Movement of villi induces endocytosis of NK1 receptors in myenteric neurons from guinea-pig ileum

Agitation of villi evokes reflexes that affect the motility of the guinea-pig small intestine. NK1 receptor endocytosis was used to investigate the possible involvement of tachykinins acting on neuronal NK1 receptors in these reflexes. Segments of guinea-pig ileum were incubated at 37°C in Krebs physiological saline containing 3×10^–6 M nicardipine, with or without agitation of the villi by gas bubbles. Gut segments were fixed after 0–75 min and processed for immunohistochemistry to reveal the NK1 receptors, following which cells were imaged by confocal microscopy. Initially, receptors were located on the surface and in the cytoplasm of myenteric neurons. In gut incubated without movement of the villi, NK1 receptors returned to the cell surface. After 45 and 60 min, NK1 receptors were detected almost exclusively at the cell surface of 83% and 97% (respectively) of nerve cells that were immunoreactive for NK1 receptors and only 12%–13% of the NK1 receptor fluorescence was located in the cytoplasm. Following the return of receptor to the cell surface, agitation of the villi caused a new wave of endocytosis of the NK1 receptors in 70%–80% of the NK1 receptor-immunoreactive neurons. The percentage of the NK1 receptor fluorescence that was in the cytoplasm increased more than 2-fold to 27±2% after 15 min villous agitation. Action potential blockade by tetrodotoxin (3×10^–7 M) prevented the internalisation of the NK1 receptor in response to villous agitation. The degree of internalisation caused by bubbling was similar to that caused by 2×10^–9 M substance P. These results indicate that, when enteric reflex circuits are activated by villous movement, tachykinins are released and cause endocytosis of the NK1 receptor in a subpopulation of myenteric neurons.     

Storage and release of labelled acetylcholine in the myenteric plexus of the guinea-pig ileum.

Guinea-pig ileum myenteric plexus-longitudinal muscle preparation was superfused with [3H]choline for 15 min either without being stimulated or during field stimulation at 0.1 or 16 Hz; the preparation was then either removed immediately or after 75- or 135-min superfusion with hemicholinium-3 (HC-3) and the total acetylcholine (ACh) and [3H]ACh contents were determined. For measuring the release of [3H]ACh the preparation was stimulated for 60 min the second time at 0.1 or 16 HZ in the presence of hemicholinium. Exposure to [3H]choline without stimulation resulted in the formation of [3H]ACh stores which were maintained in the first 75 min but decreased therafter. Labelling during stimulation at 16 Hz produced the largest and best maintained [3H]ACh content. Following labelling during 0.1-Hz stimulation, more label could be released than following labelling in the absence of stimulation. Labelling during 16-Hz stimulation did not increase any further in fool of [3H]ACh accessible to release by 0.1-Hz stimulation, but caused a 2.5 times increase in the pool from which Hz stimulation released [3H]ACh. These results suggest that two populations of cholinergic neurons exist in the myenteric plexus, one activated only by high frequency stimulation, the other by both high and low frequency stimulation.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.     

Bradykinin modulates the release of noradrenaline from vas deferens nerve terminals

To asses whether bradykinin influences the release of noradrenaline from the adrenergic varicosities of the vas deferens, tissues were loaded with 3H-noradrenaline. Upon electrical depolarization bradykinin increased in a concentration-dependent fashion, the overflow of tritium from the mouse or rat vas deferens. The 3H-overflow is dependent on the external Ca2+concentration suggesting neuronal release of 3H-noradrenaline. The present results add evidence to the hypothesis that bradykinin modulates the release of noradrenaline from peripheral sympathetic nerve terminals via the activation of a presynaptic mechanism.     

Stimulatory effect of intravenous calcium on pancreatic polypeptide secretion in man

In the present work we have examined the effect of i.v. calcium administration on the secretion of human pancreatic polypeptide (hPP) in normal subjects. The infusion of calcium glucono-galactono-gluconate, as to deliver 10 mg of calcium element per kilogram of body weight in two hours, was followed by a progressive elevation of plasma hPP which attained values two-fold those of the basal levels. This finding demonstrates that calcium behaves as a pancreatic polypeptide secretagogue in man.     

Possible existence of dopaminergic receptors on cholinergic nerve terminals in the guinea-pig Auerbach's plexus.

In the Auerbach's plexus of guinea-pig ileum lower concentrations of oxotremorine (Oxo-T) produced an increase, whereas higher concentrations of Oxo-T caused inhibition of evoked acetylcholine (ACh) release, measured by bioassay using dorsal muscle of leech. Dopamine inhibited the increase in evoked release of ACh induced by Oxo-T as a function of its concentration and this inhibitory effect was nullified in presence of pimozide, the dopamine receptor antagonist. The results demonstrate existence of presynaptic dopamine receptors having inhibitory influence on excitatory presynaptic muscarinic receptors on regulation of ACh release. However, no physiological role of dopamine could be observed on ACh release in this preparation.     

Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum.     

The effect of leucine-enkephalin on the peristaltic reflex of the isolated guinea-pig ileum

Leucine (leu)-enkephalin depresses or inhibits the peristaltic reflex of the isolated guinea-pig ileum. Opiate antagonists (naloxone and nalorphine), choline esters (acetylcholine, methacholine and carbachol), cholinomimetics (muscarine and arecoline) and polypeptides which stimulate peristalsis (eledoisin and angiotensin) antagonize the peristaltic block caused by leu-enkephalin. On the other hand, nicotinic ganglionic stimulants (nicotine and dimethylphenylpiperazine) as well as muscarinic ganglionic stimulants (McN-A-343 and AHR-602) do not restore the peristaltic reflex abolished by leu-enkephalin. Thus the inhibitory effect of leu-enkephalin is due mainly to an action on myenteric ganglia as well as on axon terminals of the myenteric plexus subserving the peristaltic reflex. The inhibitory action of leu-enkephalin may be ascribed to the opiate as well as to the cholinoceptive sites in the nervous elements in the myenteric plexus. The blocking action of leu-enkephalin is not associated with ganglionic muscarinic M-1 receptors as well as with ganglionic nicotinic receptors in the myenteric plexus of the guinea-pig isolated ileum.     

Multiple opiate receptors in guinea-pig ileum

Actions of the prototypic μ-, κ-, and σ-opiate receptor agonists, morphine (M) ketocyclazocine (K) and SKF-10,047. (S), respectively, were examined and differentiated using the guinea-pig ileum preparation. S, like M and K, depressed the electrically stimulated ileum. Naloxone antagonized the depressant actions of the prototypic drugs with different potencies. PA₂ values of naloxone for M, K, and S, respectively, were 8.81, 7.58 and 7.74. Relative cross tolerance of each prototypic drug to normorphine, a comparison standard, was also examined in morphine-pretreated ilea and quantitatively estimated as follows: (1) the median effective dose of each drug and of the standard drug normorphine were determined in the nontolerant ileum (IC50_NT) and in ilea with varying degrees of tolerance IC50_T); (2) cross-tolerance ratios (IC50_T/IC50_NT) of each drug and of normorphine were calculated for the varying degrees of tolerance; (3) cross-tolerance ratios of each drug were plotted against those of normorphine, the data were fit by a least squares straight line, and the slope of the line determined as the Relative Cross Tolerance Index (RCTI). RCTI for M was 2.21. K and S, however, had lower RCTI's of 0.44 and 0.64 respectively. In the morphine-pretreated tolerant ilea, slopes of the dose response curves of the prototypic drugs were found to differ: while M and K possessed steep and constant slopes for ilea with different degrees of tolerance, the slopes for S became shallower as ilea became more tolerant to morphine. A maximum ceiling effect of less than 50% depression was obtained for S in the most highly tolerant ilea. The above observations are consistent with possible existence of the three types of hypothesized opiate receptors in the guinea-pig ileum.     

The detection by X-ray microanalysis of noradrenaline in sympathetic nerve terminals of the rat vas deferens

Summary The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.This investigation was supported by research Fellowship No. 7/176(138)77 from the Council of Scientific & Industrial Research, New Delhi to the first author     

The calcium channel antagonist omega-conotoxin inhibits secretion from peptidergic nerve terminals

The binding of omega-conotoxin to isolated rat neurohypophysial nerve terminals, its effect on the depolarization-induced increase of cytoplasmic Ca2+ and on the potassium and electrically-induced release of vasopressin (AVP) have been studied. The results show that isolated neurosecretory nerve endings have calcium channels with a high affinity for omega-CgTx and that this toxin inhibits neurohormone release at very low concentration (IC50 = 0. 1nM). Although secretion of vasopressin is inhibited to a great extent by the toxin it is shown that a small but significant amount of the depolarization-induced AVP release is insensitive to omega-CgTx and to the dihydropyridine molecule nicardipine.     

The topography and dynamics of cholinergic transmission in the myenteric plexus-smooth muscle preparation of the guinea-pig ileum; the effect of ketocyclazocine, a kappa opiate ligand

Output of acetylcholine (ACh), neurogenic electromyogram (NEMG) and contractions of guinea-pig ileum preparations were studied during stimulation by high-frequency trains of impulses. Under control conditions the output of ACh per impulse after 2nd to 4th impulses during train stimulation (30 Hz) was higher by 20-40% than the level of ACh output during the first impulse. In the presence of ketocyclazocine (KTZ, 80 nmol x l-1) the output of ACh evoked by the first impulse was more effectively inhibited than that after impulses 2 to 4 so that the increase was higher (80-170%). NEMG, a direct consequence of the localized action of released transmitter (ACh), was recorded in the longitudinal muscle 4 and 10 mm aborally from the focal stimulation site. The incidence of NEMG responses was higher at the proximal than at the distal site and was proportional to the number of impulses in a train (100 Hz). At the distal site KTZ suppressed the appearance of NEMG responses to single impulses whereas at the proximal site its effect was much less; and so was its effect at either site during train stimulation. It is concluded that in the course of train stimulation, sites of transmission more distant from the stimulation focus were recruited, and consequently the secretion of ACh in succeeding impulses was enhanced. KTZ might preferentially inhibit the propagation of excitation by the very first impulse.     

Stimulatory action of prolactin on gonadotropin secretion in vitro

The action of prolactin (PRL) on the secretion of gonadotropin was investigated by means of a cell culture system of rat anterior pituitary gland. Anterior pituitary glands were removed from Wistar male rats, enzymatically digested and cultured. Luteinizing hormone (LH) release into medium was increased by adding PRL dose-dependently in the range between 10 ng/ml and 1 microgram/ml. This effect of PRL was further augmented by the presence of either gonadotropin-releasing hormone or estradiol. The intracellular LH concentration was also increased by PRL. PRL also caused an increase in follicle-stimulating hormone release into medium dose-dependently. In conclusion, PRL was shown to stimulate the secretion of gonadotropin at the pituitary level, thus suggesting a paracrine mode of PRL action in the anterior pituitary gland.     

Neurochemical coding of myenteric neurons in the guinea-pig antrum

Electrophysiological studies of myenteric neurons in the guinea-pig antrum suggest that different neuroactive compounds are involved in synaptic transmission. It is not known what neurotransmitters and neuropeptides are present and to what extent they colocalize. Immunohistochemical stainings were performed on whole-mount preparations of the guinea-pig antrum. Immunoreactivity for neuron-specific enolase was used as a general marker and was set at 100%. There was no overlap between cholinergic and nitrergic neurons, resulting in two separate subpopulations. The presence of choline acetyltransferase immunoreactivity was used to identify the cholinergic subset, which accounted for 56% of the cells. Immunoreactivity for nitric oxide synthase, on the other hand, was displayed in 40.7% of the neurons. Substance-P immunoreactivity was present in 37.4% of the cells and vasoactive intestinal peptide and neuropeptide Y in 21.7% and 28.6%, respectively. Small subsets of neurons had immunoreactivity for serotonin (3.9%), calretinin (6.8%) and calbindin (0.5%). Colocalization studies revealed several subgroups of neurons, containing one or more of the screened markers. Though some similarity is found in the chemical coding of the antrum compared to that of the small intestine and the corpus, remarkable differences can be seen in the occurrence of some subpopulations. Cholinergic neurons are not as predominant as in other parts of the gut, serotonin presence is doubled and some vasointestinal-peptide-positive neurons express substance P. These differences might reflect the highly specialized function of the antrum; however, the exact role of these classes remains to be established.