[12] Regulation of Na+-H+ exchange by G protein-coupled receptors

Kim A. Neve, Mary P. Rosser, Diane L. Barber

Research output: Research - peer-reviewArticle

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Abstract

Both spectrofluorometric measurements of pHi and microphysiometric measurements of pHo can be used with a number of different approaches to study regulation of NHE1 by receptors and G proteins. Both techniques are limited by the requirement for whole cells and an intact cell membrane and are therefore relatively time intensive. Activities of other receptor-regulated effectors such as adenylyl cyclase and phospholipase C can be measured rapidly in a number of samples using membrane and reconstituted preparations. In contrast, a single determination with the spectrofluorometer, including acid loading, pHi recovery, and nigericin calibration, requires approximately 45 min. Additionally, reagents commonly used for characterizing signal transduction pathways, such as membrane-impermeant guanine nucleotides and function-perturbing antibodies, are difficult to use when studying pHi in intact cells. Recently available microinjection systems (Eppendorf), designed for mammalian cells, can now be used with ratiometric imaging systems (Universal Imaging; Zeiss) to facilitate the manipulation of transduction pathways. Methods used for ratiometric imaging, including buffer compositions, fluorescent dyes, acid loading, and nigericin calibration of pHi, are identical to those described for the spectrofluorometer. Both of these techniques establish that NHE1 activity is regulated by G protein-coupled receptors independently of many other signaling pathways modulated by the receptors. The microphysiometric and fluorometric techniques yield data that are in agreement concerning the effect of PGE1 on Na+-H+ exchange and that are generally in agreement concerning the sensitivity of the response to pertussis toxin, but that differ in one important respect: endogeneous dopamine D2 receptors inhibit NHE1 in primary cultures of rat anterior pituitary cells (10), whereas recombinant D2 receptors stimulate NHE1 in C6 glioma cells and Ltk- cells (19). There are several possible explanations for this discrepancy. It could be due to differences between recombinant and endogenous D2 receptors, or differences between the cell lines and primary cultures. Alternatively, the discrepancy could be related to the use of serum-treated cells in the spectrofluorometer to assess inhibition of NHE1 activity and serum-starved cells in the microphysiometer to assess stimulation of NHE1 activity. Although G protein involvement in receptor-stimulated NHE1 activity has been demonstrated, and two constitutively active G proteins stimulate NHE1, the specific G proteins that mediate stimulation and inhibition of NHE1 by G protein-coupled receptors remain to be identified. An additional important area of research concerns the possibility that G proteins directly modulate NHE1 activity, as opposed to modulating other signaling pathways that in turn activate NHE1 by phosphorylation.

LanguageEnglish (US)
Pages225-241
Number of pages17
JournalMethods in Neurosciences
Volume25
Issue numberC
DOIs
StatePublished - 1995

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G-Protein-Coupled Receptors
GTP-Binding Proteins
Nigericin
Calibration
Acids
Membranes
Serum
Guanine Nucleotides
Dopamine D2 Receptors
Pertussis Toxin
Microinjections
Type C Phospholipases
Prostaglandins E
Fluorescent Dyes
Adenylyl Cyclases
Glioma
Signal Transduction
Buffers
Phosphorylation
Cell Membrane

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

[12] Regulation of Na+-H+ exchange by G protein-coupled receptors. / Neve, Kim A.; Rosser, Mary P.; Barber, Diane L.

In: Methods in Neurosciences, Vol. 25, No. C, 1995, p. 225-241.

Research output: Research - peer-reviewArticle

Neve, Kim A. ; Rosser, Mary P. ; Barber, Diane L./ [12] Regulation of Na+-H+ exchange by G protein-coupled receptors. In: Methods in Neurosciences. 1995 ; Vol. 25, No. C. pp. 225-241
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