Morphometry and acetylcholinesterase activity of the myenteric plexus of the wild mouse Calomys callosus

The myenteric plexus of the digestive tract of the wild mouse Calomys callosus was examined using a histochemical method that selectively stains nerve cells, and the acetylcholinesterase (AChE) histochemical technique in whole-mount preparations. Neuronal density was 1,500 +/- 116 neurons/cm2 (mean +/- SEM) in the esophagus, 8,900 +/- 1,518 in the stomach, 9,000 +/- 711 in the jejunum and 13,100 +/- 2,089 in the colon. The difference in neuronal density between the esophagus and other regions was statistically significant. The neuron profile area ranged from 45 to 1,100 microns2. The difference in nerve cell size between the jejunum and other regions was statistically significant. AChE-positive nerve fibers were distributed within the myenteric plexus which is formed by a primary meshwork of large nerve bundles and a secondary meshwork of finer nerve bundles. Most of the nerve cells displayed AChE activity in the cytoplasm of different reaction intensities. These results are important in order to understand the changes occurring in the myenteric plexus in experimental Chagas' disease.     

Projections of pelvic autonomic neurons within the lower bowel of the male rat: an anterograde labelling study

The tissues of the large intestine which receive an innervation by neurons of the major pelvic ganglia were identified following in vivo and in vitro anterograde labelling with the lipophilic tracer 1,1'didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate in the male rat. The primary target in the gut of major pelvic ganglion neurons is the myenteric plexus of the distal colon and the rectum. The serosal ganglia, on the surface of the most distal region of the rectum and the circular muscle of the distal colon and rectum were less densely innervated. The pelvic ganglia do not innervate the longitudinal muscle, submucosal blood vessels, submucosal plexus, or mucosa. The pelvic supply reaches the bowel via two groups of rectal nerves and branches of the penile nerves. All of these connections also carry the axons of viscerofugal neurons from the bowel, some of which have terminal axons in the major pelvic ganglia. Finally, the different nerves supplied different targets. In particular, while the rectal nerves carried pelvic axons supplying the myenteric plexus, circular muscle, and serosal ganglia, the penile nerves only innervated the serosal ganglia. In addition, the two groups of rectal nerves innervated slightly different regions of the bowel and provided different projection patterns. However, successful in vivo labelling was achieved in only 6/12 animals and while all in vitro experiments resulted in successful labelling, it was clear that only a proportion of pelvic projections in any given nerve were labelled. These studies have shown that the major pelvic ganglia are primarily involved in the control of motility, but not of vascular and secretomotor functions. Thus pelvic neurons do not innervate the same range of target tissues within the bowel as the prevertebral ganglia. This study has also shown that the different pathways to the gut from the major pelvic ganglia innervate different tissues, suggesting that the autonomic innervation of the gut is not homogeneous along its length.     

c-fos protein expression and ischemic changes in neurons vulnerable to ischemia/hypoxia, correlated with basic fibroblast growth factor immunoreactivity

Injuries to the brain induce rapid expression of c-fos and c-jun proto-oncogenes in neurons. The protein products (Fos and Jun) of these cellular immediate early genes are thought to regulate target genes that participate in fundamental biological responses. In recent studies of rat brain infarct we demonstrated that gliosis and angiogenesis, two of the fundamental biological responses, are related to neuronal expression of basic fibroblast growth factor (bFGF). In the present study, we explore the linkage between c-fos and bFGF genes by comparing the temporal and spatial domains of Fos and bFGF immunoreactivities (IR) in brain infarct and in transient global ischemia. We demonstrate colocalization of Fos-IR and ischemic changes in neurons at infarct periphery and in regions of "selective vulnerability" beginning 3 hours post-infarction and lasting up to 1-2 weeks. These are: cortical neurons in layers II-III and V, interneurons in hippocampal formation, cerebellar Purkinje cells, and many subcortical nuclei and brainstem nuclei. bFGF-IR appears 12-24 hours later than Fos-IR in the same region but in non-ischemic neurons and the expression persists beyond 2 weeks. Persistent and not transient c-fos expression appears to be associated with ischemic neuronal death, although some of these neurons may survive beyond 2 weeks postinfarction.     

Neurite outgrowth of striatal neurons in vitro: involvement of purines in the growth-promoting effect of myenteric plexus explants

We have shown previously that a soluble factor(s) released by the myenteric plexus promotes neurite outgrowth from postnatal striatal neurons, and that this effect was abolished by tetrodotoxin. We have now investigated the possible involvement of purines in the mediation of this neuritogenic response, by examining their effect on neurite length of striatal neurons both in co-culture with myenteric plexus explants and cultured alone. Both ATP and 2-chloroadenosine partially reversed the inhibitory effect of tetrodotoxin in co-cultures with whole myenteric plexus, while the stable ATP analogue, alpha, beta-methylene ATP, had no effect, suggesting that ATP was being broken down to adenosine before exerting its action. Further support for this view was that the ATP (P2) purinoceptor antagonist suramin did not reverse the effects of ATP, while the adenosine (P1) purinoceptor antagonist 8-(p-sulphophenyl)theophylline did antagonize the effects of ATP in tetrodotoxin-treated co-cultures. Further, both 8-(p-sulphophenyl)theophylline and adenosine deaminase reduced the effect of the myenteric plexus on striatal neurons in the absence of tetrodotoxin, and the adenylate cyclase activator forskolin completely reversed the effect of tetrodotoxin in our co-culture system. The neurite outgrowth-promoting effect of 2-chloroadenosine in tetrodotoxin-treated co-cultures was not further enhanced by a combination of neuropeptides. Serotonin and GTP were without effect on striatal neurons in the presence or absence of myenteric plexus explants. In experiments without myenteric plexus, both 2-chloroadenosine and forskolin caused a slight increase in striatal neurite length; ATP and GTP were ineffective. Basic fibroblast growth factor, nerve growth factor, neurotrophin-3 or neurotrophin-4/5 had no effect on neurite outgrowth in postnatal striatal cultures after two days in vitro. When these growth factors were added in combination with 2-chloroadenosine, the observed increase in mean neurite length did not exceed that induced by 2-chloroadenosine alone. Both 2-chloroadenosine and the ganglioside mix AGF1 increased neurite elongation of striatal neurons after two days in vitro, but an inhibition of enhanced neurite outgrowth was observed when both substances were added together. Both laminin and fibronectin were not neuritogenic for postnatal striatal neurons under our culture conditions. These observations suggest that a factor other than the growth factors tested here is involved in the promotion of striatal neurite outgrowth in co-culture with myenteric plexus explants. In summary, adenosine (probably acting through the A2 subclass of the P1 purinoceptor) leads to increased striatal neurite outgrowth in co-culture with myenteric plexus and we propose that it does so either (1) by triggering the release of a neuritogenic factor, possibly from enteric glial cells, or (2) by acting synergistically with such a growth factor. Adenosine acts via P1 purinoceptors, which leads to changes in cyclic AMP, and the response to forskolin suggests that cyclic AMP is probably involved in the events leading to increased striatal neurite outgrowth.     

Stimulation by enkephalins of D-glucose absorption in rabbit ileum

In intact tissue, DAGO ([D-Ala2, MePhe4, Gly-ol5]enkephalin; 10(-5) M; mu-ligand; addition on the serosal side) stimulated D-glucose absorption and D-glucose-dependent variations in short-circuit current (delta Isc,glu); naloxone (10(-6) M) antagonized these effects. DADLE ([D-Ala2, D-Leu5]enkephalin, mainly a delta-ligand; 10(-5) M) and (pCl-Phe4)-DPDPE ([D-pen2, p-chloro-Phe4, D-Pen5]enkephalin, a more selective delta-ligand; 10(-5) M) did not significantly stimulate delta Isc,glu (addition on the serosal side). In the absence of the muscularis and myenteric plexus or using intact tissue treated with tetrodotoxin (TTX; 3 x 10(-7) M), DAGO was unable to increase delta Isc,glu. Addition of DAGO to the mucosal side did not induce any variations in delta Isc,glu. In conclusion, DAGO is able to increase D-glucose absorption by interacting with mu-receptors located in the myenteric plexus.     

The effect of heavy maternal workload on fetal growth retardation and preterm delivery. A study among southern Thai women

The purpose of this study was to determine concurrent ovarian and endometrial prostaglandin E2 (PGE2) receptor concentrations throughout the hamster estrous cycle. The effect of progesterone (P4) on PGE2 receptor in these two tissues was also investigated during in vitro culture. Estrous cycles of mature female hamsters were monitored according to the appearance of the vaginal discharge on cycle day 1 (D1). Ovaries and uteri were removed from cyclic hamsters at 10:00 a.m. on each day of the cycle and at 6:00 p.m. on D4 (proestrus). Ovarian and endometrial cell membranes were collected and assayed for the specific PGE2 binding by Scatchard plot analysis, using seven different concentrations of 3H-PGE2 (0.72-9.1 nM) with or without the presence of unlabelled PGE2 (9.1 microM). Ovarian and endometrial tissues of the cyclic hamsters were shown to contain a saturable, specific binding site with KD=4.69+/-0.55, and 5.7+/-0.4 nM for ovary and endometrium, respectively. Relative binding activity of PGE1 for the PGE2 binding site was about 28%. PGF2alpha and PGA1 did not compete for the PGE2 binding site. In ovaries, the PGE2 receptor levels started to increase sharply in the evening of D4 and reached maximum in the morning of D1. A precipitous drop of PGE2 receptor was observed on D2 followed by gradual decreases on D3 and D4. The PGE2 receptor concentrations in endometrium were the lowest in the morning of D4, and increased thereafter until a maximal level was reached on D2. Progesterone (10 nM) augmented PGE2 receptors in ovarian but not in endometrial tissue during 24-h in vitro incubation.     

The neurochemical coding and projections of circular muscle motor neurons in the human colon

BACKGROUND & AIMS: Enteric neurons can be characterized by their chemical coding, projections, and morphology. The aim of this study was to describe the different classes of human colonic circular muscle motor neurons. METHODS: Human colonic circular muscle motor neurons were identified by retrograde tracing with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (Dil) applied to the circular muscle layer. Whole-mount preparations of the myenteric plexus were then double-labeled with antisera to choline acetyltransferase (ChAT) and/or nitric oxide synthase (NOS), or NOS and vasoactive intestinal peptide (VIP), and the position and immunoreactivity of Dil-filled neurons were recorded. RESULTS: Fifty-two percent of all Dil-filled neurons were ChAT immunoreactive, and 86% of these projected up to 11 mm orally, with 14% projecting short distances anally. Forty-eight percent of the Dil-filled neurons were NOS immunoreactive, and 77% of these projected up to 19 mm anally, with 23% projecting no more than 6 mm orally. A subpopulation of these NOS-immunoreactive motor neurons were also VIP-immunoreactive. A small population of myenteric neurons was immunoreactive for both ChAT and NOS, but none projected to the circular muscle. NOS-immunoreactive motor neurons projected for longer distances than those with ChAT immunoreactivity and were larger. CONCLUSIONS: There are two classes of human colonic motor neurons: one is excitatory (ChAT-immunoreactive) and mainly projects orally and the other is inhibitory (NOS +/- VIP immunoreactive) and projects preferentially anally.     

In vitro properties of a high affinity selective antagonist of the VIP1 receptor

A selective high affinity VIP1 receptor antagonist [Acetyl-His1, D-Phe2, Lys15, Arg16, Leu17] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP1 receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP1-, rat VIP2- or the human VIP2-recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP1 receptor. The IC50 values of 125I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP1-, rat VIP2-, human VIP1- and human VIP2 receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP1 receptor mediated stimulation of adenylate cyclase activity. The Ki values were respectively of 15 +/- 5 nM and 2 +/- 1 nM for the rat and human VIP1 receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP1 receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.