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]GTPγS) binding to G proteins was studied by in vitro autoradiography in rat and guinea pig brain. The highest stimulation of [³⁵S]GTPγS 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]GTPγS 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 (β,γ-imido[8- ³H]guanosine-5'-triphosphate) similarly to that of [³⁵S]GTPγS. Lysophosphatidic acid stimulated [³⁵S]GTPγS 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]GTPγS autoradiography provides a unique opportunity to study the spatial distribution, ontogeny, and coupling properties of these receptors.