Neural targets of endogenous dopamine in the proximal colon were investigated. In addition, the contribution of constitutively active D1-like receptors to dopaminergic inhibition of the colonic motility was explored. Our principal findings were as follows: (i) the inhibition of dopamine reuptake with GBR increasing synaptic dopamine concentrations dilated segments of rat proximal colon via the stimulation of D1-like receptors, (ii) the blockade of D1-like receptor with SCH constricted colonic segments, (iii) the effects of GBR but not SCH on the colonic motility were inhibited by L-NA or TTX, (iv) the colonic segments in which neuronal dopamine had been depleted by 6-OHDA were unresponsive to GBR but constricted by SCH. These findings suggested that enteric dopaminergic nerves stimulate nitrergic neurons expressing D1-like receptors to inhibit the colonic motility. Constitutively active D1-like receptors are expressed in cells yet to be determined, presumably non-neuronal, cells that tonically dilate the proximal colon essential for the generation of coordinated peristalsis (Figure 8).
Neuronal target of dopaminergic neurons to inhibit colonic motility
Previous studies suggest that endogenous dopamine released from enteric dopaminergic neurons stimulates inhibitory efferent neurons including nitrergic, VIP and purinergic neurons [3, 10, 17, 30].
GBR-induced dilatations of the colonic segments were prevented by SCH and absent in the colonic segment from 6-OHDA treated rats, indicating that GBR specifically acts on dopaminergic neurons. The inhibitory actions of GBR were prevented by L-NA but not the VIP receptor antagonist VIP10-28 or the P2Y1 receptor antagonist MRS, suggesting that dopaminergic neurons primarily project to nitrergic rather than VIP or purinergic neurons to maintain coordinated peristalsis in the proximal colon.
Consistently, our previous immunohistochemical study demonstrated that D1 receptor immunoreactivity is co-localized with the nitrergic nerve marker neuronal NOS in the ENS [30]. Since D1-like receptors are coupled to the Gs protein [27], D1-like receptor-mediated activation of the adenylyl cyclase-cAMP-protein kinase A pathway would enhance NO release from nitrergic neurons [11]. Thus, GBR-induced enhancement of dopaminergic neurotransmission appears to act directly on nitrergic neurons.
Roles of apo-D1-like receptors in the regulation of colonic motility
SCH (20 μM)-induced constrictions of colonic segments were not blocked by either L-NA, VIP10-28 or MRS, suggesting that the constrictions were not due to the removal of inhibitory neuronal activity. In accordance with a previous study [3], the blockade of neuronal activity with TTX also failed to inhibit the excitatory actions of SCH. Thus, it was envisaged that constitutively active D1-like receptors [38], similarly to serotonin 5-HT3/4 receptors in the colon [35], are expressed in a population of cells that are presumably non-neuronal cells. This notion was supported by the findings that, in neurodegenerative conditions, characterized by the loss of peripheral dopaminergic neurons using 6-OHDA injected rats, SCH was still capable of constricting the colonic segment.
In our previous immunohistochemical study [30], the expression of D1 receptors was not detected in colonic smooth muscle cells, interstitial cells of Cajal or platelet-derived growth factor receptor α-positive cells that form an integrated postjunctional network, referred to as the SIP syncytium [8]. Transcriptome analyses has also demonstrated that gene expression of dopamine receptors are nearly absent in the SIP syncytium [12, 18, 19].
Since TTX that blocks neuronal action potential generation does not necessarily prevent the release of neurotransmitters, spontaneously active D1-like receptors expressed at synaptic terminals of non-nitrergic, non-VIP, non-purinergic efferent neurons may be involved in the SCH-induced colonic constrictions. Since the D5 receptor gene is expressed in colonic residential macrophages [23], constitutive active D1-like receptors in these residential macrophages may well be modulating colonic contractile activity [24, 28].
Non-neuronal sources of dopamine, e.g., gastrointestinal epithelium or dendritic cells that are known to synthesize dopamine [31, 37], may also be involved in the excitatory actions of SCH. However, since the epithelium cells are anatomically remote from circular muscle layers, epithelial dopamine would not effectively diffuse into the layers without undergoing degradation or dilution. It is also unlikely that dendritic cells, immune cells, are tonically activated without a stimulant such as lipopolysaccharide.
Compensatory mechanism for enteric dopaminergic dysfunction in the regulation of colonic peristalsis
Bath-applied 6-OHDA initially abolished the generation of colonic peristalsis with a colonic dilatation presumably due to a massive leakage of dopamine from destroyed nerves. During prolonged exposure to 6-OHDA for 2 hours, the diameter was partially reduced towards its original level and peristalsis was restored at a lower frequency than control conditions, suggesting that some dopaminergic nerves were still capable of releasing dopamine to maintain peristalsis. However, further exposure to 6-OHDA for 3 hours eventually abolished peristalsis presumably due to a total lesion of dopaminergic nerves. Nevertheless, in colonic segments in which neuronal dopamine is expected be chronically depleted with the 4-week-6-OHDA treatment, peristaltic contractions remained comparable to those of normal colonic segments, implying that compensatory mechanisms have been activated to regain apparently normal colonic motility (Figure 8).
The dopaminergic neurotoxin 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine reduces the enteric dopamine in mice only by 40%, and only a half of dopaminergic neurons in the substantia nigra and striatum are degenerated [1, 20], raising the possibility that dopaminergic neurons may not be completely destroyed in 6-OHDA treated rats. Yet, the lack of colonic dilatation with GBR in the present study strongly supported the notion that enteric dopaminergic neurons are not functional in 6-OHDA treated rats. The impairment of body weight gain by first (day 0) intraperitoneal injection of 6-OHDA is likely due to dopaminergic neuronal loss causing gastrointestinal dysfunction. In contrast, second (day 7) or third (day 14) injection of 6-OHDA did not interfere with body weight gain, suggesting that a vast majority of enteric dopaminergic neurons had already been destroyed by the first 6-OHDA injection.
Since intraperitoneally injected 6-OHDA does not cross the blood-brain barrier [4], the dopaminergic neurons in the brain must be well preserved. Therefore, the loss of enteric dopaminergic neurons appears to be compensated by recruiting a new neuronal network in the proximal colon to regain apparently normal peristalsis. Such a compensatory mechanism in the colon has also been reported after the loss of other type of neurons. In colonic segments of rodents where electrical field stimulation-evoked contractions are mediated by cholinergic and tachykinergic neurons, the nerve-evoked contractions are largely diminished 7 days after the onset of trinitrobenzene sulfonic acid-induced colitis [33, 34]. The nerve-evoked contractions are apparently restored 30 days after the induction of colitis, while the contractile components mediated purinergic and opioidergic neurons become evident [33, 34].
Roles of noradrenergic neurons in the regulation of motility in isolated colonic preparations
Noradrenaline released by electrical stimulation of sympathetic nerves inhibits colonic migrating motor complexes in isolated colonic segments of mice [16]. In our isolated rat colonic preparations, the selective noradrenaline reuptake inhibitor desipramine, β-adrenoceptor antagonist propranolol or the α-adrenoceptor antagonist phentolamine did not affect the colonic motility, suggesting that constitutively released noradrenaline has a negligible role in regulating the motility. Nevertheless, the adrenergic neuron blocker guanethidine reduced the frequency of colonic peristalsis. The guanethidine-induced inhibition of peristalsis could be attributable to its transient stimulation of noradrenaline releases, but subsequent desipramine did not enhance the inhibitory action. In guanethidine-treated segments, GBR was capable of dilating the colonic segments, suggesting that enteric dopamine was not depleted. Since P2Y1 receptor blockade with MRS reduced the frequency of colonic peristalsis, guanethidine-induced inhibition of peristalsis could be due to the blockade of purines that could be released from sympathetic nerves as co-transmitter(s).
Pathological implications
In patients with Parkinson’s disease, in whom dopaminergic neurons are reduced in the both central and enteric nervous systems, constipation is a commonly seen non-motor symptom [14, 32, 36]. While the degeneration of central dopaminergic neurons also results in the impairment of colonic contractile activity [6], reductions in daily fecal output occurred at 3 weeks after the lesion of nigrostriatal dopaminergic neurons, presumably due to enteric neuronal dysfunction secondary to central dopaminergic neural loss [5]. Thus, dysfunction of enteric dopamine rather than central dopamine may have an impact on constipation in the prodromal stage of Parkinson’s disease [14, 32]. Since colonic dysmotility induced by enteric dopaminergic neural loss appears to be recovered by a compensatory mechanism, subsequent neuronal loss of central dopaminergic neurons may contribute to late stage constipation in Parkinson’s disease patients.