Involvement of enteric nerves in permeability changes due to deoxycholic acid in rat jejunum in vivo.

AIM: Stress and Clostridium difficile toxin A increase epithelial permeability in the small intestine via vagus and visceral afferents, in turn activating mucosal mast cells. Bile acids also increase epithelial permeability but it is not known if nerves or mast cells are involved in this effect in the small intestine. METHOD: In jejunum of anesthetized rats, the effects of hexamethonium and atropine on deoxycholic acid (DCA) induced fluid secretion and increase in epithelial permeability was therefore studied by determining the appearance and disappearance rates of 14C-mannitol and 51Cr-EDTA into and from a perfusion system containing 4 or 8 mm DCA and expressed as clearance. RESULTS: DCA increased net fluid transport and appearance and to a less extent disappearance rates of the probes. Hexamethonium but not atropine, chronic denervation or the NO synthase inhibitor L-NNA did significantly decrease the appearance rate and net fluid secretion. The levels of the mast cell protease II (RMCP II) in perfusate and plasma were not increased by DCA. The clearance ratio Cr-EDTA/mannitol indicates that the plasma clearance of the permeability probes is partly secondary to net fluid transport only at higher DCA concentrations. CONCLUSION: We conclude that the DCA effect on epithelial permeability is to a large part induced by intramural reflex(es) containing nicotinic receptors. The results also suggest that mast cell degranulation and NO release are not involved in the mechanism. This indicates that the nerve effect on intestinal paracellular permeability is not mediated by the mechanisms described for stress or Clostridium difficile toxin A.

Water absorption enhances the uptake of mannitol and decreases Cr-EDTA/mannitol permeability ratios in cat small intestine in situ.

BACKGROUND: Recently, we hypothesized that mannitol absorption in human intestinal permeability tests is a reflection of small intestinal water absorption and is dependent mainly on the efficiency of the countercurrent multiplier in the villi. This may affect the outcome of clinical double-sugar permeability tests. We tested the hypothesis in cats, another species with an efficient countercurrent multiplier. METHODS: The lumen-to-tissue transport of [14C]mannitol and [51Cr]EDTA was studied in in situ perfused jejunum of eight anaesthetized cats using four isotonic perfusion solutions with varying sodium and glucose content. The transport of water was monitored, and the absorption rate of the probes was calculated by their disappearance from the perfusate. RESULTS: There was a significant positive correlation between water absorption and [14C]mannitol clearance from the different perfusates (r = 0.99; P < 0.01), whereas this correlation was absent for [51Cr]EDTA clearance (r = 0.05; P = 0.95). There was also a significant negative correlation between water absorption and [51Cr]EDTA/[14C]mannitol clearance ratios (r = 0.98; P < 0.02). CONCLUSIONS: The results show a prominent effect of water absorption on mannitol uptake through pores which, also during glucose transport, exclude Cr-EDTA. The difference in water absorption from the solutions used in cat small intestine is dependent on the effectiveness of the countercurrent multiplier; we conclude that the capability of this mechanism influences mannitol absorption in vivo. Qualitatively comparable results were obtained using oral test solutions with varying NaCl and glucose concentrations in human volunteers. We propose that the functioning of the countercurrent multiplier is essential for the interpretation of double-sugar tests in clinical studies.

Effect of cholera toxin on passive transepithelial transport of 51Cr-ethylenediaminetetraacetic acid and 14C-mannitol in rat jejunum.

Intestinal fluid secretion, mainly derived from the crypts, induced, for example, by cholera toxin, decreases the passive transport of small hydrophilic molecules into the lumen. However, the effect of the fluid secretion on the passive absorption of these substances and thus on the permeability of the villus absorptive area is not known. Therefore, the transport rates of 51Cr-ethylenediaminetetraacetic acid (EDTA) and 14C-mannitol from lumen to plasma and from plasma to lumen were recorded in jejunal loops of anaesthetized rats during cholera toxin-induced fluid secretion in the absence and presence of glucose in the intestinal lumen and expressed as clearance (microL (min g)(-1)). The results showed that the cholera toxin induced fluid secretion and abolished the passive absorption of 51Cr-EDTA both in the absence and presence of luminal glucose during a high perfusion rate (0.5 mL min(-1)). The clearance of mannitol was also inhibited at the low perfusion rate (0.2 mL min-1) with the glucose-free perfusate but only reduced with the glucose perfusate. The results show that mechanisms activated by cholera toxin inhibit the passive absorption of inert hydrophilic substances. This is proposed to be mainly caused by a reduction in the accessibility of the villus epithelium to the luminal content. Furthermore, the secretion seems predominantly to inhibit the passive absorption at the basal parts of the villus while the absorption rate at the villus tips is better preserved. The results also show that the intestinal absorption and secretion of fluid takes place at different locations (villus and crypts, respectively).

Permeability of the rat small intestinal epithelium along the villus-crypt axis: effects of glucose transport.

BACKGROUND & AIMS: The aim of this study was to elucidate the permeability characteristics of the epithelium along the villus-crypt axis and investigate the effect of glucose transport on these characteristics along this axis. METHODS: The disappearance rates of (14)C-mannitol and (51)Cr-EDTA or (3)H-inulin were determined as clearance (Cl(x)) from a recirculating perfusion system of the jejunal lumen in anesthetized rats. Net fluid transport was varied over a large range by exchanging mannitol with glucose in the perfusate solution and by inhibition of nervously mediated secretory processes with hexamethonium. The perfusion rate was 0.5 or 0.2 mL/min. RESULTS: Cl(Man) enhanced significantly with increasing net fluid transport (secretion 8.50+/-1.88, to absorption 16.72+/-1.75 microL x min(-1) x g(-1)) and with glucose perfusates. Cl(Cr-EDTA) was constant irrespective of net fluid transport and was reduced to insignificant values at a perfusion rate of 0.2 mL/min. Cl(In) was not different from zero. CONCLUSIONS: The absorbing apical part of the villus contains small pores (radius, <6 A) allowing passive transport via solvent drag of, e.g., monosaccharides, whereas the pores in the crypts are large (50-60 A) and inaccessible to the luminal content. The basal part of the villus contains medium-sized pores (10-15 A) through which no solvent drag occurs. Active glucose transport in the rat mainly increases the number of small pores accessible for passive transport, whereas the size of these pores seems to stay constant.

Somatic nerve stimulation and cholera-induced net fluid secretion in the small intestine of the rat: evidence for an opioid effect.

The effects of somatic nerve stimulation on cholera toxin induced secretion was investigated in vivo in anaesthetised rats. Small intestinal secretion was induced with cholera toxin and measured by a gravimetric technique. Afferent stimulation (pulse frequency within train; 100 Hz; train duration: 50 ms; train frequency: 3 Hz) of the sciatic nerve over 30 min significantly reduced the net fluid secretion both during (P < 0.05) and after cessation of the stimulation (P < 0.01). The greatest effect was obtained immediately after the termination of the nerve stimulation when the secretion was reversed to net fluid absorption. The opioid receptor antagonist naloxone (10 mg kg(-1) i.v.) administrated during the stimulation, significantly inhibited the antisecretory effect seen after the stimulation, thus no significant difference was seen between the control period and the periods after cessation of the stimulation. The opioid receptor antagonist naloxone methiodide (10 mg kg(-1) i.v.), which does not cross the blood-brain barrier, partly inhibited the antisecretory effects but not with the same magnitude as naloxone, thus the net fluid secretion was still significantly inhibited after the stimulation (P < 0.05). We conclude that afferent stimulation of the sciatic nerve strongly inhibits the cholera toxin induced secretion in the small intestine. This inhibition involves primarily a central opioid mechanism and to a lesser extent peripheral opioid mechanism.

Substance P effects on blood flow, fluid transport and vasoactive intestinal polypeptide release in the feline small intestine.

1. Substance P (SP) infusions were given close I.A. to the feline small intestine in vivo in a dose that produced plasma concentrations of 1-5 microM. This infusion regularly evoked a net fluid secretion measured with a gravimetric technique. Concomitantly, the release into blood of vasoactive intestinal polypeptide (VIP), a putative neurotransmitter of the enteric nervous system, increased. 2. The SP-induced fluid secretion was blocked by tetrodotoxin (7 micrograms close I.A.), a blocker of fast sodium channels in excitable tissues, and hexamethonium (10 mg (kg body wt)-1, I.V.), a nicotinic receptor antagonist, suggesting that the SP effect was mediated by the enteric nervous system. In line with this it was shown that the SP-evoked release of VIP was also significantly diminished by hexamethonium. 3. Close I.A. infusions of methionine enkephalin (Met-enkephalin; 7-23 nmol min-1) or electrical stimulation of the sympathetic nerve fibres (6 Hz) to the intestine markedly diminished net fluid secretion and the release of VIP caused by SP given close I.A. 4. The cyclo-oxygenase inhibitor diclofenac (5 mg (kg body wt)-1, I.V.) or the histamine-1 receptor antagonist pyrilamine (10 mg (kg body wt)-1, I.V.) did not influence the fluid secretion caused by SP, indicating that the effects of SP were not due to the actions of prostaglandins or histamine. 5. It is proposed that SP activates a nervous reflex arch that we have shown to be activated by various luminal stimuli, including cholera toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of pH on reservoir mucosal absorption/-secretion and tubular and glomerular function. Studies in patients with a continent ileal reservoir for urinary diversion.

Ten patients with a continent ileal reservoir for urinary diversion were studied regarding transmucosal fluxes of water and electrolytes at different pH. The urine was by-passed the reservoir via a Foley catheter. The experiment set-up was designed to exclude urine from reservoir mucosa exposition during the study. Test solutions of pH 4 and pH 7 with electrolytes and a non-absorbable volume marker (PEG MW 4000) were instilled into the empty reservoir. The influence of altered pH on the flow of water and Na,+K+ and Cl- across the reservoir mucosa was analysed. The maximal concentration ability after intranasal desmopressin (Minirin), the creatinine and Na-clearance as well as the fractional Na-excretion were calculated from measurements on the by-passed urine. The results showed a secretion of water, Na+ and Cl- into the reservoir while the K+ content was constant. The mucosal flows of water and electrolytes were not affected by alterations in reservoir pH. Maximal concentration ability appeared to be reduced compared to normals. Fractional Na excretion was within normal limits.

Involvement of the myenteric plexus in the cholera toxin-induced net fluid secretion in the rat small intestine.

BACKGROUND: The enteric nervous system is responsible in vivo for most of the change in fluid transport induced by cholera toxin. The aim of the present study was to investigate the importance of the myenteric plexus in the Intramural reflex responsible for this secretion. METHODS: Long-term ablation of the myenteric plexus was achieved by serosal application of benzalkonium chloride on jejunal segments in rats. RESULTS: The treated segments without functioning myenteric plexus showed a normal net fluid absorption. Cholera toxin in this segment only induced a reduction of fluid absorption, whereas in a nontreated ileal segment it concomitantly induced a conspicuous net fluid secretion. Intravenous hexamethonium did not change the cholera toxin response in the treated jejunal segments, whereas vasoactive intestinal polypeptide elicited a marked secretion. CONCLUSIONS: Benzalkonium chloride treatment eliminated the ability of cholera toxin to induce intestinal secretion. Thus, all afferent fibers in the intramural secretory reflex activated by cholera toxin are probably conveyed via the myenteric plexus, which functions as the integrating center in the enteric nervous system. The Ussing chamber technique using stripped intestinal preparations cannot be used when studying effects of luminal secretagogues.

Transcellular fluid secretion induced by cholera toxin and vasoactive intestinal polypeptide in the small intestine of the rat.

The permeation of intravenously administered 51Cr-EDTA and [14C]mannitol to the perfused intestinal lumen was measured in anaesthetized rats together with the net intestinal fluid. Net fluid secretion was induced by cholera toxin or by intravenous infusion of vasoactive intestinal polypeptide (VIP). The plasma clearance of Cr-EDTA and mannitol was 0.9 +/- 0.1 and 1.4 +/- 0.2 microliters min-1 g-1 intestine during the control period prior to the secretion and the net fluid absorption was about 7 +/- 5 microliters min-1 g-1. Cholera toxin induced a net fluid secretion of about 30 +/- 7 microliters min-1 g-1 but the clearance did not rise but decreased significantly. The findings for VIP-induced secretion were similar. No indication of solvent drag was seen. Thus it is concluded that the fluid was secreted in channels which were smaller than the probes and we propose that the secreted fluid entered the intestinal lumen through the epithelial cells and not by the paracellular route. The decreased permeation of Cr-EDTA and mannitol from plasma to lumen during volume secretion suggest that there was a decreased mucosal permeability during the secretion. The decrease in permeability was consistent with a decrease in pore size. One explanation of the data is that the pore radius contracted from about 35 to 15 A during cholera if we assume a homogenous pore population. However, the data indicated that there was not a uniform size of the pore.(ABSTRACT TRUNCATED AT 250 WORDS)

On the involvement of tachykinin neurons in the secretory nervous reflex elicited by cholera toxin in the small intestine.

The possible involvement of tachykinins in the nervous reflex activated by exposing the intestinal mucosa to cholera toxin was investigated in cats and rats. Three types of experiments were performed. In cats the release of tachykinins into blood was followed after placing cholera toxin in the intestinal lumen. In rat experiments a tachykinin receptor antagonist (Spantide II) was given close i.a. and its effect on cholera toxin-evoked fluid secretion was studied. Finally, in rats the effect of cholera toxin on the SP contents in the intestinal mucosa was studied. No release of tachykinins could be demonstrated. Spantide II did not change the rate of cholera toxin induced secretion. The SP content in the intestinal mucosa was not influenced by placing the toxin in the intestinal lumen. Hence, no experimental evidence was obtained for the involvement of a tachykinin neuron in the intestinal secretory nervous reflex activated by cholera toxin. Based on observations reported in the literature the involvement of an acetylcholine/tachykinin neuron in the reflex is tentatively discussed.

Difference between the antisecretory mechanisms of opioids and the somatostatin analogue octreotide in cholera toxin-induced small intestinal secretion in the rat.

The antisecretory effect of morphine and the somatostatin analogue octreotide was studied on cholera toxin-induced secretion in anaesthetized rats. Small intestinal secretion was induced with cholera toxin. Morphine (6 mg/kg b.wt.) and the somatostatin analogue octreotide (3 micrograms/kg b.wt.) reduced the cholera secretion in rats whose intestines had been subjected to sympathetic denervation. This was in contrast to the secretion elicited by helodermin which was unaffected by octreotide and morphine in the presence of nicotinic ganglionic blockade. The alpha-adrenergic receptor blocker phentolamine (1-2 mg/kg b.wt. i.v.) and the inhibitor of sympathetic transmitter release guanethidine (5 mg/kg b.wt. i.v.) abolished the antisecretory effect of morphine on the cholera secretion in contrast to the antisecretory effect of somatostatin which was unaffected by the alpha-blockade. It is proposed that the antisecretory effect of morphine and octreotide on cholera toxin-induced secretion was conducted at a step prior to the activation of the secretory epithelium and that the antisecretory effect of morphine was mediated indirectly by interaction with sympathetic nerve terminals in the intestine. The findings are consistent with a model where octreotide and morphine inhibit the nervous secreto-motor reflex activated by the cholera toxin.

Actions of serotonin antagonists on cholera-toxin-induced intestinal fluid secretion.

The effects of several 5-hydroxytryptamine (5-HT) receptor antagonists were tested in rats in vivo on the intestinal fluid secretion evoked by cholera toxin. Five receptor antagonists were used, namely 2-bromolysergic acid diethylamine (2-bromo-LSD), granisetron, ketanserin, methysergide and ondansetron. The drugs were used in doses that inhibited the arterial hypertension and/or bradycardia evoked by 5-HT given i.v. Granisetron and ondansetron markedly diminished cholera-toxin-evoked secretion, whereas ketanserin was without any effect. Methysergide also diminished cholera-toxin-induced fluid secretion particularly when the drug was given as an i.v. infusion. The results are considered in relation to the pathophysiology of cholera secretion and to the current views of receptor subtypes for 5-HT. It is proposed that the receptor involved is a 5-HT3 receptor, possibly also a receptor of the 5-HT1 type. Results from experiments in which 5-HT (20 mM) was placed in the intestinal lumen to evoke an intestinal secretion suggest that the 5-HT3 receptor is located in the villus tissue. It was also demonstrated that zimeldine, an inhibitor of presynaptic 5-HT reuptake, diminished choleraic secretion, an effect that may be ascribed to a 5-HT tachyphylaxis caused by an accumulation of 5-HT in a synaptic cleft.

Antisecretory effect of splanchnic nerve stimulation on choleratoxin-induced secretion in the cat, an effect mediated at the crypts.

The experiments were performed on cats anaesthetized with alpha-chloralose. Segments of the small intestine were perfused with sodium-free hypotonic choline-mannitol solution and intestinal net fluid transport was recorded with a volumetric technique. The content of sodium and chloride in the lamina propria of the small intestinal villus was measured with an electron microprobe in freeze-dried paraffin embedded tissue. In absorbing control intestine, there was an even distribution of electrolytes along the villi. Sympathetic nerve stimulation (5 Hz, 5 ms, 5 V) did not significantly affect electrolyte distribution and net fluid transport. Intestinal secretion was elicited by pretreatment of the intestine with cholera toxin. The concentration of sodium and chloride was elevated in the apical third of the villi in intestines during the secretion since secreted sodium from the crypts was reabsorbed into the villi. Sympathetic nerve stimulation decreased the cholera secretion significantly in intestines pretreated with cholera-toxin. Furthermore, the apical gradients of sodium and chloride in the villi, caused by the reabsorbed sodium and chloride, disappeared during sympathetic nerve stimulation. It is concluded that, in the used experimental model, the antisecretory effect of sympathetic nerve stimulation was caused by inhibition of crypt secretion and not by augmented villus absorption.

Interaction between antisecretory opioid and sympathetic mechanisms in the rat small intestine.

UNLABELLED: The putative existence of an endogenous opioid antisecretory mechanism in the small intestine was tested in anaesthetized Sprague-Dawley rats. Cholera secretion was elicited with cholera toxin and net fluid secretion was measured in vivo using a gravimetric technique allowing on line registrations. Opioid blockade with naloxone (10 mg kg-1 i.v.) increased the cholera secretion significantly but had no effect on control absorption. Pretreatment with phentolamine (2 mg kg-1 i.v.) eliminated the effect of opioid blockade indicating an interaction between the opioid and the adrenergic mechanisms. The effect of naloxone on cholera secretion was unchanged after acute division of the sympathetic nerves to the intestine and removal of the adrenals. Chronic sympathetic denervation of the small intestine, on the other hand, abolished the effect of opiate blockade on the secretion. The antisecretory effect of sympathetic nerve stimulation was unchanged by opiate blockade. CONCLUSION: An intrinsic antisecretory opioid mechanism has been demonstrated in the small intestine of the rat. This endogenous opioid mechanism seems to decrease secretion indirectly via peripheral activation of the sympathetic antisecretory system.

Nerve-mediated effect of ethanol on sodium and fluid transport in the jejunum of the rat.

The hypothesis tested in this study is whether a potential harmful substance such as ethanol causes secretion in the small intestine and, if so, whether the secretion is mediated via intestinal nerve reflexes or a direct effect on the epithelium. The jejunum of anaesthetized Sprague-Dawley rats was perfused in vivo with a modified Krebs-Henseleit solution. Three per cent ethanol had no significant effect, whereas 8% ethanol in the perfusate elicited a net secretion of fluid and sodium in the intestine. This secretion was reversed by ganglionic blockade with hexamethonium (10 mg/kg intravenously). The ethanol absorption from the perfusate, on the other hand, was not affected by the ganglionic blockade. We concluded that ethanol dose-dependently caused a nerve-mediated secretion of sodium and fluid in the rat small intestine. Ethanol was probably absorbed by diffusion.

Villus and crypt electrolyte and fluid transport during intestinal secretion.

The apical parts of jejunal villi of net-absorbing intestine have been shown to contain sodium chloride concentration gradients which are associated with water absorption (Sjöqvist & Beeuwkes 1989). To determine whether these gradients are different in states of intestinal net secretion, jejunal segments of chloralose-anaesthetized cats were perfused with modified Krebs-Henseleit solutions while secretion was elicited by cholera toxin or vasoactive intestinal polypeptide (VIP). The segments were then rapidly frozen and freeze-dried, and sodium and chloride contents of the lamina propria of single villi were measured by X-ray microanalysis. The apical third of the villus was found to contain a concentration gradient of sodium and chloride when the lumen contained sodium, with no difference between secreting intestine and absorbing control intestine. When the intestine was perfused with hypotonic choline-mannitol solution, no sodium or chloride gradient was found. In this state, treatment of the intestines with secretagogues allowed development of an apical concentration gradient. This demonstrated that the absorptive function of the villus tip was unimpaired during secretion and that secretion from the crypt could supply sufficient electrolyte to allow formation of an apical gradient.

On the role of vasoactive intestinal polypeptide and tachykinins in the secretory reflex elicited by chemical peritonitis in the cat small intestine.

Peritonitis induced by serosal application of 0.1 M hydrochloric acid causes net fluid secretion via the enteric nervous system. The aim of the present study was to investigate the roles of vasoactive intestinal peptide (VIP) and tachykinins in this reflex(es). The release of tachykinins (substance P [SP], neurokinin A [NKA], neuropeptide K [NPK]) and VIP into the mesenteric circulation, net fluid transport, intestinal blood flow and sometimes motility were recorded simultaneously in extrinsically denervated jejunal segments of the cat in vivo. The release of both VIP and NKA was increased upon application of HCl to the cat jejunal serosa. Tetrodotoxin, hexamethonium and methionine enkephalin inhibited both the induced VIP release and the secretory response. The increased release of NKA was unaffected by hexamethonium. We propose that the intramural secretory reflex evoked by acid application of the serosa consists of an 'afferent' tachykinin neuron, a cholinergic interneuron and an 'efferent' VIPergic neuron innervating the secretory enterocytes.

Villous sodium gradient associated with volume absorption in the feline intestine: an electron-microprobe study on freeze-dried tissue.

Water transport in biological tissue is driven by local osmotic gradients created by accumulation of actively transported ions in tissue compartments. To localize and measure such gradients, jejunal segments from the small intestine of anaesthetized cats were perfused with modified isotonic Krebs-Henseleit electrolyte solution, and net fluid transport was measured with a volumetric technique. The segments were then rapidly frozen, freeze-dried, and prepared for X-ray micro-analysis of elemental content. Whenever the lumen perfusate contained sodium, the apical third of the villus was found to have a sodium gradient rising to a tip concentration more than twice that at the base of the villus. This sodium gradient was associated with a chloride gradient and fluid absorption. No similar potassium gradient was found. When choline replaced sodium in the intestinal lumen, no gradient of sodium chloride was found and no net fluid absorption occurred. Absorption of fluid was thus apparently coupled to absorption of sodium through creation of a local osmotic gradient in the tip of the intestinal villus.

Somatostatin and methionine-enkephalin inhibit cholera toxin-induced jejunal net fluid secretion and release of vasoactive intestinal polypeptide in the cat in vivo.

A major part of the net fluid secretion that is elicited by cholera toxin in the small intestine of the cat has been shown to be mediated by intramural nervous reflex(es). The release of vasoactive intestinal polypeptide (VIP) from the small intestine is increased by cholera toxin. We report that close intra-arterial infusions of methionine-enkephalin (met-enk) and somatostatin cause a parallel reduction in cholera toxin-induced net fluid secretion and in VIP release from the small intestine of the cat. Intestinal blood flow was slightly, but significantly increased by met-enk and not influenced by somatostatin. These results strengthen the hypothesis that VIP is involved as a neurotransmitter in the nervous reflex mediating cholera toxin-induced secretion.

Involvement of opioid and nicotinic receptors in rectal and anal reflex inhibition of urinary bladder motility in cats.

The present study was performed to investigate mechanisms involved in urinary bladder relaxation during reflex activation of the pelvic nerves in the cat. Electrical stimulation of the pelvic nerves produced a contraction of the urinary bladder (P less than 0.05) and colon (P less than 0.05). Reflex activation of the pelvic nerves by rectal distension or mechanical stimulation of the anus induced relaxation of the bladder (P less than 0.05), while a colonic contraction was elicited (P less than 0.05). Naloxone (1.5 mg kg-1, i.v.) abolished the reflex inhibition of bladder motility elicited by rectal distension or mechanical stimulation of the anus (P less than 0.05). However, the urinary bladder and colonic contraction produced by electrical stimulation of the pelvic nerves were not affected. Hexamethonium (10 mg kg-1, i.v.) or severing of the pelvic nerves completely abolished the responses of the urinary bladder and colon to electrical stimulation or reflex activation of the pelvic nerves. The results indicate that inhibitory reflexes from the rectum and anal canal to the urinary bladder are conveyed via efferents of the pelvic nerves, and involve both nicotinic and opioid receptor mechanisms.