cAMP is an important messenger for ADP-induced platelet aggregation

In rat platelets, basal cAMP levels were not changed upon stimulation with ADP and it was concluded that cAMP is not an important messenger for ADP-induced aggregation (Savi et al., Blood Coagul Fibrinolysis, 1996; 7: 249-52). In the present study, the effects of prostaglandin E(1) (PGE(1)) and ADP on human platelet aggregation, cAMP generation and VASP phosphorylation were studied. Phosphorylation of the protein kinase A (PKA) substrate VASP and inhibition of platelet aggregation by PGE(1) occurred without measurable changes in cellular cAMP levels. In addition, a marked inhibition of basal VASP phosphorylation by ADP was observed. It is concluded that cAMP determinations do not necessarily detect a possible activation or inhibition of the cAMPPKA pathway in platelets. Thus, cAMP might well be an important second messenger for ADP-induced platelet aggregation.     

cAMP is an important messenger for ADP-induced platelet aggregation

In rat platelets, basal cAMP levels were not changed upon stimulation with ADP and it was concluded that cAMP is not an important messenger for ADP-induced aggregation (Savi et al., Blood Coagul Fibrinolysis, 1996; 7: 249-52). In the present study, the effects of prostaglandin E1 (PGE1) and ADP on human platelet aggregation, cAMP generation and VASP phosphorylation were studied. Phosphorylation of the protein kinase A (PKA) substrate VASP and inhibition of platelet aggregation by PGE1 occurred without measurable changes in cellular cAMP levels. In addition, a marked inhibition of basal VASP phosphorylation by ADP was observed. It is concluded that cAMP determinations do not necessarily detect a possible activation or inhibition of the cAMPPKA pathway in platelets. Thus, cAMP might well be an important second messenger for ADP-induced platelet aggregation.     

The Toll pathway is important for an antiviral response in Drosophila

The innate immune response of Drosophila melanogaster is governed by a complex set of signaling pathways that trigger antimicrobial peptide (AMP) production, phagocytosis, melanization, and encapsulation. Although immune responses against both bacteria and fungi have been demonstrated in Drosophila, identification of an antiviral response has yet to be found. To investigate what responses Drosophila mounts against a viral infection, we have developed an in vivo Drosophila X virus (DXV)-based screening system that identifies altered sensitivity to viral infection by using DXV's anoxia-induced death pathology. Using this system to screen flies with mutations in genes with known or suggested immune activity, we identified the Toll pathway as a vital part of the Drosophila antiviral response. Inactivation of this pathway instigated a rapid onset of anoxia induced death in infected flies and increases in viral titers compared to those in WT flies. Although constitutive activation of the pathway resulted in similar rapid onset of anoxia sensitivity, it also resulted in decreased viral titer. Additionally, AMP genes were induced in response to viral infection similar to levels observed during Escherichia coli infection. However, enhanced expression of single AMPs did not alter resistance to viral infection or viral titer levels, suggesting that the main antiviral response is cellular rather than humoral. Our results show that the Toll pathway is required for efficient inhibition of DXV replication in Drosophila. Additionally, our results demonstrate the validity of using a genetic approach to identify genes and pathways used in viral innate immune responses in Drosophila.     

cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor.

Extracellular adenosine 3',5'-cyclic monophosphate (cAMP) is required for cell-type-specific gene expression in developing Dictyostelium discoideum. We have developed a microassay for the expression of these genes, using antibodies directed against their protein products. To characterize the transduction mechanism, we have used in this assay cAMP analogues that preferentially activate either the cell-surface cAMP receptor or the internal cAMP-dependent protein kinase. N6-(aminohexyl) cAMP activates the Dictyostelium cAMP-dependent protein kinase but does not bind to the cell-surface cAMP receptor and does not cause cell-type-specific gene expression. 2'-Deoxy-cAMP does not activate the cAMP-dependent protein kinase but binds to the receptor and causes cell-type-specific gene expression. Cyclic AMP-induced accumulation of prestalk mRNA in shaking cultures still occurs in the presence of caffeine, which blocks the receptor-coupled activation of adenyl cyclase. This suggests that the extracellular cAMP induction of cell-type-specific gene expression in developing Dictyostelium cells is mediated by the cell-surface cAMP receptor and that activating adenyl cyclase by this receptor is not essential. Using the N6-(aminohexyl) cAMP to competitively inhibit phosphodiesterase, we show that 30 nM cAMP is sufficient to induce prestalk or prespore gene expression.     

Extracellular loop C of NPC1L1 is important for binding to ezetimibe

Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.     

Duodenum is important for the sphincter of Oddi motor response to cholecystokinin octapeptide in conscious dogs

Background We investigated the role of the duodenum in the sphincter of Oddi response to cholecystokinin-octapeptide (CCK-OP), using conscious dogs.Methods In controls, a cannula was inserted into the duodenum opposite the papilla for retrograde manometry. In the duodenectomy group, the entire duodenum was resected, while preserving the papilla, which was implanted into the jejunum, and the cannula was placed. Sphincter motility was recorded after bolus injections of 20 and 100ng/kg of CCK-OP.Results CCK-OP at 20ng/kg produced sphincter relaxation followed by contraction in the controls, but produced no changes after duodenectomy. CCK-OP at 100ng/kg caused strong contractions followed by relaxation in the controls, but caused only contractions after duodenectomy.Conclusions (1) Relaxation and delayed contraction of the sphincter induced by 20ng/kg of CCK-OP require the presence of the duodenum; (2) early contractions of the sphincter induced by 100ng/kg of CCK-OP do not require the duodenum; (3) the duodenum plays an important role in the actions of CCK-OP on sphincter motility.     

Steroidogenic factor 1 (SF-1) is essential for ovarian development and function

The orphan nuclear receptor steroidogenic factor 1 (SF-1) was identified originally as a key regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases. Hints at considerably broader roles for SF-1 came from analyses of its expression pattern in mouse embryos. As anticipated, SF-1 was expressed in the adrenal glands and gonads from their early stages of development. Surprisingly, SF-1 also was expressed outside of the primary steroidogenic tissues in the anterior pituitary and hypothalamus. SF-1 knockout mice dramatically confirmed its multiple essential roles in vivo. These mice lacked adrenal glands and gonads, leading to adrenocortical insufficiency and male-to-female sex reversal of their internal and external genitalia. SF-1 knockout mice also had impaired pituitary expression of gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), confirming roles of SF-1 at all three levels of the hypothalamic-pituitary-gonadal axis. With some focus on the ovary, this review summarizes experiments that have defined essential roles of SF-1 in endocrine development, and highlights important areas for future studies.     

Evidence that interleukin-1 induction of synovial cell plasminogen activator is mediated via prostaglandin E2 and cAMP

Addition of the cyclooxygenase inhibitor indomethacin to human synovial cells in culture, at concentrations which completely block prostaglandin E2 (PGE2) synthesis, reversibly inhibited the interleukin-1 (IL-1) stimulation of cell-associated and extracellular plasminogen activator (PA) production. Results of mixing experiments suggested that the inhibition by indomethacin was not due to stimulation of production and/or activation of a PA inhibitor, but reflected inhibition of PA synthesis. Simultaneous addition of PGE2 or dibutyryl cAMP prevented the inhibition by indomethacin. Addition of the phosphodiesterase inhibitor, theophylline, the adenylate cyclase stimulator, forskolin, or dibutyryl cAMP caused an enhancement of the IL-1 induction of synovial cell PA. These results suggest that the IL-1 induction of synovial cell PA occurs via generation of endogenous PGE2 and cAMP.