Targeting sphingosine-1-phosphate receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-phosphate (FTY720P) are thought to be mediated via S1P1 receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a "functional antagonist" by promoting efficient internalization of S1P1 receptors. We demonstrate here that S1P1 receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

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Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

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Nitric oxide (NO), the physiological activator of soluble guanylyl cyclase (sGC), induces inhibitory effects on platelet activation via elevation of cGMP levels and stimulation of the cGMP-dependent protein kinase. YC-1, a benzylindazole derivative, was shown to activate sGC in intact platelets, resulting in inhibition of platelet aggregation. In a previous study, we demonstrated that YC-1 not only stimulates purified sGC but also potentiates the stimulatory action of submaximally effective NO and carbon monoxide (CO) concentrations. Here, we investigated the potentiating effect of YC-1 in intact platelets. YC-1 together with NO or CO led to complete inhibition of platelet aggregation at concentrations that were ineffective by themselves. Maximally effective 2, 2-diethyl-1-nitroso-oxyhydrazine (3 microM) and YC-1 (100 microM) concentrations each elevated the cGMP levels in intact platelets approximately 13-fold, and administration of the two drugs together resulted in enormous potentiation of cGMP formation, which greatly exceeded the effect on the purified enzyme and yielded a >1300-fold increase in cGMP levels. Similar results were obtained using CO instead of NO. Furthermore, YC-1 not only stimulated sGC but also inhibited cGMP-hydrolyzing phosphodiesterases in platelets. The enormous elevation of cGMP levels led to enhanced phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein. Thus, by the combination of two effects (i.e., potentiation of NO-induced sGC stimulation and phosphodiesterase inhibition), YC-1-like substances are potent activators of the sGC/cGMP pathways and are therefore interesting candidates to act as modulators of cGMP-mediated effects, especially within the cardiovascular system.

No abstract

Many of the physiological effects of the signaling molecule nitric oxide are mediated by the stimulation of the NO-sensitive guanylyl cyclase. Activation of the enzyme is achieved by binding of NO to the prosthetic heme group of the enzyme and the initiation of conformational changes. So far, the rate of NO dissociation of the purified enzyme has only been determined spectrophotometrically, whereas the respective deactivation, i.e. the decline in enzymatic activity, has only been determined in cytosolic fractions and intact cells. Here, we report on the deactivation of purified NO-sensitive guanylyl cyclase determined after addition of the NO scavenger oxyhemoglobin or dilution. The deactivation rate corresponded to a half-life of the NO/guanylyl cyclase complex of ϳ4 s, which is in good agreement with the spectrophotometrically measured NO dissociation rate of the enzyme. The deactivation rate of the enzyme determined in platelets yielded a much shorter half-life indicating either partial damage of the enzyme during the purification procedure or the existence of endogenous deactivation accelerating factors. YC-1, a component causing sensitization of guanylyl cyclase toward NO, inhibited deactivation of guanylyl cyclase, resulting in an extremely prolonged half-life of the NO/guanylyl cyclase complex of more than 10 min. The deactivation of an ATP-utilizing guanylyl cyclase mutant was almost unaffected by YC-1, indicating the existence of a special structure within the catalytic domain required for YC-1 binding or for the transduction of the YC-1 effect. In contrast to the wild type enzyme, YC-1 did not increase NO sensitivity of this mutant, clearly establishing inhibition of deactivation as the underlying mechanism of the NO sensitizer YC-1.The signaling molecule nitric oxide (NO) has been shown to play an important role in the cardiovascular and nervous systems (1-4). Most of the effects of NO are mediated by the stimulation of the NO-sensitive guanylyl cyclase (GC), 1 the enzyme that catalyzes the formation of cGMP. The subsequent increase in cGMP leads to the activation of cGMP effector proteins like cGMP-activated protein kinases, cGMP-gated Ca 2ϩ channels and cGMP-regulated phosphodiesterases. NO-sensitive GC is a heterodimeric enzyme consisting of an ␣ and ␤ subunit (5). Although four enzyme subunits (␣ 1 , ␣ 2 , ␤ 1 , ␤ 2 ) have been identified so far, only the ␣ 1 ␤ 1 and ␣ 2 ␤ 1 heterodimers have been shown to exist as catalytically active NO sensitive isoforms in vivo (6). Each of the subunits contains the homologous cyclase catalytic domain. With two different cyclase catalytic domains the GTP-converting catalytic region of GC appears to be related to the ATP-converting catalytic region of the adenylyl cyclase (AC). The crystal structure of the catalytic center of AC has been resolved (7,8). In AC, the homologous cyclase catalytic domains C1 and C2 corresponding to the ␣ and ␤ subunits of NO-sensitive GC, respectively, are arranged in a "head-to-tail"-like orientation with two pseudosymmetric region...