Determination of adenosine A2A- and dopamine D1 receptor-specific modulation of adenylate cyclase activity in rat striatum

Abstract

In the frame of current dissertation was elaborated a methodology for measurement of adenosine A2A- and dopamine D1 receptor-specific modulation of adenylate cyclase (AC) activity in rat striatum homogenate, using the determination of cyclic adenosine-3’, 5’-monophosphate concentration change. Current methodology has been used for studying relationships between rats’ behavioral particularities and D1 receptor signal transduction sensitivity in striatum and nucleus accumbens of rats. With the optimization of sample tissue preparation and reaction medium components, experimental conditions were found where the ligands of adenosine A2A and dopamine D1 receptors were able to modulate AC activity, but not the ligands of dopamine D2 receptor. The presence of D2 receptors in current tissue preparation was confirmed by [3H]raclopride binding and by inhibition of [3H]raclopride binding by other D2 receptor’s ligands. From this can be concluded that either D2 receptors don’t activate enough G proteins that inhibit AC activity or these G proteins are not functional in current system. However, ligands of A2A and D1 receptors modulated the activity of AC and also inhibited the binding of receptor-specific radioligands to these receptors in stratal sample, and furthermore, these ligands modulated both processes with similar affinity. D1 receptor-specific signal transduction was measured in striatum and nucleus accumbens of rats with high- (HE) and low (LE) exploratory activity and additionally was investigated the influence of stress on the signal transduction level of given receptor in mentioned brain areas. It was found that the HE group rats had higher level of D1 receptor-specific signal transduction in striatum than LE group rats, but in the same time no statistically significant difference was found in nucleus accumbens. Stress had statistically significant effect on D1 receptor signal transduction level in nucleus accumbens, but not in striatum. Also was investigated the relationship between the response of rats to tickling and the D1 receptor signal transduction sensitivity in nucleus accumbens. The sensitivity of D1 receptor signal transduction was not statistically different in nucleus accumbens of high- and low chirping group rats (in case of both sexes), but it was found that tickling enhances D1 receptor signal transduction level and in case of male rats the effect was larger. Käesoleva dissertatsiooni raames töötati välja metoodika, mille abil mõõta adenosiini A2A- ja dopamiini D1 retseptori poolt vahendatavat adenülaadi tsüklaasi (AC) aktiivsuse modulatsiooni roti aju juttkeha homogenaadis, kasutades selleks tsüklilise adenosiin-3’, 5’-monofosfaadi kontsentratsiooni muudu määramist. Juurutatud metoodika abil uuriti rottide erinevate käitumuslike iseärasuste seoseid D1 retseptori signaaliülekanderadade tundlikkusega nende aju juttkehas ja naalduvas tuumas. Koeproovi valmistamise viisi ja reaktsioonikeskkonna komponentide optimeerimise tulemusena leiti eksperimentaalsed tingimused, kus nii adenosiini A2A ja dopamiini D1 retseptorite ligandid olid võimelised moduleerima AC aktiivsust, kuid mitte dopamiini D2 retseptori ligandid. D2 retseptorite olemasolu antud koepreparaadis leidis kinnitust [3H]raklopriidi sidumisega ning uuritud D2 retseptori ligandide võimega efektiivselt [3H]raklopriidi sidumist inhibeerida. Sellest võib järeldada, et käesoleval juhul D2 retseptorid kas ei aktiveeri piisaval hulgal AC inhibeerivaid G valke või need G valgud ei ole antud tingimustel funktsionaalsed. A2A ja D1 retseptorite ligandid aga olid võimelised moduleerima nii AC aktiivsust kui ka inhibeerima retseptor-spetsiifiliste radioligandide sidumist antud retseptoritele juttkeha preparaadis, kusjuures nende retseptorite ligandid mõjutavad mõlemaid protsesse sarnase afiinsusega. D1 retseptori-spetsiifilist signaaliülekannet mõõdeti kõrge(HE) ja madala(LE) uudistamisaktiivsusega rottide aju juttkehas ja naalduvas tuumas ning samuti uuriti stressi mõju antud retseptori signaaliülekandele nimetatud ajuosades. Tulemusena leiti, et HE rottidel on D1 retseptori signaaliülekande efektiivsus juttkehas statistiliselt olulisel määral suurem võrreldes LE rottidega, samas aga naalduvas tuumas statistiliselt olulist erinevust ei leitud. Stress jällegi mõjutas oluliselt D1 signaaliülekannet just naalduvas tuumas, kuid mitte juttkehas. Lisaks uuriti rottide kõditamisele reageerimise seost D1 signaali ülekandega naalduvas tuumas. Tulemusena leiti, et D1 retseptori signaaliülekande tase naalduvas tuumas ei ole oluliselt erinev vähe- ja paljupiiksuvatel rottidel (nii isas- kui ka emasloomade puhul), küll aga leiti, et kõditamine suurendab D1 retseptori signaaliülekande tundlikkust ning isasloomade puhul on see mõju suurem.