Structure-Guided Design of Selective Epac1 and Epac2 Agonists

The second messenger cAMP is known to augment glucose-induced insulin secretion. However, its downstream targets in pancreatic β-cells have not been unequivocally determined. Therefore, we designed cAMP analogues by a structure-guided approach that act as Epac2-selective agonists both in vitro and in vivo. These analogues activate Epac2 about two orders of magnitude more potently than cAMP. The high potency arises from increased affinity as well as increased maximal activation. Crystallographic studies demonstrate that this is due to unique interactions. At least one of the Epac2-specific agonists, Sp-8-BnT-cAMPS (S-220), enhances glucose-induced insulin secretion in human pancreatic cells. Selective targeting of Epac2 is thus proven possible and may be an option in diabetes treatment.     

A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK

cAMP is involved in a wide variety of cellular processes that were thought to be mediated by protein kinase A (PKA). However, cAMP also directly regulates Epac1 and Epac2, guanine nucleotide-exchange factors (GEFs) for the small GTPases Rap1 and Rap2 (refs 2,3). Unfortunately, there is an absence of tools to discriminate between PKA- and Epac-mediated effects. Therefore, through rational drug design we have developed a novel cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP), which activates Epac, but not PKA, both in vitro and in vivo. Using this analogue, we tested the widespread model that Rap1 mediates cAMP-induced regulation of the extracellular signal-regulated kinase (ERK). However, both in cell lines in which cAMP inhibits growth-factor-induced ERK activation and in which cAMP activates ERK, 8CPT-2Me-cAMP did not affect ERK activity. Moreover, in cell lines in which cAMP activates ERK, inhibition of PKA and Ras, but not Rap1, abolished cAMP-mediated ERK activation. We conclude that cAMP-induced regulation of ERK and activation of Rap1 are independent processes.     

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b(0) + b(1) (1/x) with the regression coefficients b(0) and b(1,) the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1-2 months, when they reach their absolute maximum for all age periods, and then decrease.     

Effect of cGMP analogues and protein kinase G blocker on secretory activity, apoptosis and the cAMP/protein kinase A system in porcine ovarian granulosa cells in vitro

The aim of the present study was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian functions. In the first series of experiments we studied the effects of the cGMP analogues 8-pCPT-cGMP (0.001-100 nM), Rp-8-pCPT-cGMPS (0. 01-100 nM), Rp-8-Br-cGMPS (0.01-100 nM), and Rp-8-Br-PET-cGMPS (0.01-100 nM) on the release of progesterone, insulin-like growth factor I (IGF-I) and oxytocin by cultured porcine granulosa cells. In a second series of experiments, the effects of Rp-8-Br-PET-cGMPS (50 nM) and KT5822 (100 ng/ml), specific inhibitor of cGMP-dependent protein kinase (PKG), on cAMP, PKA, oxytocin and the occurrence of apoptosis in cultured cells were compared. The release of hormones and IGF-I into the culture medium was evaluated using a RIA, while the percentage of cells containing visible oxytocin, cAMP, as well as the regulatory and catalytic subunits of PKA was assessed using immunocytochemistry. Occurrence of apoptosis in these cells was detected using the TUNEL method. The stimulatory (8-pCPT-cGMP and Rp-8-pCPT-cGMPS), inhibitory (Rp-8-Br-cGMPS) and biphasic (Rp-8-Br-PET-cGMPS) effect of cGMP analogues on progesterone release was observed. All cGMP analogues used suppressed IGF-I release. All cGMP analogues decreased oxytocin release, but 8-pCPT-cGMP and Rp-8-Br-cGMPS, when given at low doses (0.01-0.1 and 1-10 nM, respectively) stimulated oxytocin output. Both, Rp-8-Br-PET-cGMPS and KT5822 increased the rate of incidence of apoptosis and percentage of cells containing immunoreactive cAMP. Both Rp-8-Br-PET-cGMPS and KT5822 decreased the proportion of cells containing immunoreactive oxytocin and regulatory subunit of PAK KT5822, but not Rp-8-Br-PET-cGMPS, increased the number of cells containing catalytic subunit of PKA. The present observations suggest the involvement of cGMP and PKG in control of the production of steroid, nonapeptide hormone, growth factor, cAMP and cAMP-dependent PKA, as well as the induction of apoptosis in porcine ovarian cells.     

αA- and αB-crystallins interact with caspase-3 and Bax to guard mouse lens development

The small heat shock protein, α-crystallin, exists in two isoforms, αA and αB, and displays strong ability against stress-induced apoptosis. Regarding their functional mechanisms, we and others have demonstrated that they are able to regulate members in both caspase and Bcl-2 families. In addition, we have also shown that αA and αB may display differential anti-apoptotic mechanisms under certain stress conditions. While αA-crystallin regulates activation of the AKT signaling pathway, αB negatively regulates the MAPK pathway to suppress apoptosis induced by UV and oxidative stress. Although previous studies revealed that αA and αB could regulate members in both caspase and Bcl-2 families, the molecular mechanism, especially the in vivo regulation still waits to be elucidated. In the present communication, we present both in vitro and in vivo evidence to further demonstrate the regulation of caspase-3 and Bax by αA and αB. First, Surface Plasmon Resonance (SPR) and yeast two-hybrid selection analysis demonstrate that αA and αB directly bind to caspase-3 and Bax with differential affinities. Second, immunohistochemistry reveals that αA and αB regulate caspase-3 and Bax at different developmental stages of mouse embryo. Third, coimmunoprecipitation shows that αA and αB form in vivo interacting complexes with caspase-3 and Bax. Together, our results further confirm that αA and αB regulate caspase-3 and Bax in vitro and in vivo to regulate lens differentiation.     

Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases

The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.