Receptors as Supramolecular Entities -

Receptors as Supramolecular Entities (eBook)

Proceedings of the Biannual Capo Boi Conference, Cagliari, Italy, 7-10 June 1981
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2013 | 1. Auflage
492 Seiten
Elsevier Science (Verlag)
978-1-4831-5550-0 (ISBN)
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Receptors as Supramolecular Entities
Advances in the Biosciences, Volume 44: Receptors as Supramolecular Entities exemplifies the concept of transmitter and cotransmitter interactions using GABA receptor as a model. The book contains papers on the interaction of sulpiride and other substituted benzamides with dopamine receptors where the result of receptor stimulation is not a stimulation of cyclic AMP formation. The text also offers a panoramic view of the concepts that are being elaborated to reach a better understanding of the function of the receptors for the most important neurotransmitters operative in brain.

Inhibitory Coupling of Dopamine Receptors to Adenylate Cyclase in Rat Anterior Pituitary


P. Onali, J.P. Schwartz and E. Costa,     Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, USA

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This chapter discusses the inhibitory coupling of dopamine receptors to adenylate cyclase in rat anterior pituitary. Many hypothalamic factors that control the release of hormones from anterior pituitary cells act via a receptor mediated regulation of membrane bound adenylate cyclase. One of these factors, dopamine (DA), is released from the hypothalamus and acts directly on the pituitary to inhibit prolactin secretion and synthesis. The chapter presents a schematic representation of the adenylate cyclase system composed of a receptor for hormones and neurotransmitters, a coupling protein (G/F protein), and the catalytic subunit. It has been proposed that following the binding of the stimulatory hormone to its receptor, the hormone-receptor complex interacts with the G/F protein. This protein has a binding site for guanine nucleotides, which is occupied by guanosine diphosphate (GDP) under resting conditions. Interaction with the hormone-receptor complex results in a displacement of GDP from its binding site by guanosine-5’-triphosphate (GTP), thereby activating the coupling protein which can then stimulate the catalytic subunit of the enzyme.

Many hypothalamic factors, which control the release of hormones from anterior pituitary cells, act via a receptor mediated regulation of membrane bound adenylate cyclase (Labrie et al., 1975). Dopamine (DA), one of these factors, is released from the hypothalamus and acts directly on the pituitary to inhibit prolactin secretion and synthesis (Macleod, 1976: Maurer, 1980). However it is still controversial whether pituitary receptors for DA are coupled to adenylate cyclase, because previous studies have shown either no effect (Zor et al., 1969: Schmidt and Hill, 1977: Spano et al., 1978), stimulation (Ann et al., 1979) or inhibition (DeCamilli et al., 1979) of pituitary adenylate cyclase by DA. Our approach to this problem has been to investigate the effect of DA on the activation of adenylate cyclase of rat anterior pituitary by various factors which are known to interact specifically with the different molecular components of the cyclase system.

Figure 1 shows a schematic representation of the adenylate cyclase system composed of a receptor for hormones and neurotransmitters, a coupling protein (G/F protein) and the catalytic subunit. It has been proposed (Cassel and Selinger, 1977) that following the binding of the stimulatory hormone to its receptor, the hormone–receptor complex interacts with the G/F protein. This protein has a binding site for guanine nucleotides, which is occupied by GDP under resting conditions. Interaction with the hormone–receptor complex results in a displacement of GDP from its binding site by GTP, thereby activating the coupling protein which can then stimulate the catalytic sub–unit of the enzyme. The system is turned off by the hydrolysis of GTP to GDP which apparently is catalyzed by a GTPase associated with the coupling protein. This protein is thereby returned to the inactive state with a consequent decrease in the activity of the catalytic subunit. Although the sequence of events may be more complex than described here, according to this model the adenylate cyclase system can be activated in three different ways: via stimulation of the membrane receptor(s) by specific endogenous ligands; by direct action on the coupling protein; and by direct action on the catalytic subunit. We have tested the effect of DA following stimulation of the pituitary adenylate cyclase activity by each of these three mechanisms.

Fig. 1 Schematic representation of the adenylate cyclase system. H: hormone or neurotransmitter; R: surface receptor; G/F: coupling protein; AC: adenylate cyclase catalytic subunit

Effect of DA on the Stimulation of Adenylate Cyclase by Endogenous Ligands in Anterior Pituitary of Male Rats


As a model of adenylate cyclase activation by ligands of receptors coupled with the enzyme we chose the enzyme activity stimulated by vasoactive intestinal peptide (VIP). Previous studies have indicated that this peptide acts selectively on the mammotrophs of anterior pituitary. In vitro, VIP stimulates only the release of prolactin from the adenohypophysis (Rotsztejn et al., 1980: Samson et al., 1980) and is a potent activator of pituitary adenylate cyclase (Borghi et al., 1979). Figure 2 shows that VIP elicited a concentration–dependent increase of adenylate cyclase activity in homogenates of male rat anterior pituitary. When DA was added to the reaction mixture, the stimulation of the enzyme activity by VIP was reduced. This inhibition was dependent on the concentration of DA (Fig. 3) and reached a maximum at 10 μM when approximately 50% of the stimulated activity was inhibited. The apparent IC50 was 4 × 10−7M. DA failed to change the basal adenylate cyclase activity of the enzyme stimulation by prostaglandin E1(Onali et al., 1981), which does not stimulate prolactin release in vitro (Macleod and Lehmeyer, 1970: Drouin and Labrie, 1976).

Fig. 2 DA inhibition of the adenylate cyclase activity stimulated by VIP. VIP was tested at the reported concentrations alone () and in the presence of 10 μM DA (), Adenylate cyclase stimulation was calculated by subtracting the control values for each compound. Control values, expressed as pmoles of cAMP formed in 10 min/mg prot (means±S.E.M.) were: basal activity, 70±2.8; DA, 69±5.2. Anterior pituitaries from male rats (170–250 g) were homogenized manually in ice cold buffer (1:30 w/v) containing 10 mM HEPES(pH 7.4 at 4°C), 1 mM dithiothreitol (DTT), 1 mM EGTA and 0.32 M sucrose. The homogenate was then centrifuged at 400 xg for 5′. Unless otherwise specified, adenylate cyclase activity was assayed at 30°C for 10′ in a reaction mixture (150 μl) containing 53 mM HEPES/NaOH (pH 7.4 at 30°C), 0.3 mM EGTA, 1 mM DTT, 2 mM MgCl2, 1 mM cyclic AMP, 0.5 mM 1–methyl–3–iso–butylxanthine, 10 μM bacitracin, 50 μg of bovine serum albumin, 10 μM GTP, 0.5 mM [α−32P]ATP (25–50 cpm/pmol), 5 mM creatine phosphate (sodium salt), 100 U/ml creatine phosphokinase and 50 μl of 400 xg supernatant (150–200 g of protein). [32P]Cyclic AMP was isolated by sequential chromatography on Dowex 50W–X4 and alumina according to Salomon et al. (1976). Protein was assayed according to the method of Bohlen et al (1973).

Fig. 3 Dose–dependent inhibition of VIP–sensitive adenylate cyclase by dopamine. DA was tested alone and in the presence of 0.1 μM VIP. The VIP stimulation of the enzyme was determined by subtracting the respective control value. Inhibition is expressed as a percentage of the enzyme stimulation produced by VIP. Control values as pmoles of cAMP/mg prot/10 min (mean±S.E.M.) were: basal activity, 55±1.2; 0.1 μM VIP, ll8.0±2.3. The enzyme activity assayed in the presence of DA alone was not significantly different from the basal activity. (N=6).

The inhibitory effect of DA was antagonized by a number of DA receptor blockers (Onali et al., 1931). Figure 4 shows that (−) sulpiride, a DA antagonist with some selectivity for the D–2 DA receptors (Trabucchi et al., 1975: Kebabian and Calne, 1979: Theodorou et al., 1980), was able to counteract the DA inhibition in a concentration–dependent manner. The less active enantiomer, (+) sulpiride, was a poor antagonist. These results indicate that the inhibitory effect of DA was mediated through stimulation of specific DA receptors and support the view that these receptors are coupled in an inhibitory manner to the adenylate cyclase system (DeCamilli et al., 1979).

Fig. 4 Antagonism of DA inhibition of VIP–sensitive adenylate cyclase by (+) and (−) sulpiride. Both antagonists were tested at the indicated concentrations alone and in the presence of 0.1 μM VIP or VIP + 10−5M DA. The stimulation elicited by VIP was calculated by subtracting the respective control values; the effect of DA is reported as a percentage of the stimulation obtained by VIP alone.

Since the coupling of stimulatory receptors to this enzyme is regulated by guanine nucleotides (Rodbell, 1980), we investigated whether GTP is also required for the inhibitory effect of DA. Figure 5 shows that GTP stimulated the basal enzyme activity and also potentiated the stimulation of adenylate cyclase by VIP. In the absence of added GTP, DA did not inhibit the stimulation of the enzyme by VIP, but the inhibition because progressively larger as the concentration of the nucleotide was increased. (Fig. 5 is shown on next page).

Fig. 5 GTP–dependency of dopamine...

Erscheint lt. Verlag 22.10.2013
Sprache englisch
Themenwelt Sachbuch/Ratgeber Natur / Technik Naturführer
Medizin / Pharmazie Gesundheitsfachberufe
Medizin / Pharmazie Medizinische Fachgebiete Pharmakologie / Pharmakotherapie
Naturwissenschaften Biologie Zoologie
Naturwissenschaften Chemie
Technik
ISBN-10 1-4831-5550-1 / 1483155501
ISBN-13 978-1-4831-5550-0 / 9781483155500
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