Role of G-proteins in the regulation of organic osmolyte efflux from isolated rat renal inner medullary collecting duct cells

Abstract

Hypotonic shock (change of osmolality from 600 mosmol to 300 mosmol by lowering NaCl concentration) increases the release of organic osmolytes from isolated inner medullary collecting duct (IMCD) cells in the following sequence: taurine > betaine > sorbitol > myo-inositol > glycerophosphorylcholine (GPC). The role of G-proteins in regulating the hypotonicity-induced efflux was analysed by exposing cells to various concentrations of a G-protein inhibitor, pertussis toxin (PTX; 20-200 ng/ml), and a Gialpha-protein stimulator, mastoparan (10-50 microM). PTX diminished the hypotonic release of sorbitol and betaine by 43.2+/-9. 5% and 32.2+/-7.8% (n = 5), respectively. Efflux of GPC, myo-inositol and taurine was not significantly altered. Mastoparan (10 microM) increased osmolyte release under isotonic conditions such that release of betaine was increased 3.8-fold and that of sorbitol 2.1-fold, while GPC, myo-inositol and taurine effluxes were only slightly augmented. Under hypotonic conditions, mastoparan stimulated betaine release (1.86+/-0.2-fold, n = 5) but not that of sorbitol. As tested in connection with sorbitol and betaine release, the effect of mastoparan was abolished by PTX, but not the A23187-evoked sorbitol release. Like mastoparan, arachidonic acid increased the release of sorbitol and betaine under isotonic conditions, but under hypotonic conditions it only increased the release of betaine. As to the role of intracellular Ca2+, hypotonic shock evoked an intracellular Ca2+ peak which could be prevented by PTX. Mastoparan increased intracellular Ca2+ under isotonic conditions, whether the extracellular Ca2+ concentration was low or high. The results indicate that G-proteins are involved in regulating sorbitol and betaine efflux from IMCD cells. The G-proteins regulating sorbitol release are probably involved in generating the proper intracellular Ca2+ signal. Betaine efflux, which is independent of intracellular Ca2+, might be regulated by a G-protein-stimulated release of arachidonic acid. Thus, probably several G-proteins are involved in controlling organic osmolyte efflux from IMCD cells.

Physiology of Cell Volume Regulation in Vertebrates

“…Their sn-2 ester bonds are hydrolyzed by members of the PLA 2 family, providing membrane-bound lyso-phospholipids and free fatty acids for downstream signaling. Released arachidonic acid is in itself a bioactive lipid, which can directly activate transport pathways for, e.g., K ϩ (450, 749) and organic osmolytes (491,868). In contrast, at higher concentrations, arachidonic acid directly inhibits swelling-activated K ϩ , Cl Ϫ , and taurine efflux (316, 489,672,881).…”

“…Furthermore, the volume-sensitive taurine efflux pathway does not respond to diet-induced adaptive regulation as does TauT, supporting the notion that the swelling-induced taurine efflux pathway is different from TauT (1052). The swelling-induced taurine efflux increases exponentially with reduction of extracellular tonicity (364, 492,772,786,868), and it has been estimated that a 20% decrease in the osmolarity is required for activation of taurine efflux in, e.g., NIH3T3 cells (786). The volume-sensitive taurine efflux pathway has the properties of a diffusion pathway permeable to various organic osmolytes (taurine Ͼ sorbitol Ͼ choline Ͼ thymidine ϾϾ sucrose) (316) and is sensitive to an array of inhibitor compounds (DIDS, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, tamoxifen, polyunsaturated fatty acids) (see Table 6 and Refs.…”

Volume Regulation of Thick Ascending Limb of Henle Cells: Significance of Organic Osmolytes

“…It was proven for the rat inner medullary collecting duct that changes in ion conductivity are of minor importance in the maintenance of cell volume [6, 7]. The organic osmolytes (sorbitol, inositol, betaine, glycerophosphorylcholine (GPC), and amino acids) are most important in the osmoregulation of collecting duct cells [8, 9, 10, 11]. Similar to the collecting duct cells, it was possible to describe a so-called ‘osmotic gap’ by means of measuring the intra- and extracellular ion content in the thick ascending limb.…”

Small molecule-enrichment analysis in response to osmotic stimuli in the intervertebral disc

“…Davis et al (1992) suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway. Besides, inhibitors of G proteins have been shown to blunt swelling-induced osmolyte efflux (Ruhfus et al, 1996), as well as increase intracellular Ca 2+ concentration (Arora et al, 1994) and mitogen-activated protein kinase (MAPK) activity (Noe et al, 1996). These results suggest that G proteins do regulate some effects of cell volume.…”