| Dopamine (DA), catecholamine neurotransmitter, is synthesized from tyrosine hydroxylated by tyrosine hydroxylase (TH). It is not only an established neurotransmitter in the central nervous system, but also an important enteric neurotransmitter and an important modulator of enteric function. DA receptors are cell surface receptors and belong to a large family of guanine nucleotides regulatory (G) protein-coupled receptors. They have been categorized into two major subtypes based largely on central nervous system studies, designated D1-like (DA-1) and D2-like (DA-2). Now at least five DA receptors subtypes have been identified, including two D1-like (D1 and D5 receptors, known also in rodents as D1A and D1B sites) and three D2-like receptor genes (D2, D3 and D4). DA receptors are widely expressed in brain as well as in extracerebral structures such as the heart, blood vessels, carotid body, kidney, adrenal gland, parathyroid gland and gastrointestinal tract. It is associated with distinct functions including motor behavior, regulation of blood flow, gastrointestinal motility and secretion, mucosal "defence", and pancreatic exocrine secretion. Defective tissue DA production and/or DA receptor function have been reported in Parkinson's disease (PD), human essential hypertention and inanimal genetic hypertension models, gastrointestinal dysfunction.PD often occurs at elderly people. In digestive system chronic constipation and dysphagia is the main symptom. The former is both a common and an early symptom. Some studies demonstrated constipation in PD shows slow colonic transit, decreased phasic rectal contraction and paradoxical sphincter contraction on defecation. Others showed dopaminergic neurons in the enteric system are defected in PD with choric constipation. These studies suggest that abnormal regulation of DA is associated with constipation in PD. There have been only a few studies on the effect of DA on colonic ion transportation, however, the results are highly inconsistent.In order to understand possible cause of constipation in PD patients, the present study aimed to investigate the role of DA on rat distal colonic electrolyte transport and to determine the type of receptors and the intracellular signaling mechanisms involved in the response.Healthy adult male SD rats were used. Segments of distal colon about 5 cm were removed. Stripped epithelial sheets were mounted in Ussing chambers connected to a voltage current clamp and changes in the short circuit current (μA/cm2) were measured continuously as an index of electrogenic ion transfer with different blockers, inhibitors, activators, antagonists and agonists. The results are given as arithmetic means ± S.E.M.. Statistical analyses were performed by one-way ANOVA, paired sample t-test and independent sample t-test. A P value less than 0.05 was assumed to denote a significant difference.Indomethacin (10 μM), TTX (1 μM), or both, when added to the serosal side, potential difference (PD) changed to -1.5 ± 0.3 raV, -2.1 ± 0.2 mV, -2.8 ± 0.6 mV, basal current (BC) to 12.4 ± 2.1 μA/cm2, 22.3 ± 2.5 μA/cm2, 26.5 ± 5.1 μA/cm2, transepithelial resistance (TR) to 125.4 ±11.6 Ωm2, 95.3 ± 7.2 Ωcm2, 102.2 ± 7.4 Ω cm2. DA, added to the serosal bathing solution, rapidly decreased Isc in a concentration dependent manner with an EC50 of 20.06 μM. The delta Isc produced by a single maximal dose of DA had the same magnitude as that produced by successive addition of smaller doses, reaching the samefinal concentration. Mucosal DA had no effect. Pretreatment with indomethacin (10μM) or indomethacin and TTX (1 μM) did not alter DA-induced Isc .But pretreatment with TTX reduced it by 36.7% (P<0.05). Different segments of distal colonic mucosa had different responses to DA, with remarkable response observed in the segments close to anus. The Isc induced by DA could not be observed when apical Cl- or bilateral Cl- or HCO3- was replaced. Apical addition of epithelial Na+ channel blocker amiloride (10 μM), non-selective K+channel blocker Ba2+(5 mM) and calcium-dependent CI" channel blocker DIDS (100 μM) didn't significantly affect DA-induced Isc decrease. However, non-specific Cl- channels blocker DPC (1 mM) or glibenclamide (1 mM) could inhibit DA-induced Isc response by 70.9% (P<0.001) and 77.0% (P<0.001). Basolateral addition of DPC (1 mM) or glibenclamide (1 mM) inhibited the effect of DA by 73.0% or completely blocked it. The Isc response was also attenuated by 86.1% when pretreated with bumetanide (100 μM), an inhibitor of Na+-K+-2Cl- cotransportor or K+-Cl-cotransportor (P<0.001), but it was insensitive to DIDS (200 μM), an inhibitor of Na+-HCO3- cotransportor and Cl-/HCO3- exchanger (P>0.05). Basolateral application of SCH-23390 (0.01-10 μM), D1-like receptor antagonist, or sulpiride (10 μM, 50 μM) and nemonapride(l μM, 10 μM), D2-like receptor antagonist had no effect on the response of colonic mucosa to DA. D1-like receptor agonist SKF-38393 (1-20 μM) and D2-like receptor agonist quinpirole (1~l00μM) could induce Isc increase and had no change in Isc, and had no effect on succedent DA response. The signaling study showed that bilateral addition of MDL-12330A (10 μM), an inhibitor of adenylate cyclase, combined with H89 (10 μM), a PKA inhibitor, significantly inhibited DA-induced Isc (70.9% reduction, P< 0.001). Apical addition of BAPTA-AM (100 μM), Ca2+ chaletor, inhibited DA response by 69.6 % (P<0.001).In conclusion, the results suggested that DA could stimulate rat distal colonic Cl-absorption in a concentration dependent manner with prominent response observed in the segments close to anus. The signal transduction mechanisms involved the activation of either cAMP-PKA or Ca2+ pathway, which may not be mediated by D1-like or D2-likereceptors. |
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