Characterization of 5-HT1A receptor-mediated []GTPγS binding in rat hippocampal membranes
Introduction
Activation of central 5-HT1A receptors elicits a variety of well-characterized biological and cellular responses including stimulation of hormone secretions from the anterior pituitary gland, hypotension and bradycardia, neuronal hyperpolarization, activation of an inwardly rectifying K+ current and inhibition of forskolin-stimulated adenylyl cyclase as examples (Zifa and Fillion, 1992). These responses occur after agonist binds to the seven transmembrane G protein-coupled 5-HT1A receptor (Hoyer et al., 1994) to activate the Gi and/or Go protein subtypes (Harrington et al., 1988; Zgombick et al., 1989; Okuhara and Beck, 1994; Oleskevich, 1995). Currently the primary methods available to provide functional assessment of central 5-HT1A receptor activity at the cellular level require intact cells for electrophysiological studies or adenylyl cyclase assays. Recently there have been numerous reports of measuring agonist-stimulated []GTPγS binding, the initial step in signal transduction following G protein activation, as an index of receptor stimulation for a wide variety of neurotransmitters. To date, the only applications of GTPγS binding to study 5-HT1A receptor function have been in heterologous expression systems using cloned human 5-HT1A receptors (Newman-Tancredi et al., 1996a, Newman-Tancredi et al., 1996b, Newman-Tancredi et al., 1997a; Stanton and Beer, 1997). It was the purpose of these studies to develop and characterize 5-HT1A receptor-mediated GTPγS binding in rat hippocampal membranes as a measure of 5-HT1A receptor function using frozen tissue homogenates. We have demonstrated that GTPγS binding can be used to assess 5-HT1A receptor function in membrane preparations of both hippocampus and frontal cortex and thus provides a novel ex vivo method to investigate the initial cellular event in transmission of an extracellular signal to an intracellular response.
Section snippets
Membrane preparation
Hippocampi were obtained from two sources. For some experiments frozen tissue was purchased from Pel-Freez Biologicals (Rogers, AR). Other tissue was obtained from male Sprague–Dawley rats purchased from Harlan Industries (Indianapolis, IN). Rats were killed by decapitation, the brains placed in ice-cold 0.9% NaCl for 1–3 min, the hippocampi dissected free-hand and placed in a vial on ice. The tissue was weighed and frozen at −70°C. Upon defrosting the tissue was homogenized using a Tekmar
Effects of GDP on agonist-stimulated GTPγS binding in hippocampus and frontal cortex
The effects of graded concentrations of GDP on GTPγS binding were determined in two experiments. The data presented in the inset of Fig. 1 demonstrate that GDP causes a marked reduction in basal (i.e. agonist-independent) GTPγS binding in hippocampus and frontal cortex. Furthermore, there is a direct relationship between the GDP concentration in the incubation mix and the stimulation of GTPγS binding produced by 1 μM of the full 5-HT1A receptor agonist R(+)-8-OH-DPAT in both brain regions (Fig.
Discussion
Experiments were designed to characterize 5-HT1A receptor mediated []GTPγS binding in rat hippocampal membranes. The data suggest that the non-selective agonists 5-HT and 5-CT stimulate GTPγS binding through activation of more than just the 5-HT1A receptor: (1) they produce responses greater than those produced by the full, selective 5-HT1A receptor agonist R(+)-8-OH-DPAT and (2) the effects of 5-HT were not completely abolished by the selective 5-HT1A receptor antagonist WAY 100,635. In
Acknowledgements
These studies were conducted while Dr. Alper was a Visiting Scientist at Eli Lilly and Company on sabbatical leave from the University of Kansas School of Medicine. Dr. Alper was supported in part by grants from the NIH (NS 29765) and a Career Reorientation Award from the Kansas Affiliate of the American Heart Association (KS-96-CR-41).
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