Pyloric sphincter tone plays an important role in the regulation of gastric emptying. Non-adrenergic, non-cholinergic (NANC) innervation to the pylorus is predominantly inhibitory and mediates relaxation of the sphincter (Anuras et al. 1974). A high density of NOS-immunopositive nerve cells and fibres has been demonstrated in the pylorus (Ekblad et al. 1994), and significant reduction of NOS activity of the pylorus has been demonstrated in infantile hypertrophic pyloric stenosis (Vanderwinden et al. 1992). It has been shown that transgenic mice with homozygous depletion of the nNOS gene develop grossly enlarged stomachs with hypertrophy of the pyloric sphincter (Huang et al. 1993). Thus, gastric outlet obstruction is associated with the lack of NO-generating neurons in the pylorus. Previous studies have shown that NO biosynthesis inhibitors delay gastric emptying in rats (Plourde et al. 1994; Ishiguchi et al. 2000a). These observations suggest NANC relaxation and gastric emptying in the pylorus is mediated by NO.
The relaxation of the pylorus following gastric distension is a crucial factor in expelling gastric contents to the duodenum. Although it has been well established that the motility of the pylorus is under vagal control (Allescher et al. 1988), the mechanism of pyloric relaxation in response to gastric distension remains unclear.
Symptoms of gastroparesis include postprandial nausea, epigastric pain, burning, bloating, early satiety, excessive eructation, anorexia and vomiting (Webb & Fogel, 1995). Although associated with many diseases, the most frequent cause of gastroparesis is diabetes mellitus. About one-half of patients with insulin- or non-insulin-dependent diabetes have delayed gastric emptying (Webb & Fogel, 1995). Recent data suggest that not only autonomic neuropathy, but also hyperglycaemia per se, contribute to the pathogenesis of disordered gastric motility. Improved glycaemic control in diabetic patients is associated with improvements in delayed gastric emptying and its symptoms (Jones et al. 1995), and acute hyperglycaemia has been shown to inhibit gastric acid production, trypsin secretion and bile salt output in response to a test meal in human volunteers (MacGregor et al. 1976; Lam et al. 1997). Acute hyperglycaemia also causes a reversible impairment of motility in various regions of the gastrointestinal (GI) tract, including stomach (Barnett & Owyang, 1988), jejunum (de Boer et al. 1993), colon (Chey et al. 1995) and gall bladder (Gielkens et al. 1998).
The mechanisms by which acute hyperglycaemia impairs GI motility have not been elucidated. Impaired motility induced by acute hyperglycaemia does not result from reactive endogenous hyperinsulinaemia, because hyperinsulinaemia per se does not inhibit GI motility (Chey et al. 1995; Gielkens et al. 1997). Gastric emptying is delayed in rat models of diabetes (Chang et al. 1997; Yamano et al. 1997) and in normal rats with acute elevations in blood glucose concentrations (Chang et al. 1996). In these models, gastric emptying is delayed because of increased outflow resistance at the level of the pylorus, and in advanced diabetes in humans, improperly timed pyloric contractions of abnormal intensity and duration lead to pylorospasm and functional outlet obstruction (Mearin et al. 1986).
Insulin-dependent diabetes is characterized by marked hyperphagia and reduced thermogenesis. As the hypothalamus appears to be important in regulating food intake and energy balance, these energetic and neuroendocrine disturbances of diabetes may be mediated by changes in specific hypothalamic neurons and neurotransmitters. Neuropeptide Y (NPY), a 36-amino-acid peptide originally isolated from porcine brain, is present in high concentrations in the central nervous system, particularly in the hypothalamus, limbic brain regions, cerebral cortex and various brain stem nuclei (Humphreys et al. 1992). NPY concentrations in the central hypothalamus are significantly increased in diabetic rats (Williams et al. 1988), and hypoinsulinaemia increases hypothalamic NPY levels (Malabu et al. 1992). Alteration of hypothalamic NPY levels, which has potent effects on hypothalamo-pituitary function (Humphreys et al. 1992), may contribute to certain neuroendocrine disturbances in diabetes mellitus.
The aims of the present study are three-fold: (1) to investigate the neural mechanisms mediating the pyloric relaxation in response to gastric distension under euglycaemic conditions; (2) to investigate whether hyperglycaemia inhibits gastric distension-induced pyloric relaxation; (3) to test the hypothesis that the inhibitory effects of hyperglycaemia on gastric distension-induced pyloric relaxation involve actions of NPY in the central nervous system.
Using rats anaesthetized with ketamine and xylazine, we have demonstrated that: (1) gastric distension-induced pyloric relaxation is mediated via the vagus nerve and NO release from the myenteric plexus in euglycaemia; (2) acute hyperglycaemia significantly inhibits gastric distension-induced pyloric relaxation; and (3) acute hyperglycaemia stimulates NPY release at the hypothalamus and that the inhibitory effects of hyperglycaemia on gastric distension-induced pyloric relaxation are restored by central administration of NPY antibody and Y1 antagonist. These results suggest that the Y1 receptor subtype may play a dominant role in mediating hyperglycaemia-induced inhibition on pyloric relaxation. These observations may help to clarify the manner in which acute hyperglycaemia causes impaired gastric emptying.
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Traditionally, disordered motility in diabetes mellitus has been attributed to irreversible autonomic nerve damage (Keshavarzian et al. 1987). However, recent observations indicate that hyperglycaemia causes a reversible impairment of motility in various regions of the GI tract (Barnett & Owyang, 1988; Fraser et al. 1991). It has been shown that gastric emptying is delayed in diabetic rats (Chang et al. 1997; Yamano et al. 1997) and normal rats with hyperglycaemia (Chang et al. 1996). These observations suggest that hyperglycaemia itself has an inhibitory effect on gastric emptying.
The antral pump and pyloric opening are of paramount importance for gastric emptying of solids. Large solid particles are retained in the stomach by pyloric closure and are retropelled and triturated in the antral mill (Minami & McCallum, 1984). In the emptying state, strong antral contractions are regularly associated with inhibition of pyloric motility. Abnormal pyloric motility has been demonstrated in diabetes and hyperglycaemia in humans (Mearin et al. 1986), and it has been proposed that stimulation of localized pyloric contractions and inhibition of antral contractions contribute to the delayed gastric emptying induced by hyperglycaemia (Fraser et al. 1991). In the present study, we investigated the effects of hyperglycaemia on the pyloric relaxation in response to gastric distension in rats anaesthetized with xylazine and ketamine.
Gastric distension-induced pyloric relaxation was significantly reduced by vagotomy, suggesting mediation by the vagus nerve. In contrast, gastric distension-induced pyloric relaxation was not affected by spinal cord transection or guanethidine and only slightly reduced by splanchnectomy. As it has been shown that some vagal fibres pass to the duodenum and stomach along with sympathetic fibres in the mesenteric nerves (Richards et al. 1996), the inhibitory effects of splanchnectomy on gastric distension-induced pyloric relaxation may be due to the partial damage of vagal fibres around the coeliac ganglia.
Vagal afferent fibres arise in the mucosa or muscle layer of the GI tract. These afferent receptors transmit sensory information to the central nervous system and play an important role in the vago-vagal reflex. It has been generally accepted that tension receptors are located in the serosa/muscle layers, and chemoreceptors are located in the mucosa (Grundy, 1988). Gastric distension activates vagal afferent fibres. Grundy (1988) has demonstrated that gastric distension provokes the firing of vagal afferents and Traub et al. (1996) have shown that gastric distension promotes c-Fos expression at the nucleus of the solitary tract, a centre of vagal afferents.
In the present study, gastric distension-induced pyloric relaxation was significantly reduced by hexamethonium and l-NAME. We have reported that pyloric relaxation in response to electrical stimulation of vagal efferents is significantly reduced by l-NAME and almost completely abolished by hexamethonium (Ishiguchi et al. 2000a). These findings suggest that pyloric relaxation is under the control of NO release from the myenteric plexus. Our present study suggests that gastric distension-induced pyloric relaxation is mediated predominantly via a vago-vagal reflex. It is also suggested that the release of NO from the myenteric plexus mediates gastric distension-induced pyloric relaxation.
Intravenous infusion of d-glucose significantly inhibits gastric distension-induced pyloric relaxation (Fig. 3A). This effect was not due to hyperosmolarity, since i.v. infusion of mannitol did not have any effect (Fig. 3B). In contrast, vagal stimulation-induced pyloric relaxation is not affected by hyperglycaemia. Similarly, NANC relaxation of the pylorus is also not affected by increasing concentration of glucose in vitro. These results suggest that the site of action of hyperglycaemia is neither the vagal efferent nor the myenteric plexus. Furthermore, inhibitory effects on gastric distension-induced pyloric relaxation were observed following an i.c.v. injection of d-glucose, suggesting that the inhibitory effect of hyperglycaemia is mediated via the central nervous system.
Glucose-sensitive neural elements exist in the hypothalamus and the nucleus of the solitary tract (Oomura & Yoshimatsu, 1984). In the ventromedial hypothalamic nucleus, approximately 40 % of cells responded to changes in blood glucose over a range of concentrations from 3.6 to 17 mm, by increasing their firing rate with increasing concentrations of glucose (Silver & Erecinska, 1998).
NPY binding sites are seen in a variety of areas, including cortex, hypothalamus, pons and medulla oblongata. Results from binding studies have characterized six distinct subtypes of receptors. Two of these, Y1 and Y2, are both found in large quantities in the dorsal vagal complex (DVC) of the medulla. Y1 and Y2 receptors are found both pre- and post-junctionally in the nervous system (Humphreys et al. 1992; Penner et al. 1993; Chen et al. 1997; Yoneda et al. 1998).
Aramakis et al. have shown that NPY, Y1 and Y2 agonists produce multiple effects (increased, decreased and biphasic changes) on single neuron discharge rates in the paraventricular nucleus in vitro (Aramakis et al. 1996). Intracerebroventricular injection of both Y1 and Y2 agonists suppress growth hormone secretion in rats (Suzuki et al. 1996). i.c.v. injection of NPY and Y1 agonist has been shown to decrease basal gastric acid output in anaesthetized rats, suggesting that gastric acid output is mediated by NPY via the Y1 receptor (Penner et al. 1993). In contrast, other investigators have demonstrated that i.c.v. injections of NPY increase basal and meal- stimulated gastric and pancreatic secretion in conscious dogs (Geoghegan et al. 1993).
Microinjection of NPY and a Y1 agonist into DVC increased bile secretion in a dose-dependent manner in anaesthetized rats, while microinjection of a Y2 agonist inhibited bile secretion (Yoneda et al. 1998). Chen et al. have shown that a Y2 agonist applied to the DVC suppressed gastric motility in thyrotropin-releasing hormone (TRH)-stimulated conditions, while the agonist had no effects on gastric motility under basal conditions. In contrast, a Y1 agonist had no effect on TRH-stimulated gastric motility, while the Y1 agonist strongly stimulated gastric motility under basal conditions (Chen et al. 1997). There appears to be different effects following either stimulation or inhibition of NPY receptor subtypes (Y1 and Y2), depending on species, tissues and anaesthesia.
It remains unclear which NPY receptor subtypes regulate pyloric relaxations in rats. Our study has shown that i.c.v. injections of NPY and a Y1 agonist significantly inhibited gastric distension-induced pyloric relaxation. In contrast, an i.c.v. injection of a Y2 agonist significantly enhanced gastric distension-induced pyloric relaxation. This suggests that Y1 and Y2 receptor subtypes mediate inhibitory and excitatory effects on gastric distension-induced pyloric relaxation, respectively.
In the present study, it is possible that i.c.v. injections of NPY may have inhibited gastric distension-induced pyloric relaxation by non-specific leakage of the injected NPY into the systemic circulation. In order to address this possibility, we studied gastric distension-induced pyloric relaxation with systemic i.v. administration of NPY. Intravenous administration of NPY (3 nmol per rat) immediately caused phasic contractions of the rat pylorus. A previous study has shown that peripheral administration of NPY (500 pmol kg−1) increased duodenal and colonic intraluminal pressure in rats (Wager-Page et al. 1993). It is suggested that the stimulatory effects of systemic NPY on GI motility were mediated by postjunctional mechanisms (Wager-Page et al. 1993). Systemic NPY administration caused pyloric contractions but had no effect on gastric distension-induced pyloric relaxation, and therefore, the effects of NPY in this regard appear to be mediated through its actions on the central nervous system.
Williams and colleagues have demonstrated that NPY concentrations in the hypothalamus are significantly increased within 3 weeks of sustained hyperglycaemia in streptozotocin (STZ)-induced diabetic rats, and elevated concentrations of NPY in the hypothalamus in diabetic rats have been suggested to be responsible for diabetic hyperphagia (Williams et al. 1988). The present study demonstrates that NPY concentrations in the hypothalamus were also significantly increased following acute hyperglycaemia in rats.
In the present study we have also shown that i.c.v. administration of NPY antibody and of the Y1-selective antagonist BIBP 3226 abolishes the inhibitory effects of hyperglycaemia on gastric distension-induced pyloric relaxation. These data therefore suggest that the Y1 receptor subtype may play a dominant role in mediating hyperglycaemia-induced inhibition of pyloric relaxation.
It is concluded that hyperglycaemia stimulates NPY release in the hypothalamus and inhibits vagal activity via the hypothalamic NPY Y1 receptor in anaesthetized rats. Reduced vagal efferent activity in the setting of acute hyperglycaemia decreases release of NO from the myenteric plexus and results in impaired pyloric relaxation and delayed gastric emptying.
The present study suggests that the hyperglycaemia associated with diabetes mellitus may have acute effects on gastric emptying. These effects are in part mediated by the actions of NPY in the central nervous system. The deleterious effects of hyperglycaemia on gastric motility emphasize the importance of rigorous metabolic control in the management of diabetes.