Jin-Xia Zhu

Reduced expression of choline acetyltransferase in vagal motoneurons and gastric motor dysfunction in a 6-OHDA rat model of Parkinson's disease.

Parkinsons disease (PD) has been characterized by dopaminergic neuron degeneration in the substantia nigra (SN) accompanied by pathology of the dorsal motor nucleus of the vagus (DMV). PD patients have often experienced gastrointestinal dysfunctions, such as gastroparesis. However, the mechanism underlying these symptoms in PD patients is not clear. In the present study, we investigated alterations of cholinergic and catecholaminergic neurons in the DMV and gastric motor function in rats microinjected with 6-hydroxydopamine (6-OHDA) bilaterally into the SN (referred to as 6-OHDA rats) and explored possible mechanisms. A strain gauge force transducer was used to record gastric motility in vivo. Expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) was evaluated by immunofluorescence and western blot analysis. Acetylcholine (Ach) content was measured using ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) analysis. After treatment with 6-OHDA for 6weeks, 6-OHDA rats exhibited decreased ChAT and enhanced TH expression in the DMV and decreased Ach content in the gastric muscular layer. Delayed gastric emptying and impaired gastric motility in vivo were observed in 6-OHDA rats. The results of the present study indicated that decreased ChAT and enhanced TH expression in the DMV may be correlated with the development of delayed gastric emptying and impaired gastric motility, which may be partly due to the decreased Ach release from the vagus.

The role of the vagal pathway and gastric dopamine in the gastroparesis of rats after a 6‐hydroxydopamine microinjection in the substantia nigra

Gastroparesis is a common non‐motor system symptom of Parkinsons disease (PD). However, the mechanism responsible for the gastric motor abnormality is not clear. We previously reported on the impaired gastric motility in 6‐hydroxydopamine (6‐OHDA) rats, which were treated with a bilateral microinjection of 6‐OHDA in the substantia nigra (SN). We hypothesize that the enhanced dopamine system and reduced acetylcholine (Ach) in gastric tissues might contribute to the delayed gastric emptying observed in PD.

Dopamine receptor D1 mediates the inhibition of dopamine on the distal colonic motility

The motility of distal colon could be inhibited by dopamine (DA), yet, the involved receptor is controversial according to the published reports. The goal of present study was to investigate DA receptor(s) mediated inhibition of DA on the colonic motility in rat. The contraction of isolated colon strips was assessed through isometric force transducer. The expressions of DA receptors in distal colon were detected through immunofluorescence and Western blot. DA concentration in colonic smooth muscle was measured by liquid chromatography/mass spectrometry. The results showed that DA inhibited the spontaneous motility of distal colon in a dose-dependent manner with EC50 8.3 μM. Tetrodotoxin increased colonic contractive frequency, but failed to affect the inhibition of DA on the colonic motility. Pretreatment with SCH-23390, an antagonist of dopaminergic receptor D1, shifted the dose-response curve to the right with EC50 of DA 37 μM. However, blocking dopaminergic receptor D2 with sulpiride, had no effect. The immunoreactivity of D1 and D2 were detected in the distal colon including myenteric plexus and smooth muscle. Acute cold-restraint stress (CRS) enhanced spontaneous contraction of rat distal colon, which was more sensitive to DA compared with control. Moreover, DA content and D1 expression in smooth muscle layer were increased under CRS condition. In conclusion, D1 in the smooth muscle is mediated DA inhibition on the spontaneous contraction of rat distal colon. The increased DA content and D1 receptor expression in the smooth muscle layer could be a compensatory effect under CRS condition to balance the enhanced colonic motility.

Expression of NKCC2 in the rat gastrointestinal tract

Abstract  NKCC2, an isoform of Na+–K+–2Cl− cotransporter, is principally present in the kidney and plays a critical role in salt reabsorption. Expression of NKCC2 has been found in the apical membrane of intestinal epithelial cells in a number of marine fish, however, details for expression in the mammalian gastrointestinal tract are lacking. RT‐PCR, Western blotting and immunohistochemistry were used to study the expression and localization of NKCC2 in the rat gastrointestinal tract. We found that mRNA transcripts, protein and immunoreactivity (IR) for NKCC2 were expressed in the stomach, small and large intestine of adult rats. NKCC2 IR was localized to the base of the gastric glands, intestinal epithelia, myenteric and submucosal plexuses. NKCC2 IR was expressed strongly in the apical membranes and weakly in the basolateral membranes of intestinal epithelial cells. In the enteric nervous system, NKCC2 IR was widely distributed and localized to enteric neurons with cholinergic, calretinin and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to non‐cholinergic secretomotor neurons in the submucosal plexus. In conclusion, this study for the first time clearly detected the expression of NKCC2 in the gastrointestinal tract of a mammalian species. Expression of NKCC2 in gastrointestinal epithelial cells suggested that this cation chloride cotransporter might be involved in gastrointestinal ion transport. Expression of NKCC2 in enteric neurons might contribute to the accumulation of Cl− and a more depolarized ECl− in enteric neurons.

Expression and activation of β-adrenoceptors in the colorectal mucosa of rat and human

Background  The functions of the distal colon are regulated by local and extrinsic neural pathways. In previous studies, we have found that dopamine (DA) and norepinephrine (NE) could evoke colonic ion transport by activating β‐adrenoceptors. The present study aims to investigate the segmental differences in expression and activation of β‐adrenoceptors in the distal colon in physiological and pathophysiological conditions.

β-Adrenoceptors, but not dopamine receptors, mediate dopamine-induced ion transport in late distal colon of rats

Dopamine, an important modulator in the gastrointestinal system, induces concentration-dependent transepithelial ion transport in the distal colon of the rat, as shown by a decrease in the short-circuit current, and acts in a segmentally dependent manner. However, the receptor(s) that mediates dopamine-induced ion transport is unknown. We have investigated the receptor mechanisms underlying dopamine-induced colonic ion transport by means of short-circuit current recording, real-time polymerase chain reaction, and Western blotting analysis, plus gene transfection and enzyme-linked immunosorbance assay. mRNA transcripts of adrenoceptors (α, β) and dopaminergic receptors (D1 and D2) were detected in the rat late distal colonic mucosa, with β2 displaying the highest expression. A similar result was found in human colorectal mucosa (equivalent of late distal colon in rat). Pretreatment with a β1-adrenoceptor antagonist (CGP-20712A) and a β2-adrenoceptor antagonist (ICI 118,551) inhibited the dopamine-induced short-circuit current response by 52.59% and 92.51%, respectively. However, neither dopamine D1 receptor antagonist SCH-23390 nor dopamine D2 receptor antagonist sulpiride blocked the effect of dopamine. Protein expression of both β1- and β2-adrenoceptors was found in the mucosa of rat distal colon and human sigmoid colon and rectum. Dopamine significantly increased intracellular cAMP levels in COS-7 cells transfected with β1- or β2-adrenoceptors. Thus, β-adrenoceptors (mainly β2-adrenoceptors), but not dopamine receptors, mediate dopamine-induced ion transport in the late distal colon of the rat. This extends our knowledge of the late distal colon (rats) or colorectum (human) and provides further experimental evidence that might aid the prevention, diagnosis, and clinical therapy of human colorectal diseases.

Changes in brain G proteins and colonic sympathetic neural signaling in chronic-acute combined stress rat model of irritable bowel syndrome (IBS)

The role of the brain-gut axis interaction in the pathogenesis of irritable bowel syndrome (IBS) is not well understood. To examine this possibility, a novel rat model of IBS subjected to both chronic and acute stress (CAS) was established. G proteins play a crucial role in the pathophysiology of depression. The alpha 2A adrenoceptor (alpha(2A)-AR) and the norepinephrine reuptake transporter (NET) determine the sympathetic signal activity. It is conceivable that stress may induce brain G proteins, colonic alpha(2A)-ARs, and NET abnormal expression, which may be responsible for the abnormalities in IBS. Colonic motility, visceral sensation, and secretion were assessed by counting fecal pellets, abdominal muscle contractions in response to colorectal balloon distension (CRD), and short-circuit current study, respectively. Western blot analysis was used to investigate the expression of G proteins, alpha(2A)-ARs, and NET. Compared with control animals, the colonic epithelial secretion, fecal pellets, and numbers of abdominal muscle contraction induced by CRD were significantly higher in both acute stress only (AS) and CAS rats. However, the G proteins, alpha(2A)-AR, and NET expression changed differently in AS and CAS rats. We showed that exposure to either AS or CAS would cause the increase of secretion, motility, and sensation, but the change of protein expression in brain-gut axis was different. It may be responsible for the pathogenesis of IBS.

Alteration of enteric monoamines with monoamine receptors and colonic dysmotility in 6-hydroxydopamine-induced Parkinson's disease rats.

Constipation is common in Parkinsons disease (PD), in which monoamines (dopamine [DA], norepinephrine [NE], and 5-hydroxytryptamine [5-HT]) play an important role. Rats microinjected with 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra (SN) exhibit constipation, but the role of monoamines and their receptors is not clear. In the present study, colonic motility, monoamine content, and the expression of monoamine receptors were examined using strain gauge force transducers, ultraperformance liquid chromatography tandem mass spectrometry, immunofluorescence, and Western blot. The 6-OHDA rats displayed a significant reduction in dopaminergic neurons in the SN and a decreased time on rota-rod test and a marked decrease in daily fecal production and fecal water content. The amplitude of colonic spontaneous contraction was obviously decreased in 6-OHDA rats. Blocking D1-like receptor and β3-adrenoceptor (β3-AR) significantly reduced the inhibition of DA and NE on the colonic motility, respectively, whereas the 5-HT and 5-HT4 receptor agonists promoted the colonic motility. Moreover, DA content was increased in the colonic muscularis externa of 6-OHDA rats. The protein expression of β3-ARs was notably upregulated, but 5-HT4 receptors were significantly decreased in the colonic muscularis externa of 6-OHDA rats. We conclude that enhanced DA and β3-ARs and decreased 5-HT4 receptors may be contributed to the colonic dysmotility and constipation observed in 6-OHDA rats.

No Direct Projection is Observed from the Substantia Nigra to the Dorsal Vagus Complex in the Rat

BACKGROUND Parkinsons disease (PD) is a neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons in the substantia nigra (SN). Destruction of the SN can lead to gastric dyskinesis accompanied by decreased expression of choline acetyltransferase (ChAT) and increased expression of tyrosine hydroxylase (TH) in the dorsal vagus complex (DVC), which includes the dorsal motor nucleus of the vagus (DMV) and nucleus tractus solitarius (NTS). However, it is unclear if the SN and DVC are directly connected. OBJECTIVE To investigate the neural projection from the SN to the DVC in rats. METHODS Retrograde and anterograde tracing techniques combined with double-labeling immunofluorescence technique were used. RESULTS Destruction of the SN significantly decreases ChAT immunoreactivity (IR) and increases TH-IR in the DVC. After injection of the retrograde tracer fluoro-gold (FG) into the DVC, FG-labeled neurons were observed in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamus (LH), inferior olive (IO), and locus coeruleus (LC). No FG-positive cells were observed in the SN or striatum. Furthermore, after injection of anterograde tracer biotinylated dextran amine (BDA) into the SN, BDA-positive fibers were observed in the caudate putamen (Cpu), globus pallidus (GP), LC, and LH but not in the DVC. CONCLUSION This study is the first to demonstrate that neurons in the SN do not directly innervate the DVC in rats. The DVC might be indirectly innervated by the SN through the hypothalamus and/or the LC. These data provide important morphological insights into the potential mechanism underlying the gastroparesis observed in PD patients.

Emodin induces chloride secretion in rat distal colon through activation of mast cells and enteric neurons

BACKGROUND AND PURPOSE Emodin (1,3,8‐trihydroxy‐6‐methylanthraquinone) is an active component of many herb‐based laxatives. However, its mechanism of action is unclear. The aim of the present study was to investigate the role of mast cells and enteric neurons in emodin‐induced ion secretion in the rat colon.