Short-term regulation of Na+/K+ adenosine triphosphatase by recombinant human serotonin 5-HT1A receptor expressed in HeLa cells.

Abstract

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 Ko5 from 2.8±0.3 to 1.8±0.3 mM potassium without a significant change in apparent V,,,. The EC50 for the transport effect was -3 uM 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-HTIA 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. (J. Clin.

Antagonistic actions of renal dopamine and 5‐ hydroxytryptamine: increase in Na+, K+‐ATPase activity in renal proximal tubules via activation of 5‐HT1A receptors

“…However, our results agree with those of Renal 5-HT and Na',K -ATPase activity (Middleton et al, 1990) while showing stimulation of Na',K+-ATPase by 8-OH-DPAT in HeLa cells expressing the human 5-HTIA. The notion that stimulation of Na',K+-ATPase activity is accomplished by activation of the 5-HTlA receptor subtype is further supported by the fact that the effect of 8-OH-DPAT is abolished in a concentration-dependent manner by the specific 5-HTIA receptor antagonist, (+)-WAY 100135.…”

The recombinant 5‐HT1A receptor: G protein coupling and signalling pathways

“…Interestingly when the human 5‐HT 1A receptor was expressed in Xenopus oocytes, it was shown to activate PLC ( Ni et al ., 1997 ). In aggregate, the work of several laboratories has shown that the 5‐HT 1A receptor couples to PI‐PLC in HeLa cells ( Boddeke et al ., 1992 ; Harrington et al ., 1994 ; Middleton et al ., 1990 ), in Ltk − cells ( Liu & Albert, 1991 ), in Xenopus oocytes ( Ni et al ., 1997 ), and in BALB/c‐3T3 cells ( Abdel‐Baset et al ., 1992 ), but only weakly or not at all in CHO cells ( Cowen et al ., 1997 ; Newman‐Tancredi et al ., 1992 ; Raymond et al ., 1992 ), Cos‐7 cells ( Fargin et al ., 1989 ), NIH‐3T3 cells ( Varrault et al ., 1992a ), or GH 4 C 1 cells ( Liu & Albert, 1991 ).…”

“…Activation of protein kinase C (PKC) occurs in HeLa cells ( Middleton et al ., 1990 ; Raymond et al ., 1989 ), and depends upon phospholipase C activation ( Fargin et al ., 1989 ). This effect is probably mediated by G protein βγ subunits, and almost certainly depends upon the expression of βγ‐regulatable PLC.…”

“…In addition to regulating channels, the 5‐HT 1A receptor has been shown to activate several active ion transport processes. In HeLa cells, the receptor stimulates Na + ‐dependent phosphate uptake through a PKC‐mediated pathway ( Raymond et al ., 1989 ; 1990 ), and Na + /K + ‐ATPase through a Ca 2+ ‐mediated pathway ( Middleton et al ., 1990 ). Both pathways probably depend upon G βγ ‐mediated stimulation of PLC.…”

[Ca2+]i determines the effects of protein kinases A and C on activity of rat renal Na+,K+‐ATPase

“…Studies on microsomal NKA preparations from various tissues and species (Limas et al 1973; Tria et al 1974; Braughler & Corder, 1978; Lingham & Sen, 1982; Tung et al 1990; Bertorello et al 1991; Lopina et al 1995) have revealed an inhibitory effect of cAMP or PKA on NKA. Incubation of HeLa cells (Middleton et al 1990) or mouse LTK − cells (Horiuchi et al 1993) with cAMP is accompanied by a decrease in NKA enzymatic activity. In contrast, injection or superfusion of Xenopus oocytes with cAMP has a clear stimulatory effect on the pump current (Vasilets & Schwarz, 1992).…”