Inhibition of cyclic GMP‐dependent protein kinase‐mediated effects by (Rp)‐8‐bromo‐PET‐cyclic GMPS

Abstract

1 The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)-and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromoflphenyl-1,N2-ethenoguanosine 3',5'-cyclic monophosphorothioate, ((Rp)-and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 6 The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 gM). The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/ PKG-dependent mechanism. 7 The [3H]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cyclic GMPS. Moreover, the forskolin concentrationrelaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG-over PKA-mediated effects.8 The results obtained indicate that (Rp)-8-bromo-PET-cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

“…8). These results are consistent with a previous study showing that Rp-PET can inhibit cGMPactivated cGKI in intact cells under certain conditions (24). Considering the 46-fold higher lipophilicity (14) and the 4-fold higher concentration of Rp-PET versus 8-Br-cGMP used in our experiment, it appears that the inhibitory potential of Rp-PET in intact cells is quite moderate and that it must be present in large excess to inhibit the effect of a strong agonist such as 8-Br-cGMP.…”

“…3B, lower panel), respectively. These K i values were similar to K i values reported previously for the inhibition of the cGKI isozymes by Rp-PET in vitro (24). Interestingly, inhibition of cGMP-activated cGKI␣ by Rp-PET was less complete than inhibition of cGKI␤ (Fig.…”

“…For instance, the cGMP analogue 8-Br-PET-cGMP (PET) is a cGKI agonist (23), whereas Rp-8-Br-PET-cGMPS (Rp-PET) (see Fig. 1B) competitively inhibits activation of purified cGKI by cGMP (24). Rp-PET is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies (14,19).…”

Endogenous Type II cGMP-dependent Protein Kinase Exists as a Dimer in Membranes and Can Be Functionally Distinguished from the Type I Isoforms

“…Finally our observation that R p -cGMPS analogs, with some degree of specificity, can inhibit cGK II-controlled functions in intact tissues, as well as cGK I action described earlier (36,37), indicates that these compounds are more generally applicable as tools to demonstrate the involvement of cGK I or II in specific physiological processes. For example, in a separate study of the mechanism of action of heat-stable enterotoxin (STa) in rat intestinal epithelium, R p -8-pCPT-cGMPS appeared also able to inhibit the STa-induced Cl Ϫ secretion (15).…”

Pulmonary PKG-1 is upregulated following chronic hypoxia

“…This is consistent with the current study in that we effectively inhibited pulmonary vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats with mechanistically distinct inhibitors of PKG-1. Rp-8-Br-PET-cGMPS is the most potent and selective PKG inhibitor amongst the (Rp)-phosphorothioate cGMP analogs and antagonizes signaling by binding to PKG 1␣ or -␤ and prohibiting the conformational change of the enzyme required for its activation (5). In contrast, KT-5823, one of several K-252b derivatives that mediate inhibition by competing with ATP, is highly selective for PKG.…”