In vitro autoradiographic visualization of guanosine-5'-O-(3-[35S]thio)triphosphate binding stimulated by sphingosine 1-phosphate and lysophosphatidic acid.
Sphingosine 1-phosphate or lysophosphatidic acid activation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to G proteins was studied by in vitro autoradiography in rat and guinea pig brain. The highest stimulation of [35S]GTPgammaS binding by sphingosine 1-phosphate was observed in the molecular layer of the cerebellum. Marked stimulation was observed in most forebrain areas, including neocortex and striatum. With the exception of the substantia gelatinosa and nucleus of the solitary tract, sphingosine 1-phosphate-enhanced binding was weaker in the brainstem and spinal cord. Lysophosphatidic acid-enhanced labeling was only observed in white matter areas. The G protein inhibitor 5'-p-fluorosulfonylbenzoyl guanosine completely inhibited lysophosphatidic acid-enhanced [35S]GTPgammaS binding but only partially sphingosine 1-phosphate-enhanced binding. N-Ethylmaleimide abolished binding stimulated by both agonists. Sphingosine 1-phosphate enhanced labeling by another GTP analogue (beta,gamma-imido[8-3H]guanosine-5'-triphosphate) similarly to that of [35S]GTPgammaS. Lysophosphatidic acid stimulated [35S]GTPgammaS binding in the olfactory bulb, glia limitans, and cortical subventricular zone of 1-day-old rats, whereas enhanced labeling was not observed in the latter area of 5-day-old rats. Sphingosine 1-phosphate stimulated binding in the cortical and striatal subventricular zones and olfactory bulb in 1- and 5-day-old rats. In the absence of radioligand for sphingosine 1-phosphate and lysophosphatidic acid receptors, [35S]GTPgammaS autoradiography provides a unique opportunity to study the spatial distribution, ontogeny, and coupling properties of these receptors.
Waeber
C
C
Massachusetts General Hospital and Harvard Medical School, Charlestown 02129, USA.
Chiu
M L
ML
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000819:agonists / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006168:Guinea Pigs; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008246:Lysophospholipids / Q000378:metabolism*; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013462:Sulfur Radioisotopes
D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D013462:Sulfur Radioisotopes; C060506:sphingosine 1-phosphate; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D013110:Sphingosine; D005033:Ethylmaleimide