Organic cation transporter mRNA and function in the rat superior cervical ganglion.
Reuptake of extracellular noradrenaline (NA) into superior cervical ganglion (SCG) neurones is mediated by means of the noradrenaline transporter (NAT, uptake 1). We now demonstrate by single-cell RT-PCR that mRNA of the organic cation transporter 3 (OCT3, uptake 2) occurs in rat SCG neurones as well. Furthermore, our RT-PCR analyses reveal the presence of mRNA for novel organic cation transporters 1 and 2 (OCTN1 and OCTN2), but not for OCT1 or OCT2 in the ganglion. Making use of the NAT as a powerful, neurone-specific transporter system, we loaded[3H]-N-methyl-4-phenylpyridinium ([3H]-MPP+) into cultured rat SCG neurones. The ensuing radioactive outflow from these cultures was enhanced by desipramine and reserpine, but reduced (in the presence of desipramine) by the OCT3 inhibitors cyanine 863, oestradiol and corticosterone. In contrast, cyanine 863 enhanced the radioactive outflow from cultures preloaded with [3H]-NA. Two observations suggest that a depletion of storage vesicles by cyanine 863 accounts for the latter phenomenon: first, the primary radioactive product isolated from supernatants of cultures loaded with [3H]-NA was the metabolite [3H]-DHPG; and second, inhibition of MAO significantly reduced the radioactive outflow in response to cyanine 863. The outflow of [3H]-MPP+ was significantly enhanced by MPP+, guanidine, choline and amantadine as potential substrates for OCT-related transmembrane transporters. However, desipramine at a low concentration essentially blocked the radioactive outflow induced by all of these substances with the exception of MPP+, indicating the NAT and not an OCT as their primary site of action. The MPP+-induced release of [3H]-MPP+ was fully prevented by a combined application of desipramine and cyanine 863. No trans-stimulation of [3H]-MPP+ outflow was observed by the OCTN1 and OCTN2 substrate carnitine at 100 microM. Our observations indicate an OCT-mediated transmembrane transport of [3H]-MPP+. Amongst the three OCTs expressed in the SCG, OCT3 best fits the profile of substrates and antagonists that cause trans-stimulation and trans-inhibition, respectively, of [3H]-MPP+ release.
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CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-N GENE-Y GENE-N CHEMICAL GENE-N GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y CHEMICAL GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL
MPP+ MPP+ [3H]-MPP+ desipramine [3H]-MPP+ [3H]-MPP+ [3H]-MPP+ noradrenaline NA [3H]-N-methyl-4-phenylpyridinium [3H]-NA [3H]-MPP+ desipramine reserpine desipramine oestradiol corticosterone [3H]-NA MAO noradrenaline transporter OCT [3H]-DHPG transmembrane transporters NAT NAT OCT OCTN1 OCTN2 OCT OCTs OCT3 organic cation transporter 3 noradrenaline OCT3 novel organic cation transporters 1 and 2 OCTN1 OCTN2 OCT1 OCT2 NAT OCT3 Organic cation transporter [3H]-MPP+ MPP+ guanidine choline amantadine desipramine