Paneth cells of the human small intestine express an antimicrobial peptide gene.

Mucosal surfaces of several organ systems are important interfaces for host defense against microbes. Recent evidence suggests that antimicrobial peptides contribute to the defense of these surfaces. Defensins are one family of antimicrobial peptide, but their known distribution in humans has been limited to four members found in cells of myeloid origin. We sought to determine if the human defensin family was more complex. We found that the family of human defensins is diverse and is not restricted to expression in leukocytes. Southern blot and genomic clone analyses reveal that numerous defensin-related sequences are present in the human genome. A gene for a new human defensin family member was characterized. This gene, designated human defensin-5, is highly expressed in Paneth cells of the small intestine. This is the first example of an antimicrobial peptide gene expressed in an epithelial cell in humans. The data support the hypotheses that epithelial defensins equip the human small bowel with a previously unrecognized defensive capability which would augment other antimicrobial defenses.     

The mechanism of motilin excitation of the canine small intestine

Close intraarterial injections of motilin to the small intestine of the anaesthetized dog produce prolonged phasic contractions. Tetrodotoxin infused intraarterially blocked field stimulated contractions and abolished the response to motilin as did treatment with a combination of hexamethonium and atropine. Atropine alone increased the dose of motilin required to induce responses. Hexamethonium alone similarly increased the dose of motilin required in the jejunum, but not for the ileum. These results suggest that motilin acts to contract small intestine by stimulation of intrinsic excitatory nerves, some of which are post-ganglionic cholinergic and some of which are not, but are activated by a pathway with a nicotinic synapse. The ED₅₀ for ileal contractions was greater than that for the jejunum and the time to reach maximum contractions longer suggesting a decreased responsiveness of the lower small intestine to motilin as compared to the upper gastro-intestinal tract. These results and the lesser quantity of immunoreactive motilin in the ileum than in the jejunum may explain the lack of relationship of the activity front of the migrating motor complex in the lower small intestine to venous motilin concentrations.     

Motor effects of gastrin releasing peptide (GRP) and bombesin in the canine stomach and small intestine

Close intraarterial injections of synthetic porcine gastrin releasing peptide (GRP) or bombesin stimulated contractions in the stomach and inhibited ongoing contractile activity in the small intestine of anaesthetized dogs. Contractile activity of the circular muscle was recorded by serosal strain gauges and phasic activity when desired was elicited by local field stimulation or intraarterial motilin injections. In the stomach (corpus and antrum) following tetrodotoxin blockade of field-stimulated contractions, the contractile response to either peptide was not present, suggesting that stimulation of receptors on nerves initiated contractions in the stomach. Similarly, in the small intestine, the inhibitory response was eliminated by tetrodotoxin suggesting a neural receptor. Pre-treatment with reserpine did not alter the inhibitory response, either in the presence or absence of atropine, therefore, adrenergic inhibitory mechanisms did not appear to be involved. The concentration of bombesin producing 50% inhibition of field stimulation (ED50) was increased following treatment with the putative M1 muscarinic antagonist, pirenzipine suggesting activation of M1 cholinergic inhibitory receptors by bombesin. After blockade by atropine of field-stimulated contractions and the contractile response to intraarterial acetylcholine, the ED50 for bombesin inhibition of motilin contractions was increased. After muscarinic blockade, the residual inhibitory response of GRP/bombesin may involve activation of a neural non-cholinergic non-adrenergic inhibitory mechanism. These results suggest that GRP and bombesin act to alter motility in the dog in vivo by affecting neural activity.     

Receptors for neurotensin in canine small intestine.

We have previously characterized the neurotensin receptors on the circular smooth muscle (CM) of the canine small intestine (1). In the present studies, using radioligand binding technique, neurotensin receptors were localized on the membranes from deep muscular (DMP) and the submucous plexus while no binding was observed on either the longitudinal smooth muscle or myenteric plexus membranes. The high affinity binding sites (Kd 0.1-0.2 nM) on DMP membranes were similar to those on CM; the low affinity component was of much lower affinity (Kd approximately 40 nM). DMP had 4-6 times higher density of binding sites than the CM. The recognition properties of DMP receptors were similar to those on the CM and reduced sulfhydryl groups were required for the binding activity. The action of neurotensin on the contractility of the canine small intestine, therefore, appears to be through a direct action on the circular smooth muscle and through the prejunctional action on the DMP neurons through distinct receptors. Thiol groups in the neurotensin receptors may be important for the receptor function.     

VIP and PHI coexist with an NPY-like peptide in intramural neurones of the small intestine

Vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and neuropeptide Y (NPY) are neuropeptides present in all layers of the small intestine. NPY-immunoreactive fibres in the gut seem to derive from two sources. One population is of extramural (sympathetic) origin and contains noradrenaline, another is of intramural origin and does not contain noradrenaline. In the present study of mouse, rat and pig, immunocytochemistry showed immunoreactive PHI to coexist completely with immunoreactive VIP. This was predictable, since VIP and PHI derive from the same precursor. In addition, however, VIP and PHI were found to coexist with immunoreactive NPY in non-adrenergic (but not in adrenergic) nerve fibres and nerve cell bodies. This coexistence was unexpected, since the VIP precursor does not contain NPY-like sequences.     

Epithelial distribution of neural receptors in the guinea pig small intestine

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.     

Longitudinal and circumferential spike patches in the canine small intestine in vivo

In an open-abdominal anesthetized and fasted canine model of the intact small intestine, the presence, location, shape, and frequency of spike patches were investigated. Recordings were performed with a 240-electrode array (24 x 10, 2-mm interelectrode distance) from several sites sequentially, spanning the whole length of the small intestine. All 240 electrograms were recorded simultaneously during periods of 5 min and were analyzed to reconstruct the origin and propagation of individual spikes. At every level in the small intestine, spikes propagated in all directions before stopping abruptly, thereby activating a circumscribed area termed a "patch." Two types of spikes were found: longitudinal spikes, which propagated predominantly in the longitudinal direction and occurred most often in the duodenum, and a second type, circumferential spikes, which propagated predominantly in the circular direction and occurred much more frequently in the jejunum and ileum. Circumferential spikes conducted faster than longitudinal spikes (17 +/- 6 and 7 +/- 2 cm/s, respectively; P < 0.001). Circumferential spikes originated in >90% of all cases from the antimesenteric border, whereas longitudinal spikes were initiated all around the circumference of the intestinal tube. Finally, the spatial sequence of spike patches after the slow wave was very irregular in the upper part of the intestine but much more regular in the lower part. In conclusion, spikes and spike patches occur throughout the small intestine, whereas their type, sites of origin, extent of propagation, and frequencies of occurrence differ along the length of the small intestine, suggesting differences in local patterns of motility.     

Calcium channel binding in nerves and muscle of canine small intestine

Using [3H]-nitrendipine (Nit) and [125I]-omega conotoxin (w-CTX), the cellular and subcellular distribution of calcium channel subtypes in the homogenates of canine small intestinal circular muscle was studied. Nit. bound to the membranes from the circular smooth muscle cells (PM) and to the synaptosomal membranes from the deep muscular plexus (DMP); the Kd and Bmax values of Nit binding from these two sources were similar (Kd 0.4 +/- 0.16 nM and 0.77 +/- 0.24 nM; Bmax 206 +/- 22 and 192 +/- 39 fmol/mg of protein in DMP and PM respectively). w-CTX, however, bound only to the DMP (Kd 18.41 +/- 7.5 pM, Bmax 265 +/- 36 fmol/mg of protein). In DMP, nifedipine (10(-6) M) failed to interact with the binding of w-CTX; similarly, no modulation of Nit binding with unlabelled w-CTX (10(-7) M) could be detected. Therefore w-CTX and Nit binding sites represent two distinct, non-interactive and differentially distributed binding sites in canine small intestine.     

Cholecystokinin-dependent selective inhibitory effect on 'minute rhythm' in the ovine small intestine

Cholecystokinin (CCK) can exert multiple actions on intestinal motility but its effect on the small-intestinal 'minute rhythm' (MR) is virtually unknown. Therefore, the electrical activity from the abomasal antrum, duodenal bulb, duodenum, jejunum and ileum was continuously recorded in six sheep before, during and after slow intravenous administration, of three doses each, of cholecystokinin-octapeptide (CCK-OP) and cerulein. In four of these sheep, two additional electrodes and the strain gauge force transducer were also inserted in the duodenum. Chronic experiments were performed in the fasted and non-fasted animals and saline or CCK peptides were injected during phases 1, 2a or 2b of the duodenal migrating myoelectric complex (MMC). The administration of both CCK peptides in various doses evoked an inhibitory effect mostly in the duodenal bulb, except for the lowest dose of cerulein. The effects of 20 times greater doses of CCK-OP than that of cerulein were more pronounced. The introduction of both CCK peptides during phase 1 of the MMC produced no marked or significant response. In non-fasted animals, the effects of both hormonal peptides, given during phase 2b of the MMC, were often stronger than those given during phase 2a, while in fasted animals the effects of CCK peptides, administered in the course of phases 2a and 2b of the MMC, were similar. Both higher doses of CCK peptides increased the number of spike bursts within the given MR pattern in the duodenum and decreased the incidence of MR mostly in the duodenal bulb. The inhibitory effects of both CCK peptides on the bulbar MR exhibited a dose-response character, though the lowest dose often evoked the slight stimulatory response. It is concluded that CCK principally exerts an inhibitory effect upon the MR in the duodenal bulb and modifies the MR in the duodenum by increasing the spike burst number in a given MR pattern. Both these actions of CCK peptides seem to be physiological. There is a positive relationship between the intensity of the refractory period and the demonstrated effect of CCK in the duodenum.     

5-Hydroxytryptophan modulates postprandial motor patterns of canine proximal small intestine

The aim of the study was to clarify whether 5-hydroxytryptophan (5-HTP) stimulates the postprandial motor pattern of the duodenum in a similar way as that of the adjacent jejunal segment in dogs. Computerized analysis of motor patterns recorded by closely spaced strain gauges focused on the temporal and spatial distribution of the contractions. Results indicate that 5-HTP increased the incidence and the length of the spread of contraction waves after both an acaloric and a nutrient meal in the duodenum as well as in the adjacent jejunal segment. Effects were more pronounced after the nutrient than after the acaloric meal. After the nutrient meal, but not after the acaloric meal, 5-HTP additionally enhanced the number of both duodenal and jejunal contractions per minute and increased the force of duodenal contractions. The acaloric meal induced significant differences in the motor patterns between the duodenum and the adjacent jejunum. 5-HTP abolished these differences owing to a relatively stronger stimulation of duodenal motility. 5-HTP did not affect gastric emptying of both meals. We conclude (i) that 5-HTP is a potent stimulator of propagated contractions both in the duodenum and the adjacent jejunal segment and (ii) that intestinal motor patterns can be regulated independently of gastric emptying.     

Neuronal involvement in the effect of an antisecretory factor-derived peptide on induced secretion in the porcine small intestine

The antisecretory factor is a protein inhibiting enterotoxin-induced intestinal inflammation and hypersecretion. We studied the signaling pathway of three antisecretory factor-derived peptides (A1, A3 and A4) in the proximal and distal porcine small intestine. In vivo (ligated loops), only A3 significantly reduced the cholera toxin-induced fluid accumulation and only in proximal loops. A3 and A4 reduced Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the proximal segment, whereas A1 and A3 reduced the Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the distal segment. The secretory response to intraluminally added 5-hydroxytryptamine was not significantly inhibited by the peptides. The amount of intraluminal 5-hydroxytryptamine accumulated in cholera toxin-stimulated loops from the proximal segment was significantly reduced by A3. In vitro,the effect of A3 on secretagogue-induced increases in short-circuit current was recorded from proximal small intestine by the Ussing chamber technique. A3 decreased the tetrodotoxin sensitive effect of substance P. The in vivo results suggest that the antisecretory effect may involve inhibition of the local release of 5-hydroxytryptamine induced by cholera toxin, but not inhibition of secretory reflexes induced by 5-hydroxytryptamine. The in vitro results suggest that the effect of A3 lies beyond the surface epithelium, and involves mucosal neuronal structures.     

A slow wave frequency complex of the canine small intestine during the fasting state

The electrical activity of the duodenum and proximal jejunum was studied in conscious healthy dogs implanted with unipolar silver electrodes. A computerized method was used for the calculation of the mean frequency of the slow wave for each consecutive minute of the electromyographic signal. A "slow wave frequency complex" was identified in the fasted animals. It was characterized by an increase of the mean frequency of the slow wave which ranged, from one dog to another, between 1 and 3 cycles/min. The complex lasted about 30 min. It consisted of two distinct phases: a phase of increasing frequency of the slow wave which lasted about one-third of the total duration of the complex and a phase of progressive return of the frequency to its precomplex value. Each phase III of the migrating myoelectric complex occurring in both the duodenum and the jejunum was associated with one slow wave frequency complex. The phase III began a few minutes before the start of the slow wave frequency complex and ended a few minutes before the slow wave frequency reached its maximum. Ectopic phase IIIs which occurred in the jejunum but not in the duodenum were not associated with slow wave frequency complexes. The slow wave frequency complex was never seen in the fed dogs.     

Neurotensin receptors on circular smooth muscle of canine small intestine: role of disulfide bridges

We have studied the effect of sulfhydryl agents on the binding of 125I-tyr3-neurotensin to the purified plasma membranes from circular smooth muscle and on the in vitro response of circular muscle strips of canine small intestine to neurotensin. Dithiothreitol (DTT) enhanced the binding by about 80%. Cysteine (a reductant) also enhanced the binding while cystine (an oxidant) reduced the binding to the similar extent. DTT stimulated the tissue in the organ bath and abolished the stimulatory response to low concentrations of neurotensin. The stimulatory response to acetylcholine was not altered by DTT. The implications of the role of disulfide bridges in the neurotensin response is discussed.     

Alpha 2-adrenergic receptors on the circular smooth muscle of canine small intestine.

We have studied the distribution and properties of alpha 2-adrenergic receptors in the circular muscle layer (containing deep muscular plexus) of canine small intestine. Using radioactivity labelled rauwolscine, we located the binding sites to the neuronal membranes supporting the prejunctional action of alpha 2-adrenergic agents in the gut. Moreover, although the functional data to suggest the existence of postjunctional alpha 2-adrenergic receptors coupled to contraction are not available so far, we measured a substantial number of rauwolscine binding sites on the smooth muscle plasma membranes. Scatchard and Hill analyses of the saturation data were indicative of the presence of a single high affinity site (Hill coefficient 0.996) with a KD value of 8.8 nM and the maximum number of binding sites (Bmax) of 313 fmol/mg of protein. Competition studies suggested the presence of multiple subtypes of alpha 2-adrenoceptors.     

Localization of calcitonin gene-related peptide in the small intestine of various vertebrate species

Summary Calcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.     

Galanin mediated inhibitory nervous modulation of cutaneous vascular reactions

Noxious stimulation induces local inflammatory responses in a variety of mammals but these reactions are only faint in avian species. The possibility that endogenous galanin inhibits neurogenic vascular responses in avians was tested in the wing skin of anaesthetized pigeons. Intraarterial infusion of nanomolar concentrations of the specific galanin antagonist M35 dose dependently enhanced the small mustard oil induced increase of skin blood flow measured by means of a Laser Doppler Imager. Similarly, the small transient vasodilatation following electrical stimulation of a cutaneous nerve was also enhanced by M35. The effect of M35 was not observed after chronic denervation. Coperfusion of M35 dose dependently augmented the histamine and bradykinin induced plasma extravasation revealed by skin microdialyses, but this effect was abolished in the chronically denervated skin. However, chronic denervation per se enhanced the plasma extravasation induced by histamine but not by bradykinin and this effect was diminished by coperfusion of galanin. The results suggest an inhibitory modulation of cutaneous neurogenic inflammatory reactions by endogenous galanin in the pigeon.