Comparison of G-protein selectivity of human 5-HT2C and 5-HT1A receptors.

We compared the ability of human 5-HT2C and 5-HT1A receptors to couple to selected G proteins expressed in insect Sf9 cells through simultaneous infection with recombinant baculoviruses. We also examined the coupling of G proteins to these same receptors in membranes derived from the Sf9 cells using in situ reconstitution with purified G proteins. Our data show that unoccupied 5-HT2C and 5-HT1A receptors can attain an activated conformation that is stabilized by interaction with specific G proteins. While high-affinity agonist binding to the 5-HT2C receptor was increased to a greater extent by Galphaq than by Galphai2, the high-affinity agonist binding to the 5-HT1A receptor was preferentially enhanced by Galphai2 coexpression. When the two 5-HT receptors were expressed in cells also expressing G proteins, both 5-HT2C and 5-HT1A receptors appear to activate Galphai2 in preference to Galphaq. In contrast, in situ reconstitution data show that 5-HT2C receptors robustly activate Galphaq and marginally activate Galphao or Galphai, whereas 5-HT1A receptors only marginally activate Galphaq and robustly activate Galphao and Galphai. These results suggest that the overexpression of receptor and potential G-protein coupling partners in Sf9 cells may lead to erroneous conclusions as to the signaling selectivity of receptors.

Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines.

The extrusion of protons is considered a very general parameter of the activation of many kinds of membrane or intracellular molecules, such as receptors, ion channels, and enzymes. We found that pepstatin A caused a reproducible, concentration-related increase in the extracellular acidification rate in two microglial cell lines, Ra2 and 6-3. Washing abolished pepstatin A-induced acidification immediately. However, pepstatin A did not cause the extracellular acidification in other cell types, such as CHO, C6 glioma, and NIH3T3 cells. These observations strongly suggest that pepstatin A interacts with certain membrane proteins specific to both Ra2 and 6-3 cells from outside. N-methylmaleimide and N,N'-dicyclohexylcarbodiimide, inhibitors of H(+)-ATPase, were found to reduce pepstatin A-induced response strongly, while bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, vanadate, a P-type H(+)-ATPase inhibitor, and NaN3, an F1 ATPase inhibitor, virtually did not. 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of Na(+)/H(+) exchanger isoform 1, greatly enhanced pepstatin-induced response, while amiloride did not. Zn(2+), a voltage-dependent proton channel blocker, did not affect pepstatin-induced response neither. Staurosporine, a nonspecific inhibitor of protein kinase C, inhibited pepstatin A-induced response, while chelerythrine, more selective inhibitor of protein kinase C, greatly enhanced it. H-7 and H-8 did not affected the response. These findings suggest that pepstatin A induces extracellular acidification in microglia cell lines, Ra2 and 6-3, through an N-methylmaleimide- and N,N'-dicyclohexylcarbodiimide-sensitive, but bafilomycin A1-insensitive, ATPase, which seems to be distinct from protein kinase C-dependent process.

How does prolonged caloric restriction ameliorate age-related impairment of long-term potentiation in the hippocampus?

Prolonged dietary restriction has been reported to suppress age-induced phenomena. In order to investigate how prolonged caloric restriction reduces age-related deterioration of hippocampal synaptic transmission, we compared the levels of major hippocampal polyunsaturated fatty acids, arachidonic acid and docosahexaenoic acid between 4- and 26-month-old rats. The Ca(2+) responses upon perfusion of NMDA or 30 mM K(+) between 4- and 26-month-old rats with prolonged dietary restriction were also compared using the fluorescent probe Fura-2. A decrease in membrane arachidonic acid is thought to be a major causal factor in the age-related impairment of long-term potentiation. Long-term caloric restriction seems to increase arachidonic acid levels regardless of age. However, there is no significant difference of hippocampal arachidonic acid levels between in freely feeding 4- and 26-month-old rats. Similar results were obtained from the measurement of hippocampal docosahexaenoic acid levels. Under caloric restriction, the 500 microM N-methyl-D-aspartate-induced Ca(2+) response was greatly reduced by aging, while the 30 mM K(+)-induced Ca(2+) response was not affected. In our preliminary data, the amplitude of the population spike after tetanic stimulation did not differ between 4- and 26-month-old rats under caloric restriction, while 50 microM of 2-amino-5-phosphonovaleric acid, a N-methyl-D-aspartate antagonist, markedly inhibited a potentiation of the population spike in 4-month-old rats, but with negligible inhibition in 26-month-old rats. From these results, an age-related impairment of hippocampal excitatory synaptic transmission may not be solely due to the reduction of membrane arachidonic acid. Caloric restriction might prevent age-related reduction in hippocampal synaptic transmission by enhancing non-N-methyl-D-aspartate mechanisms.

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways.

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.