Short-term regulation of Na+/K+ adenosine triphosphatase by recombinant human serotonin 5-HT1A receptor expressed in HeLa cells.
Agonist occupancy of the cloned human serotonin (5-HT)1A receptor expressed in HeLa cells stimulates Na+/K+ ATPase activity as assessed by rubidium uptake. The purpose of the study was to determine which of the receptor-associated signaling mechanisms was responsible for this effect. 5-HT stimulated Na+/K+ ATPase 38% at 2 mM extracellular potassium, an effect characterized by a decrease in apparent K0.5 from 2.8 +/- 0.3 to 1.8 +/- 0.3 mM potassium without a significant change in apparent Vmax. The EC50 for the transport effect was approximately 3 microM 5-HT. The response was pertussis toxin-sensitive but did not involve inhibition of adenylate cyclase, as stimulation of Na+/K+ ATPase by 5-HT was observed in the presence of excess dibutyryl cAMP. Protein kinase C was not required for the response since short-term incubation with the phorbol esters phorbol 12 myristate, 13 acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) did not mimic the 5-HT effect. Moreover, 5-HT increased Na+/K+ ATPase activity after inactivation of protein kinase C by overnight incubation with PMA. 5-HT and the sesquiterpene lactone thapsigargin increased cytosolic calcium in this cell model, and the EC50 for 5-HT corresponded with that for stimulation of Na+/K+ ATPase. Both thapsigargin and A23187, a calcium ionophore, also increased Na+/K+ ATPase activity in a dose-responsive fashion. The response to 5-HT, thapsigargin, and A23187 was blocked by conditions that removed the cytosolic calcium response. By two-dimensional gel electrophoresis, we established evidence for a calcium-sensitive but protein kinase C-independent signaling pathway. We conclude that the 5-HT1A receptor, which we have previously shown to stimulate phosphate uptake via protein kinase C, stimulates Na+/K+ ATPase via a calcium-dependent mechanism. This provides evidence for regulation of two separate transport processes by a single receptor subtype via different signaling mechanisms.
Middleton
J P
JP
Duke University Medical Center, Department of Medicine, Durham, North Carolina 27710.
Raymond
J R
JR
Whorton
A R
AR
Dennis
V W
VW
D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D010750:Phosphoproteins / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D011985:Receptors, Serotonin / Q000502:physiology*; D011994:Recombinant Proteins; D012413:Rubidium / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology*; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014162:Transfection
D010750:Phosphoproteins; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D013729:Terpenes; D012701:Serotonin; D000001:Calcimycin; D003994:Bucladesine; D019284:Thapsigargin; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium; D002118:Calcium