Sphingosine, W-7, and Trifluoperazine Inhibit the Elevation in Cytosolic Calcium Induced by High K+Depolarization in Synaptosomes

Abstract: A possible role for protein kinases in the regulation of free cytosolic Ca²⁺ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca²⁺ (monitored with the Ca²⁺-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K⁺. The ability of various drugs to inhibit the cytosolic Ca²⁺ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca²⁺ induced by high K⁺ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 m/W ), staurosporine (1μ M ), and K252_a(1μ M ), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca²⁺ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca²⁺ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca²⁺ increases after depolarization.     

Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Sphingosine and FTY720 modulate pacemaking activity in interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca²⁺ activated Cl⁻ channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720- P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.     

Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note

A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.     

Cloning of rat ABCA7 and its preferential expression in platelets

We cloned the full-length cDNA of a rat orthologue of ABCA7 (rABCA7) from rat platelets. The cDNA of rABCA7 is 6510bp in length and encodes a protein of 2170 amino acids. The amino acid sequence of rABCA7 exhibits homology to those of mouse ABCA7 (92.5% identical in amino acid sequence) and human ABCA7 (76.6%). We obtained two clones of monoclonal antibodies against rABCA7 recognizing different epitopes. Analysis of CHO cells stably expressing rABCA7 by confocal laser-scanning microscopy indicated that rABCA7 is mainly located in the plasma membrane. Western blot analysis of rat tissues revealed that rABCA7 was preferentially expressed in platelets and that its apparent molecular mass was 250kDa. This is the first report of the tissue distribution of rABCA7 at the protein level and is the first reported case of ABC transporters being expressed in platelets, suggesting their important role in platelet function.