Rui-g Fan

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.

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.

Distribution of dopamine receptors D1- and D2-immunoreactive neurons in the dorsal motor nucleus of vagus in rats

The dorsal motor nucleus of vagus (DMV) plays an important role in the regulation of gastrointestinal function. Dopamine (DA) exerts potent neuromodulatory effects on the motoneurons in the DMV via dopamine receptors (DRs). However, the distribution of DRs and their neurochemical phenotypes in the DMV are unclear. In the present study, the distribution of DRs D1- and D2-immunoreactive (IR) neurons and their neurochemical phenotypes in the DMV of rats were investigated using a double-labeling immunofluorescence technique combined with confocal microscopy. The results indicated that a considerable quantity of D1 and D2 was expressed throughout the DMV. A large amount of choline acetyltransferase (ChAT)-IR and a few tyrosine hydroxylase (TH)-IR neurons were observed in the DMV. Nearly all of the neurons were also D1-IR and D2-IR. In conclusion, the present study demonstrates the wide distribution of D1 and D2 in the cholinergic and catecholaminergic neurons in the DMV of rats. The DRs might play an important role in the regulation of DA on the activity of cholinergic and catecholaminergic neurons in the DMV.

Distinctive expression and cellular distribution of dopamine receptors in the pancreatic islets of rats

Activation of the dopamine (DA) D2 receptor inhibits glucose-stimulated insulin secretion in isolated rodent islets in vitro; however, no information is available regarding the cellular localization of DA receptors (DRs, including D1-D5 receptors) in pancreatic islets in situ. We investigate the protein expression and cellular localization of five types of DRs in pancreatic islets by means of Western blotting and double-labeling immunofluorescence in both normal control and alloxan-induced type 1 diabetes model (T1DM) rats. In control rats, D1 immunoreactivity (-IR) was distributed in the core of the islet and co-localized with insulin-IR, D2-IR was peripherally distributed and found only in somatostatin-immunoreactive cells and D5-IR was co-localized with glucagon-IR and pancreatic polypeptide-IR. No IR for either the D3 or D4 receptor was observed in rat islets. The protein level of the D1 receptor was reduced in T1DM rats (D1/D-glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 0.63 ± 0.05 in control rats compared with 0.16 ± 0.03 in T1DM rats, n = 8, P < 0.05) but no significant alteration was detected in the protein expression of either the D2 receptor (D2/GAPDH, 0.48 ± 0.04 compared with 0.43 ± 0.04, n = 8, P = 0.42) or the D5 receptor (D5/GAPDH, 0.50 ± 0.04 compared with 0.47 ± 0.04, n = 8, P = 0.58). The present study is the first clear demonstration of the protein expression and cellular localization of the D1, D2 and D5 receptors in rat pancreatic islets and provides crucial morphological evidence for further investigations of the underlying mechanism regarding the DA regulation of pancreatic endocrine function.

Upregulation of β1-adrenoceptors is involved in the formation of gastric dysmotility in the 6-hydroxydopamine rat model of Parkinson's disease.

Gastrointestinal dysmotility is one of the nonmotor symptoms of Parkinsons disease (PD). Gastroparesis and upregulated β-adrenoceptors (β-ARs) have been reported in rats with bilateral microinjection of 6-hydroxydopamine (6-OHDA) in the substantia nigra, but the underlying mechanism is unclear. The aim of the current study is to investigate the role of β-ARs in gastroparesis in 6-OHDA rats. Gastric motility was studied through strain gauge measurement. Immunofluorescence, real-time reverse transcription-polymerase chain reaction and Western blotting were performed to examine the expression of β-ARs. Norepinephrine (NE) inhibited gastric motility in a dose-dependent fashion in both control and 6-OHDA rats, but much stronger adrenergic reactivity was observed in the 6-OHDA rats. The inhibition of gastric motility by NE in both control and 6-OHDA rats was not affected by tetrodotoxin, a neural sodium channel blocker. Blocking β1-AR or β2-AR did not affect the inhibition of strip contraction by NE in control rats, but β1-AR blockage obviously enhanced the half maximal inhibitory concentration value of NE in 6-OHDA rats. Selective inhibition of β3-AR blocked the effect of NE significantly in both control and 6-OHDA rats. The protein expression of β1-AR, but not β2-AR and β3-AR in gastric muscularis externa was increased significantly in 6-OHDA rats. In conclusion, β3-AR involves the regulation of gastric motility in control rats, whereas the upregulation of β1-AR is responsible for enhanced NE reactivity in 6-OHDA rats and therefore is involved in the formation of gastroparesis. The effect of both β1-AR and β3-AR on gastric motility is independent of the enteric nervous system.

Alterations in TH- and ChAT-immunoreactive neurons in the DMV and gastric dysmotility in an LPS-induced PD rat model.

To study movement disorder in Parkinsons disease (PD), an animal model of PD can be created by injecting lipopolysaccharide (LPS) into the substantia nigra of rats. In addition to body movement disorders, patients with PD often experience gastrointestinal (GI) dysfunction, such as gastroparesis. However, the underlying mechanism of these disorders remains unclear. The dorsal motor nucleus of vagus (DMV) is a well-known visceral nucleus that regulates GI function. The present study investigated alterations in DMV neurons and gastric motility in rats with LPS-induced PD (LPS-PD rats). Gastric motility was recorded using a strain gauge force transducer in vivo. The distributions of tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-positive neurons in the DMV were determined using immunofluorescence and confocal laser microscopy. Our results indicated that in LPS-PD rats, the number of neurons in the substantia nigra, including neurons with TH immunoreactivity, was markedly reduced, although glial cell proliferation was clearly observed. However, enhanced TH immunoreactivity and decreased ChAT immunoreactivity were found in the DMV. Furthermore, weakened gastric motility was recorded in anesthetized LPS-PD rats. In conclusion, rats with LPS-induced PD exhibited gastric dysmotility with an alteration in DMV neurons. This PD model may be used to study autonomic nervous system disorders that are often observed in patients with early-stage PD.

Altered expression of dopamine receptors in cholinergic motoneurons of the hypoglossal nucleus in a 6-OHDA-induced Parkinson's disease rat model.

Parkinsons disease (PD) is a common neurodegenerative disorder that is often associated with weak tongue motility. However, the link between the degenerated dopaminergic neurons in the substantia nigra (SN) and lingual dysfunction remains unclear. In the present study, we investigated the localization of dopamine receptor 1 (D1) and dopamine receptor 2 (D2) and alternations in their expression in cholinergic motoneurons of the hypoglossal nucleus (HN) using double-label immunofluorescence, Western blotting and semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR) in rats that received microinjections of 6-hydroxydopamine bilaterally into the SN (6-OHDA rats). The results revealed that a large population of choline acetyltransferase immunoreactive (ChAT-IR) neurons was distributed throughout HN and that almost all of the ChAT-IR motoneurons were also D1-IR and D2-IR. Several tyrosine hydroxylase (TH)-IR profiles were observed in a nonuniform pattern near the ChAT-IR, D1-IR or D2-IR somas, suggesting potent dopaminergic innervation. In the 6-OHDA rats, TH immunoreactivity in the SN was significantly decreased, but food residue was increased and treadmill occupancy time was shortened. In the HN, protein expression of TH and D2 was increased, whereas that of ChAT and D1 was decreased. A similar pattern was observed in mRNA levels. The present study suggests that dopamine may modulate the activity of cholinergic neurons via binding with D1 and D2 in the HN. Changes in the expression of ChAT, TH, D1 and D2 in the HN of 6-OHDA rats might be associated with the impaired tongue motility in PD. These findings should be further investigated.

New perspectives of vesicular monoamine transporter 2 chemical characteristics in mammals and its constant expression in type 1 diabetes rat models.

Vesicular monoamine transporter 2 (VMAT2) has been exploited as a biomarker of β-cell mass in human islets. However, a current report suggested no immunoreactivity of VMAT2 in the β cells of rat islets. To investigate the cellular localization of VMAT2 in islets further, the pancreatic tissues from monkeys and humans were compared with those of rats and mice. The study was performed using among-species comparisons and a type 1 diabetes model (T1DM) for rats by Western blotting, double-label immunofluorescence, and confocal laser scanning microscopy. We found that VMAT2-immunoreactivity (IR) was distributed peripherally in the islets of rodents, but was widely scattered throughout the islets of primates. Consistent with rodent islets, VMAT2-IR did not exist in insulin (INS)-IR cells but was abundantly present in glucagon (GLU)-IR and pancreatic polypeptide (PP)-IR cells in monkey and human islets. VMAT2-IR had no colocalization with INS-IR in any part of the rat pancreas (head, body, and tail). INS-IR cells were reduced dramatically in T1DM rat islets, but no significant alteration in the proportion of VMAT2-IR cells and GLU-IR cells was observed. Furthermore, a strong colocalization of VMAT2-IR with GLU-IR was distributed in the peripheral regions of diabetic islets. For the first time, the current study demonstrates the presence of VMAT2 in α cells and PP cells but not in β cells in the islets of monkeys and humans. This study provides convinced morphologic evidence that VMAT2 is not present in β cells. There needs to be studies for new markers for β cell mass.

Dopamine enhances duodenal epithelial permeability via the dopamine D5 receptor in rodent

The intestinal barrier is made up of epithelial cells and intercellular junctional complexes to regulate epithelial ion transport and permeability. Dopamine (DA) is able to promote duodenal epithelial ion transport through D1‐like receptors, which includes subtypes of D1 (D1R) and D5 (D5R), but whether D1‐like receptors influence the duodenal permeability is unclear.