Dual action of serotonin on local excitatory and inhibitory neural circuits regulating the corticotropin-releasing factor neurons in the paraventricular nucleus of the hypothalamus.

Serotonergic neurons originating from the raphe nuclei have been proposed to regulate corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVH). Since glutamate- and γ-aminobutyric acid (GABA)-containing neurons, constituting the hypothalamic local circuits, innervate PVH CRF neurons, we examined whether they mediate the actions of serotonin (5-hydroxytryptamine [5-HT]) on CRF neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) or spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in PVH CRF neurons, under whole cell patch-clamp, using the CRF-modified yellow fluorescent protein (Venus) ΔNeo mouse. Serotonin elicited an increase in the frequency of sEPSCs in 77% of the cells and a decrease in the frequency of sIPSCs in 71% of the cells, tested in normal medium. Neither the amplitude nor decay time of sEPSC and sIPSC was affected, thus the site(s) of action of serotonin may be presynaptic. In the presence of tetrodotoxin (TTX), serotonin had no significant effects on either parameter of sEPSC or sIPSC, indicating that the effects of serotonin are action potential-dependent, and that the presynaptic interneurons are largely intact within the slice; distant neurons may exist, though, since some 20%-30% of neurons did not respond to serotonin without TTX. We next examined through what receptor subtype(s) serotonin exerts its effects on presynaptic interneurons. DOI (5-HT2A/2C agonist) mimicked the action of serotonin on the sIPSCs, and the serotonin-induced decrease in sIPSC frequency was inhibited by a selective 5-HT2C antagonist RS102221. 8-OH-DPAT (5-HT1A/7 agonist) mimicked the action of serotonin on the sEPSCs, and the serotonin-induced increase in sEPSC frequency was inhibited by a selective 5-HT7 antagonist SB269970. Thus, serotonin showed a dual action on PVH CRF neurons, by upregulating glutamatergic- and downregulating GABAergic interneurons; the former may partly be mediated by 5-HT7 receptors, whereas the latter by 5-HT2C receptors. The CRF-Venus ΔNeo mouse was useful for the electrophysiological examination.

Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

BACKGROUND: Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. EXPERIMENTAL APPROACH: Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. RESULTS: In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. CONCLUSIONS: Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.