Stable GDP analog-induced inactivation of Gi proteins promotes cardiac adenylyl cyclase inhibition by guanosine 5′-(βγ-imino) triphosphate and muscarinic acetylcholine receptor

Abstract

Low concentrations of GDP and its stable analog guanosine 5'-O-(2-thio)diphosphate (GDP beta S) have been shown to stimulate adenylyl cyclase activity in canine cardiac sarcolemmal membranes independent from a phosphate transfer reaction. The mechanism of this stimulation was further examined. The stable GTP analog guanosine 5'-(beta gamma-imino)triphosphate (Gpp(NH)p) increased basal adenylyl cyclase activity and inhibited forskolin-stimulated activity with EC50 (half-maximal effective concentration) values of 0.7 mumol/l and 10 nmol/l, respectively. In the presence of GDP beta S (5 mumol/l), which increased basal activity by about 150%, addition of Gpp(NH)p inhibited adenylyl cyclase activity by up to 50% with an EC50 value of 40 nmol/l. Activation of cardiac muscarinic acetylcholine receptors by carbachol amplified this Gpp(NH)p-induced inhibition of GDP beta S-stimulated adenylyl cyclase activity. The stimulatory effect of GDP beta S and the inhibitory effect of Gpp(NH)p on GDP beta S-stimulated adenylyl cyclase activity were both attenuated by increasing the Mg2+ concentration or substituting Mn2+ for Mg2+ in the assay. Furthermore, both effects were strongly reduced or abolished upon pretreatment of the sarcolemmal membranes with a low concentration of the SH reagent N-ethylmaleimide (10 mumol/l). These results suggest that the stimulatory effect of GDP beta S on basal adenylyl cyclase activity in canine cardiac sarcolemmal membranes is caused by inactivation of G(i) proteins, which are then rendered susceptible to activation by Gpp(NH)p and inhibitory receptors.

Guanine Nucleotide Exchange-Independent Activation of GsProtein by β2-Adrenoceptor

“…Other explanations such as GDP-induced disinhibition of G i protein (Piacentini et al, 1996;Lutz et al, 2002) or conversion of GDP to GTP do not explain the present results as a whole. However, there are still two compelling arguments that suggest that the guanine nucleotide exchange is a requirement for G␣ activation and that GTP is the activating nucleotide.…”

“…Early observations that G s or G i could be activated in the presence of GDP (Iyengar and Birnbaumer, 1979;Schneyer et al, 1984;Harding and Harris, 1986;Quist et al, 1992;Piacentini et al, 1996;Lutz et al, 2002) have mostly been attributed to the conversion of GDP to GTP during the experiments (Kimura and Schimada, 1983;Kikkawa et al, 1990;Kowluru et al, 1996). Such a conversion must be nearly complete and almost instantaneous to be an explanation for the present results, because GTP and GDP supported the cyclase activation with almost identical potencies in the presence or absence of agonist.…”

“…A number of experiments in the literature show that G s (or G i ) protein can indeed be activated in the presence of GDP (Iyengar and Birnbaumer, 1979;Schneyer et al, 1984;Harding and Harris, 1986;Quist et al, 1992;Piacentini et al, 1996;Seifert et al, 1998, Hatley et al, 2003Lutz et al, 2002). These reports include observations dating from late 1970s to the present day.…”

Spontaneous release of GDP from G i proteins and inhibition of adenylyl cyclase in cardiac sarcolemmal membranes

“…Previous studies on various membrane systems, including cardiac membranes, have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphorylation-resistant analog, GDPβS (Harding and Harris 1986;Niroomand et al 1993;Piacentini et al 1996;Quist et al 1992;Schneyer et al 1984). The mechanism of this stimulatory action has not been resolved yet.…”

Can a GDP-Liganded G-Protein Be Active?: Fig. 1.

“…For example, Ug ur et al (2005) report that GDP or GDP␤S enhanced agonist-stimulated adenylyl cyclase activity. A stimulatory effect of GDP and GDP␤S on adenylyl cyclase activity has been reported before, but this was interpreted as an inactivation of nucleotide free G␣ i from an active conformation that inhibits adenylyl cyclase (Piacentini et al, 1996;Lutz et al, 2002). Although the findings of Ug ur et al (2005) cannot be fully explained by a GDP-induced disinhibition of G i , the data do not unequivocally rule out that possibility.…”