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Inhibition of Activity of GABA Transporter GAT1 by delta-Opioid Receptor |
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Here we investigated regulation of GABA transporter GAT1 by deltaOR in rats and in Xenopus oocytes. |
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The function of GAT1 was analyzed in terms of Na+-dependent [3H]GABA uptake as well as GAT1-mediated currents. |
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Coexpression of deltaOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. |
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Activation of deltaOR further reduced the rate of GABA uptake as well as GAT1-mediated current. |
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Coexpression of muOR, as well as muOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. |
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Inhibition of GAT1-mediated current by activation of deltaOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. |
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We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia. |
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Therefore, we next examined the interaction between GAT1 and muOR or deltaOR using oocyte as a simplified and well-controlled system. |
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To investigate regulation of GAT1 by opioid receptor, we used the Xenopus oocytes with heterologously expressed GAT1 and opioid receptor as a model system. |
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Only oocytes with heterologously expressed GAT1 showed significant [3H]GABA uptake (see Figure 3(a)). |
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Corresponding amounts of cRNA for the rat Na pump, ralpha2beta, had no significant effect on the GAT1-mediated uptake (Figure 2). |
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The reduced uptake in oocytes expressing both the GAT1 and the deltaOR compared to those expressing GAT1 alone cannot be attributed to background activation of deltaOR since treatment with the opioid receptor antagonist naloxone (1 muM) could only slightly reverse the inhibition (Figure 3(a)). |
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Functional expression of deltaOR and muOR was confirmed in the absence of GAT1 activity by application of 100 nM of the deltaOR or muOR agonists, DPDPE or DAMGO, respectively, and by the resulting activation of the endogenous Ca2+-activated channels (data not shown, see also); it is worth to mention that the voltage dependencies of the currents induced by DPDPE and DAMGO differ from each other indicating that different signaling pathways are activated. |
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Since the heterologous expression of GAT1 with deltaOR, but not muOR, exhibited significant effect on GABA uptake, we focused the rest of our study on deltaOR. |
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Effect of Opioid Receptor Coexpression on GAT1-Mediated Steady-State Current |
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Dependence of GAT1-mediated current on membrane potential was determined in voltage-clamp experiments as the difference of membrane current in the presence and the absence of 100 muM GABA. |
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The steady-state GAT1-mediated current was reduced in the oocytes expressing GAT1 when 10 ng cRNA for deltaOR were coinjected (Figure 4(b)) to a similar extent as the GABA uptake (compared with Figure 3(b)). |
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As found for the uptake, activation of deltaOR by application of DPDPE (100 nM) led to further inhibition of GAT1-mediated current by about 50%. |
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Because DPDPE could induce a Ca2+-dependent current (see above), the effect of DPDPE on GAT1-mediated current was, therefore, determined in the presence of DPDPE as the difference of current in the presence and absence of GABA. |
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Similar to the findings with the GABA uptake, coexpression of muOR had no significant effect on the GAT1-mediated steady-state current (Figure 4(b)), and activation of the receptor by 100 nM DAMGO did not significantly influence the current (Figure 4(c)). |
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Also activation of the endogenous acetylcholine (ACh) receptor by 100 muM ACh did not affect the GAT1-mediated current (data not shown). |
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Effect of Opioid Receptor Coexpression on GAT1-Mediated Transient Current |
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The coexpression of deltaOR obviously led to a reduction in the number of functioning transporters by about 30% which can only partially account for the reduction of GABA uptake (about 70%, see Figure 3(b)) or of GAT1-mediated current (about 75%, see Figures 4(b) and 4(c)). |
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Neither the GAT1-mediated steady-state currents nor the transient charge movements were significantly affected when the ralpha2beta Na+ pump was coexpressed with the GAT1. |
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Western blot analysis shows that coexpression of deltaOR does not significantly affect the band intensity for GAT1 (Figure 6(a)). |
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Activation of delta-Opioid Receptor Inhibits GAT1-Mediated Current in PAG Neurons |
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The above data have demonstrated that deltaOR interferes with GAT1 activity; in particular, activation of the opioid receptor inhibits GAT1 function. |
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Steady-state currents were determined during superfusion of the brain slice with different solutions; in a typical experiment sequence was with SGABA representing solution with GABA and SGABA+inhibitor solution with additional GAT1-specific inhibitor tiagabine or NNC711. |
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To determine GAT1-mediated current, the currents in SGABA before and after application of the inhibitor were averaged, and the current in the presence of the inhibitor was subtracted to obtain the current component sensitive to the specific inhibitor. |
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Thereafter, solutions in the presence of the deltaOR against DPDPE were applied: Correspondingly, the GAT1-mediated current was determined as the difference of current in the absence and presence of the GAT1 inhibitor. |
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For the GAT1-mediated current averaged data are presented in Figure 7(b). |
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The result revealed strong inhibition of GAT1-mediated current in response to application of the deltaOR-specific agonist DPDPE (Figure 7). |
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The nearly complete inhibition of total GAT1-mediated current indicates that in the brain slices all transport modes of GAT1 were blocked. |
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Hence, these data on PAG slices are consistent with the DPDPE-induced reduction of GAT1-mediated GABA uptake and current observed in oocytes. |
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For further investigation we have expressed GAT1 of mouse brain together with mouse deltaOR and muOR in Xenopus oocytes. |
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When GAT1 and the deltaOR of mouse brain were coexpressed in Xenopus oocytes by coinjection of cRNA for GAT1 (40 ng) and different amounts for deltaOR (0, 5, 10, 20, 40 ng), increasing amounts of the coinjected cRNA of deltaOR led to decreased rate of GAT1-mediated GABA uptake (Figure 2); the dependency was arbitrarily fitted by with half-maximum inhibition of the rate at K 1/2 = 10.7 ng and n = 1.7. |
| Evidence Sentence: |
To investigate regulation of GAT1 by opioid receptor, we used the Xenopus oocytes with heterologously expressed GAT1 and opioid receptor as a model system. |
