Controlled ingestion of kaolinite (5%) modulates enteric nitrergic innervation in rats.

We have previously shown that kaolinite slowed down gastric emptying and intestinal transit and induced changes in enteric mechanical activities. As gastric emptying and intestinal transit have been shown to be regulated by nitric oxide (NO), the effect of an imposed ingestion of kaolinite on enteric nitrergic innervation was determined. Kaolinite has also been shown to increase plasmatic levels of leptin. Therefore, the responses of enteric neurons in the presence of leptin after kaolinite ingestion were determined, and a possible role of nitrergic neurons was evaluated in rats using organ bath technique. Our results showed that kaolinite modulates activities of enteric nerves at 14 days of ingestion. Exogenous l-NNA produced a decrease in nerve stimulation (NS)-induced relaxation in both jejunum and colon of control groups. At 14 days of kaolinite ingestion, this effect of l-NNA was significantly reduced only in the jejunum. Although l-NNA did not affect NS-induced contraction in jejunum and colon of control animals, it increased the amplitude of the NS-induced contraction in the colon of rats at 14 days of kaolinite ingestion. Leptin inhibitory effects on ENS in the jejunum were also altered at 14 days of ingestion. These differences were masked in the presence of l-NNA. Our data give evidence that changes in mechanical activities induced by kaolinite might be due to alterations in inhibitory (nitrergic and/or other) innervation at 14 days of kaolinite ingestion and to modifications of leptin effects on the responses to intramural nerve stimulation.

Kaolinite ingestion facilitates restoration of body energy reserves during refeeding after prolonged fasting.

Clay consumption is a spontaneous behavior currently observed in animals and humans, particularly during undernutrition. Often regarded as intestinal care products, ingested clays also enhance food efficiency, notably by increasing intestinal lipid uptake. Clay complementation could then optimize the reconstitution of energy reserves in animals with low lipid stocks consecutive to intensive fasting. The aim of this study was therefore to observe the effects of voluntarily kaolinite complementation during the refeeding of fasted rats to determine whether body mass, food uptake, lipid and mineral contents as intestinal morphology and protein profile were modified. This study examined two types of refeeding experiments after prolonged fasting. Firstly, rats with ad libitum access to food were compared to rats with ad libitum access to food and kaolinite pellets. Animals were randomly put into the different groups when the third phase of fasting (phase III) reached by each individual was detected. In a second set of experiments, rats starting phase III were refed with free access to food and kaolinite pellets. When animals had regained their body mass prior to fasting, they were euthanized for chemical, morphological, and proteomic analyses. Although kaolinite ingestion did not change the time needed for regaining prefasting body mass, daily food ingestion was seen to decrease by 6.8% compared with normally refed rats, without affecting lipid composition. Along the intestinal lining, enterocytes of complemented animals contained abundant lipid droplets and a structural modification of the brushborder was observed. Moreover, the expression of two apolipoproteins involved in lipid transport and satiety (ApoA-I and ApoA-IV) increased in complemented rats. These results suggest that kaolinite complementation favors intestinal nutrient absorption during refeeding despite reduced food uptake. Within the intestinal lumen, clay particles could increase the passive absorption capacity and/or nutrient availability that induce mucosal morphological changes. Therefore, clay ingestion appears to be beneficial for individuals undergoing extreme nutritional conditions such as refeeding and limited food supplies.

Effects of controlled ingestion of kaolinite (5%) on food intake, gut morphology and in vitro motility in rats.

Geophagia is found in various animal species and in humans. We have previously shown that spontaneously ingested kaolinite interacts with the intestinal mucosa modifies nutrient absorption and slows down gastric emptying and intestinal transit in rats in vivo. However, the precise mechanisms involved are not elucidated. The aim of this work was to investigate the effects of controlled kaolinite ingestion on food intake, gut morphology and in vitro motility in rats. Male Wistar rats were fed with 5% kaolinite in standard food pellets during 7, 14 and 28 days. Body mass and food consumption were measured daily. Intestinal morphological and proteomic analyses were conducted. The length of mucosal lacteals was evaluated. Plasmatic levels of leptin and adiponectin were determined. Finally, organ bath studies were conducted to evaluate smooth muscle contractility. Food consumption was significantly increased during the first two weeks of kaolinite ingestion without any mass gain compared to controls. Kaolinite induced weak variations in proteins that are involved in various biological processes. Compared to control animals, the length of intestinal lacteals was significantly reduced in kaolinite group whatever the duration of the experiment. Leptin and adiponectin plasmatic levels were significantly increased after 14 days of kaolinite consumption. Changes in spontaneous motility and responses to electrical nerve stimulation of the jejunum and proximal colon were observed at day 14. Altogether, the present data give evidence for a modulation by kaolinite-controlled ingestion on satiety and anorexigenic signals as well as on intestinal and colonic motility.

Interactions between ingested kaolinite and the intestinal mucosa in rat: proteomic and cellular evidences.

Although some of the effects of clay ingestion by humans and animals, such as gastrointestinal wellness and the increase in food efficiency are well known, the underlying mechanisms are not yet fully understood. Therefore, the interactions between the intestinal mucosa and kaolinite particles and their effects on mucosal morphology were observed using light microscopy (LM), transmission electron microscopy (TEM), conventional (CSEM) and environmental (ESEM) scanning electron microscopy combined with an EDX micro-analysis system. Kaolinite consumption, given with free access to rats, varied considerably from one animal to the other but was regular through time for each individual. Some kaolinite particles appeared chemically dissociated in the lumen and within the mucus barrier. Aluminium (Al) originating from ingested clay and present in the mucus layer could directly cross the intestinal mucosa. A significant increase in the thickness of the villi with large vacuoles at the base of the mucosal cells and a decrease in the length of enterocyte microvilli characterized complemented animals. The proteomic analyses of the intestinal mucosa of complemented rats also revealed several modifications in the expression level of cytoskeleton proteins. In summary, kaolinite particles ingested as food complement interact with the intestinal mucosa and modify nutrient absorption. However, these data, together with the potential neurotoxicity of Al, need further investigation.

Clay ingestion enhances intestinal triacylglycerol hydrolysis and non-esterified fatty acid absorption.

Consumption by animals and humans of earthy materials such as clay is often related to gut pathologies. Our aim was to determine the impact of kaolinite ingestion on glucose and NEFA transport through the intestinal mucosa. The expression of hexose transporters (Na/glucose co-transporter 1 (SGLT1), GLUT2, GLUT5) and of proteins involved in NEFA absorption (fatty acid transporter/cluster of differentiation 36 (FAT/CD36), fatty acid transport protein 4 (FATP4) and liver fatty acid binding protein (L-FABP)) was measured (1) in rats whose jejunum was perfused with a solution of kaolinite, and (2) in rats who ate spontaneously kaolinite pellets during 7 and 28 d. Also, we determined TAG and glucose absorption in the kaolinite-perfused group, and pancreatic lipase activity, gastric emptying and intestinal transit in rats orally administered with kaolinite. Glucose absorption was not affected by kaolinite perfusion or ingestion. However, kaolinite induced a significant increase in intestinal TAG hydrolysis and NEFA absorption. The cytoplasmic expression of L-FABP and FATP4 also increased due to kaolinite ingestion. NEFA may enter the enterocytes via endocytosis mainly since expression of NEFA transporters in the brush-border membrane was not affected by kaolinite. After uptake, rapid binding of NEFA by L-FABP and FATP4 could act as an intracellular NEFA buffer to prevent NEFA efflux. Increased TAG hydrolysis and NEFA absorption may be due to the adsorption properties of clay and also because kaolinite ingestion caused a slowing down of gastric emptying and intestinal transit.

Effect of acetic acid or trypsin application on rat colonic motility in vitro and modulation by two synthetic fragments of chromogranin A.

The hypothesis that Chromogranin A (CgA)-derived peptides are involved in mechanisms modulating altered colonic motility was tested. Rat distal colonic strips were studied using an organ bath technique. Acetic acid (AA)-induced effects were characterized on spontaneous mechanical activities (SMA) in the presence of CgA4-16 or CgA47-66. In preparations with mucosa, AA induced a transient hyperactivity followed by a decrease in tone. The first phase is sensitive to tetrodotoxin (TTX) and capsaicin. The second phase was sensitive to BAYK8644 but insensitive to L-nitro-arginine-methyl-ester (L-Name)/apamin together. CgA4-16 or CgA47-66 alone produced no change on SMA. The administration of CgA4-16 prior to AA increased the duration of the excitatory component and reduced tone inhibition. CgA47-66 prior to AA only decreased duration of the excitatory phase. In preparations without mucosa, AA decreased tone. This effect was sensitive to BAYK8644 and CgA4-16. Trypsin decreased basal tone. This effect was suppressed by TTX, BAYK8644 or L-Name/apamin and were reduced by CgA4-16. AA-induced effects on rat colonic motility in vitro may be mediated through activation of primary afferents and an action at L-Type calcium channels. CgA-derived peptides are shown to decrease AA-induced effects on motility.

The effect of a chromogranin A-derived peptide (CgA4-16) in the writhing nociceptive response induced by acetic acid in rats.

The nociceptive effects of i.p administration of a synthetic peptide (CgA4-16) derived from chromogranin A (CgA) were studied on a model of inflammatory (somato-visceral) pain. Inflammatory mediators participate in controlling the activity of enterochromaffin cells that store and release chromogranins. Adult male Wistar rats were injected i.p with diluted acetic acid (AA) to induce abdominal writhes. Pharmacological agents were injected prior to CgA4-16 and/or AA together. While i.p CgA4-16 alone did not produce any effect, the peptide increased the number of abdominal constrictions induced by i.p AA administration in a dose-related manner. To determine the possible mechanisms involved in CgA4-16 produced pronociceptive effect, i.p diltiazem or indomethacin were tested. The pronociceptive effect induced by CgA4-16 was blocked by pretreatment of either substance. I.p administration of CGRP, substance P (SP) or capsaicin evoked dose-related abdominal writhing. CgA4-16, 20 min prior to CGRP or capsaicin, potentiated the nociceptive effects induced by CGRP or capsaicin, but not those induced by SP. Taken together, these data suggest for the first time that a CgA-derived peptide may modulate inflammatory pain.

A role for chromogranin A (4-16), a vasostatin-derived peptide, on human colonic motility. An in vitro study.

The hypothesis that CgA-derived peptides may be involved in mechanisms modulating motility was tested. Human colonic smooth muscles were studied using an organ bath technique. Acetic acid (AA) effects were characterized on spontaneous mechanical activities (SMA) and on responses to transmural nerve stimulation (NS). AA induced a significant decrease in tone and abolished SMA; this effect was insensitive to either TTX or L-NAME/apamin. The AA-induced inhibitory effects were significantly reduced in the presence of CgA4-16. This effect was insensitive to TTX or L-NAME/apamin. Furthermore, AA-induced effects were blocked in the presence of BAYK8644 and CgA4-16 together. The inhibitory effect of nifedipine was delayed in the presence of CgA4-16. NS induced a triphasic response. Only the excitatory components were reduced in the presence of AA. This effect was dose-related and remained unchanged in the presence of CgA4-16 alone, but was blocked in the presence of simultaneous administration of CgA4-16 and L-NAME/apamin. AA application induced inhibition of human colon motility in vitro. This effect may be mediated through an action on L-type calcium channels. CgA4-16 may display a protective role, which prevents the inhibition of motility due to AA to occur, by acting on both smooth muscle and afferent terminals.

Effects of a chromogranin-derived peptide (CgA 47-66) in the writhing nociceptive response induced by acetic acid in rats.

Chromogranin A (CgA) is an acidic protein identified within a large variety of endocrine cells. Colocalized with catecholamines in chromaffin cells, CgA is a prohormone precursor of small biologically active peptides. Vasostatin (CgA 1-76) is the most conserved fragment of CgA and chromogranin A 47-66 peptide (CgA 47-66) possesses potent antimicrobial activities. The aim of this study was to test the hypothesis that CgA 47-66 may be involved in mechanisms modulating nociception. Thus, we used acetic acid (AA) which produces a delayed inflammatory response and episodes of abdominal writhing, a marker of pain, when injected intraperitoneally (i.p.) to rats. Administration (i.p.) of CgA 47-66 induced specific opposite dose-dependent effects depending on concentration. That is, CgA 47-66 below 0.5 mg/kg produced antinociceptive effects, whereas at 2 mg/kg it produced a marked pronociceptive effect. The latter effect was blocked by diltiazem and indomethacin. CgA 47-66-induced antinociceptive effects on AA-induced responses were reversed when the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF 9-41 was i.p. injected to animals prior to AA and CgA 47-66 administration. The administration of i.p. calcitonin gene-related peptide (CGRP) or substance P (SP) evoked dose-dependent abdominal writhing; this effect was abolished when CgA 47-66 was injected. The present data suggest, for the first time, that a fragment of CgA, CgA 47-66, possesses potent antinociceptive effects at low doses. Although the mechanism triggered by this peptide is unknown, CRF receptors are likely to be involved.

Functional evidence for a role of GABA receptors in modulating nerve activities of circular smooth muscle from rat colon in vitro.

In the enteric nervous system, activation of neuronal GABA(A)- and GABA(B)-receptors has been shown to modulate neuronal activity. The consequences of this modulation depend on the location in the gastrointestinal tract or the animal species studied. These data illustrate the complexity of GABA-induced effects. Furthermore, the GABA(C)-receptor has been identified in a neuroendocrine cell line suggesting a modulating role of this third type of GABA receptor in intestinal functions. Therefore, the modulating role of GABA-receptor agonists was determined in circular preparations of rat distal colon during electrical nerve stimulation (NS) in vitro. Mechanical response to NS was characterized by a relaxation followed at the end of the stimulation by an off-contraction. In normal Krebs solution (basal conditions), muscimol and baclofen, respectively GABA(A)- and GABA(B)-agonists, induced a significant increase of the electrically induced off-contraction. The GABA(C) agonist, CACA, showed no significant effect on the response to NS. Excitatory effects of muscimol on the off-contraction were abolished in the presence of atropine. Furthermore, in the presence of atropine, muscimol increased the amplitude of the electrically induced relaxation; similarly the baclofen-induced increase of off-contraction amplitude was significantly lower than that observed in control conditions. Baclofen and muscimol effects on the off-contraction were abolished in the presence of hexamethonium or guanethidine. Furthermore, muscimol and baclofen did not induce any significant change on the response to NS in the presence of L-NAME and apamin together. Thus, it seems that in rat distal colon, GABA regulates significantly both excitatory (through GABA(A)- and GABA(B)-receptors) and inhibitory (through GABA(A)-receptors) neuronal activities. We also gave evidence for a possible interplay between GABAergic intrinsic neurons and adrenergic nerve terminals. Finally, it is shown for the first time the presence of the GABA vesicular transporter (VIAAT) around myenteric ganglia of rat colon.