The human 5-HT1A receptor expressed in HeLa cells stimulates sodium-dependent phosphate uptake via protein kinase C.
Regulation of phosphate uptake was studied in HeLa cell lines after transfection with DNA encoding the human 5-HT1A receptor. Phosphate uptake was saturable and greater than 90% sodium-dependent, with Vmax approximately 30-35% without changing Km. Treatment with 5-HT or the 5-HT1A-specific agonist 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene increased Vmax approximately 40% without affecting Km. This effect was blocked by pretreatment with the 5-HT1 antagonists, methiothepine and spiperone, or pertussis toxin. Surprisingly, the stimulation was not secondary to an inhibition of adenylyl cyclase because 5-HT stimulated phosphate uptake approximately 20% in the presence of 1 mM 8-Br-cAMP. Rather, the primary pathway linked to the stimulation of phosphate uptake involved activation of protein kinase C because (i) 5-HT measurably activated protein kinase C in these cells, (ii) activators of protein kinase C (phorbol esters and diacylglycerol analogues) stimulated phosphate uptake in these cells (iii) the half-maximal doses for 5-HT-induced phosphatidylinositol hydrolysis and stimulation of phosphate uptake were virtually equivalent, and both effects were equally sensitive to pertussis toxin, and (iv) the stimulation was markedly attenuated in cells made deficient in protein kinase C. These results demonstrate that the stimulation of phosphatidylinositol hydrolysis by the 5-HT1A receptor can generate physiologically measurable effects on cellular transport and suggest that such accessory pathways may play a prominent role in signal transduction.
Raymond
J R
JR
Department of Medicine Nephrology, Duke University Medical Center, Durham, North Carolina 27710.
Fargin
A
A
Middleton
J P
JP
Graff
J M
JM
Haupt
D M
DM
Caron
M G
MG
Lefkowitz
R J
RJ
Dennis
V W
VW
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology; D002794:Choline / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D015536:Down-Regulation; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D012701:Serotonin / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D010710:Phosphates; D011985:Receptors, Serotonin; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D012701:Serotonin; D014660:Vasoactive Intestinal Peptide; D003994:Bucladesine; D012964:Sodium; D000242:Cyclic AMP; D011493:Protein Kinase C; D002794:Choline