The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.     

Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain.

Evidence is accumulating which supports a role for ATP in the initiation of pain by acting on P2X receptors expressed on nociceptive afferent nerve terminals. To investigate whether these receptors play a role in temporomandibular (TMJ) pain, we studied the presence of functional P2X receptors in rat TMJ by examining the nociceptive behavioral response to the application of the selective P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-meATP) into the TMJ region of rat. The involvement of endogenous ATP in the development of TMJ inflammatory hyperalgesia was also determined by evaluating the effect of the general P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on carrageenan-induced TMJ inflammatory hyperalgesia. Application of alpha,beta-meATP into the TMJ region of rats produced significant nociceptive responses that were significantly reduced by the co-application of lidocaine N-ethyl bromide quaternary salt, QX-314, (2%) or of the P2 receptor antagonist PPADS. Co-application of PPADS with carrageenan into the TMJ significantly reduced inflammatory hyperalgesia. The results indicate that functional P2X receptors are present in the TMJ and suggest that endogenous ATP may play a role in TMJ inflammatory pain mechanisms possibly by acting primarily in these receptors.     

The suppressive effect of sphingosine 1-phosphate on monocyte-endothelium adhesion may be mediated by the rearrangement of the endothelial integrins α5β1 and αvβ3

BACKGROUND: Sphingosine 1-phosphate (S1P), known to play important roles in vascular biology, is a bioactive lysophospholipid mediator that maintains endothelial integrity via its cell-surface receptors (S1Ps). In this in vitro study, we aimed to examine the role of S1P in monocyte-endothelium adhesion, which is an important event in the pathophysiology of atherosclerosis. METHODS AND RESULTS: S1P pretreatment of human umbilical vein endothelial cells (ECs), but not U937 cells, effectively suppressed U937-EC adhesion independently from the expression of adhesion molecules, namely ICAM-1, VCAM-1, and E-selectin. This S1P-induced suppressive effect was inhibited by the blockage of S1P(1) and S1P(3) receptors and the specific inhibitors of G(i) protein, Src family proteins, phosphatidylinositol 3-kinase, and Rac1, indicating involvement of these key downstream pathways. Moreover, the RGD peptide and antibodies, which neutralize adhesion via alpha(5)beta(1) and alpha(v)beta(3), effectively inhibited U937-EC adhesion with a degree similar to S1P pretreatment. Both an adhesion assay and flow-cytometric analysis demonstrated that U937 cells adhered through integrins alpha(5)beta(1) and alpha(v)beta(3) expressed on the apical surface of monolayer ECs, and S1P shifted the localization of these integrins from the apical surface to the basal surface. CONCLUSIONS: From the present results, we propose that S1P may contribute to the maintenance of vascular integrity and the regulation of atherogenesis through the rearrangement of endothelial integrins.     

Spinal cord protein interacting with C kinase 1 is required for the maintenance of complete Freund’s adjuvant-induced inflammatory pain but not for incision-induced post-operative pain

Protein interacting with C kinase 1 (PICK1) is a PDZ-containing protein that binds to AMPA receptor (AMPAR) GluR2 subunit and protein kinase Cα (PKCα) in the central neurons. It functions as a targeting and transport protein, presents the activated form of PKCα to synaptic GluR2, and participates in synaptic AMPAR trafficking in the nervous system. Thus, PICK1 might be involved in many physiological and pathological processes triggered via the activation of AMPARs. We report herein that PICK1 knockout mice display impaired mechanical and thermal pain hypersensitivities during complete Freund’s adjuvant (CFA)-induced inflammatory pain maintenance. Acute transient knockdown of spinal cord PICK1 through intrathecal injection of PICK1 antisense oligodeoxynucleotide had a similar effect. In contrast, knockout and knockdown of spinal cord PICK1 did not affect incision-induced guarding pain behaviors or mechanical or thermal pain hypersensitivities. We also found that PICK1 is highly expressed in dorsal horn, where it interacts with GluR2 and PKCα. Injection of CFA into a hind paw, but not a hind paw incision, increased PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn. These increases were absent when spinal cord PICK1 was deficient. Given that dorsal horn PKCα-mediated GluR2 phosphorylation at Ser880 and GluR2 internalization contribute to the maintenance of CFA-induced inflammatory pain, our findings suggest that spinal PICK1 may participate in the maintenance of persistent inflammatory pain, but not in incision-induced post-operative pain, through promoting PKCα-mediated GluR2 phosphorylation and internalization in dorsal horn neurons.     

Upregulation of P2X3 receptors by neuronal calcium sensor protein VILIP-1 in dorsal root ganglions contributes to the bone cancer pain in rats

Primary and metastatic cancers that affect bone are frequently associated with severe and intractable pain. The mechanisms underlying the development of bone cancer pain are largely unknown. In this study, we first demonstrated that a functional upregulation of P2X3 receptors in dorsal root ganglion (DRG) neurons is closely associated with the neuronal hyperexcitability and the cancer-induced bone pain in MRMT-1 tumor cell–inoculated rats. Second, we revealed that visinin-like protein 1 (VILIP-1), a member of visinin-like proteins that belong to the family of neuronal calcium sensor proteins is responsible for the observed upregulation of P2X3 receptors in DRG neurons. The interaction between the amino terminus of VLIP-1 and the carboxyl terminus of the P2X3 receptor is critical for the surface expression and functional enhancement of the receptor. Finally, overexpression of VILIP-1 increases the expression of functional P2X3 receptors and enhances the neuronal excitability in naive rat DRG neurons. In contrast, knockdown of VILIP-1 inhibits the development of bone cancer pain via downregulation of P2X3 receptors and repression of DRG excitability in MRMT-1 rats. Taken together, these results suggest that functional upregulation of P2X3 receptors by VILIP-1 in DRG neurons contributes to the development of cancer-induced bone pain in MRMT-1 rats. Hence, P2X3 receptors and VILIP-1 could serve as potential targets for therapeutic interventions in cancer patients for pain management. Pharmacological blockade of P2X3 receptors or knockdown of VILIP-1 in DRGs would be used as innovative strategies for the treatment of bone cancer pain.     

Chemokines and chemokine receptors during activation and deactivation of monocytes and dendritic cells and in amplification of Th1 versus Th2 responses

Chemokines are a superfamily of small cytokines which are generally chemotactic for leukocytes. The general features of chemokines and their receptors are reviewed. Recent evidence indicates that receptor expression dictates the spectrum of action of chemokines, as shown recently for Th1 and Th2 cells. Chemokines represent amplification loops of polarized Th1 and Th2 responses. Receptor expression is tightly regulated during differentiation, activation, and deactivation of mononuclear phagocytes and dendritic cells. Thus, regulation of receptor expression is crucial as a set point of the chemokine system.     

Pharmacological control of the human gastric histamine H2 receptor by famotidine:comparison with H1, H2 and H3 receptor agonists and antagonists

Abstract. Histamine 0–1 µM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 µM. The histamine dose-response curve was mimicked by the H₃ receptor agonist (R) α-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H₂, H₁ and H₃ receptor antagonists, according to the following order of potency IC₅₀:famotidine (0.3 µM) > triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (β₂-type), PGE₂ and VIP. The Schild plot was linear for famotidine (P < 0.01), with a regression coefficient r= 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H₂ receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H₂ receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) α-MeHA and thioperamide are two selective ligands at histamine H₃ receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H₃ receptors in the regulation of gastric secretion is proposed.     

Role of CB1 and CB2 cannabinoid receptors in the development of joint pain induced by monosodium iodoacetate

Joint pain is a common clinical problem for which both inflammatory and degenerative joint diseases are major causes. The purpose of this study was to investigate the role of CB1 and CB2 cannabinoid receptors in the behavioral, histological, and neurochemical alterations associated with joint pain. The murine model of monosodium iodoacetate (MIA) was used to induce joint pain in knockout mice for CB1 (CB1KO) and CB2 cannabinoid receptors (CB2KO) and transgenic mice overexpressing CB2 receptors (CB2xP). In addition, we evaluated the changes induced by MIA in gene expression of CB1 and CB2 cannabinoid receptors and μ-, δ- and κ-opioid receptors in the lumbar spinal cord of these mice. Wild-type mice, as well as CB1KO, CB2KO, and CB2xP mice, developed mechanical allodynia in the ipsilateral paw after MIA intra-articular injection. CB1KO and CB2KO demonstrated similar levels of mechanical allodynia of that observed in wild-type mice in the ipsilateral paw, whereas allodynia was significantly attenuated in CB2xP. Interestingly, CB2KO displayed a contralateral mirror image of pain developing mechanical allodynia also in the contralateral paw. All mouse lines developed similar histological changes after MIA intra-articular injection. Nevertheless, MIA intra-articular injection produced specific changes in the expression of cannabinoid and opioid receptor genes in lumbar spinal cord sections that were further modulated by the genetic alteration of the cannabinoid receptor system. These results revealed that CB2 receptor plays a predominant role in the control of joint pain manifestations and is involved in the adaptive changes induced in the opioid system under this pain state.     

Mineralization of three‐dimensional osteoblast cultures is enhanced by the interaction of 1α,25‐dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors

1α,25‐Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein‐2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three‐dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild‐type (WT), Pdia3‐silenced (Sh‐Pdia3) and VDR‐silenced (Sh‐VDR) pre‐osteoblastic MC3T3‐E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε‐caprolactone)–collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro‐computed tomography (μCT) showed that the mineralized volume of untreated Sh‐Pdia3 and Sh‐VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh‐VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacological control of the human gastric histamine H2 receptor by famotidine: comparison with H1, H2 and H3 receptor agonists and antagonists

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.     

Purinergic receptors: new targets for the treatment of gout and fibrosis

Adenosine triphosphate is involved in many metabolic reactions, but it has also a role as a cellular danger signal transmitted through purinergic receptors (PRs). Indeed, adenosine 5′‐triphosphate (ATP) can bind to PRs which are found in the membrane of many cell types, although the relative proportions of the receptor subtypes differ. PRs are classified according to genetic and pharmacological criteria and especially their affinities for agonists and their transduction mechanism (i.e. as metabotropic P2YRs or ionotropic P2XRs). Extracellular ATP release by activated or necrotic cells may activate various PRs and especially P2X7R, the best‐characterized PR, on immune cells. P2X7R is known to regulate the activation of the Nod‐like receptor (NLR)‐family protein, NLRP3 inflammasome, which permit the release of IL‐1β, a potent pro‐inflammatory cytokine. The P2X7R/NLRP3 pathway is involved in many inflammatory diseases, such as gout, and in fibrosis diseases associated with inflammatory process, liver or lung fibrosis. Some authors imaging also a real promising therapeutic potential of P2X7R blockage. Thus, several pharmaceutical companies have developed P2X7R antagonists as novel anti‐inflammatory drug candidates. Clinical trials of the efficacy of these antagonists are now underway. A better understanding of the P2X7R/NLRP3 signalling pathways permits the identification of targets and the development of a new class of drugs able to inhibit the fibrogenesis process and collagen deposition.     

The expression of the solute carriers NTCP and OCT-1 is regulated by cholesterol in HepG2 cells

Drug disposition and response are greatly determined by the activities of drug-metabolizing enzymes and transporters. While the knowledge in terms of CYP enzymes and efflux ABC transporters (such as MDR1, P-glycoprotein) is quite extensive, influx transporters are increasingly being unveiled as key contributors to the process of drug disposition. There is little information on the regulation of these proteins in human cells, especially as regards the effect of endogenous compounds. In this study, we analysed the expression of CYP3A4 and three uptake transporters NTCP (SLC10A1), OATP-A/OATP1A2 (SLCO1A2) and OCT-1 (SLC22A1) in HepG2 cells following treatment with cholesterol. While CYP3A4 and OATP1A2 expression was unaffected, cholesterol treatment led to increased levels of NTCP and OCT-1 mRNAs. Alterations in the functional characteristics and/or expression levels of drug transporters in the liver may conceivably contribute to the variability in drug oral bioavailability often observed in the clinical settings.     

Involvement of α2‐adrenoceptors,imidazoline, and endothelin‐A receptors in the effect of agmatine on morphine and oxycodone‐induced hypothermia in mice

Potentiation of opioid analgesia by endothelin‐A (ET_A) receptor antagonist, BMS182874, and imidazoline receptor/α₂‐adrenoceptor agonists such as clonidine and agmatine are well known. It is also known that agmatine blocks morphine hyperthermia in rats. However, the effect of agmatine on morphine or oxycodone hypothermia in mice is unknown. The present study was carried out to study the role of α₂‐adrenoceptors, imidazoline, and ET_A receptors in morphine and oxycodone hypothermia in mice. Body temperature was determined over 6 h in male Swiss Webster mice treated with morphine, oxycodone, agmatine, and combination of agmatine with morphine or oxycodone. Yohimbine, idazoxan, and BMS182874 were used to determine involvement of α₂‐adrenoceptors, imidazoline, and ET_A receptors, respectively. Morphine and oxycodone produced significant hypothermia that was not affected by α₂‐adrenoceptor antagonist yohimbine, imidazoline receptor/α₂ adrenoceptor antagonist idazoxan, or ET_A receptor antagonist, BMS182874. Agmatine did not produce hypothermia; however, it blocked oxycodone but not morphine‐induced hypothermia. Agmatine‐induced blockade of oxycodone hypothermia was inhibited by idazoxan and yohimbine. The blockade by idazoxan was more pronounced compared with yohimbine. Combined administration of BMS182874 and agmatine did not produce changes in body temperature in mice. However, when BMS182874 was administered along with agmatine and oxycodone, it blocked agmatine‐induced reversal of oxycodone hypothermia. This is the first report demonstrating that agmatine does not affect morphine hypothermia in mice, but reverses oxycodone hypothermia. Imidazoline receptors and α₂‐adrenoceptors are involved in agmatine‐induced reversal of oxycodone hypothermia. Our findings also suggest that ET_A receptors may be involved in blockade of oxycodone hypothermia by agmatine.     

SH2B1沉默对胃癌SGC-7901细胞增殖、凋亡及PI3K/AKT通路的影响

目的研究沉默SH2B1基因表达对胃癌SGC-7901细胞增殖、凋亡及3-磷酸肌醇激酶(PI3K)/蛋白质丝氨酸苏氨酸蛋白激酶(AKT)通路的影响。方法体外培养胃癌SGC-7901细胞,采用SH2B1 siRNA转染SGC-7901细胞作为研究组,采用国际通用的与所有基因均无同源序列的non-target siRNA转染作为阴性对照组(NC组),以未经处理的胃癌SGC-7901细胞作为空白对照组(BC组),48h后收集各组转染成功细胞,采用荧光定量聚合酶链反应(qRT-PCR)检测SH2B1 mRNA表达情况,采用蛋白质印迹法(Western Blot)检测SH2B1蛋白表达情况;采用细胞计数试剂盒(CKK-8)检测细胞增殖情况,采用流式细胞仪检测细胞凋亡,同时检测Ki67、增殖细胞核抗原(PCNA)、Caspase-9、PI3K、AKT、p-AKT蛋白表达情况。结果研究组SGC-7901细胞SH2B1蛋白及mRNA表达量较BC组和NC组明显降低,差异有统计学意义(P<0.05);转染后,siRNA组SGC-7901细胞增殖明显受到抑制、平板克隆形成率较BC组和NC组明显降低,凋亡率明显升高,差异均有统计学意义(P<0.05);与BC和NC组比较,研究组SGC-7901细胞PI3K、p-AKT蛋白、Ki67及PCNA蛋白呈低表达,差异有统计学意义(P<0.05),Caspase-9蛋白呈高表达,AKT蛋白表达差异无统计学意义(P>0.05)。结论沉默SH2B1基因表达可能通过抑制PI3K/ATK信号通路激活,抑制SGC-7901细胞增殖,促进凋亡。     

The interaction between β1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system

Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three‐dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS‐induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS‐induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS‐activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS‐activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB. Copyright © 2012 John Wiley & Sons, Ltd.     

Lack of TRPV1 inhibits cystitis-induced increased mechanical sensitivity in mice

Transient receptor potential vanilloid 1 (TRPV1) is highly expressed in primary afferent neurons. Tissue damage generates an array of chemical mediators that activate and sensitize afferent nerve fibers, and sensitization of afferent nerve fibers plays an important role in development of visceral pain. We investigated participation of TRPV1 in visceral pain associated with bladder inflammation induced in mice by systemic treatment with cyclophosphamide or intravesical instillation of acrolein. The effects of experimental cystitis on bladder function (an indicator of visceral pain) and the threshold of response to mechanical or thermal stimuli of the hind paws were investigated using TRPV1 knock-out (KO) and congenic wild-type (WT) mice. We found that cystitis induced bladder mechanical hyperreactivity and increased mechanical sensitivity of hind paws in WT, but not in TRPV1 KO mice. Lack of functional TRPV1 did not inhibit development of histological evidence of bladder inflammation, or increased expression of mRNAs for nerve growth factor, endothelial nitric oxide synthase, cyclooxygenase-2 and bradykinin receptors in urothelium. Cystitis did not affect the threshold of response to thermal stimuli in WT or KO mice. These results suggest that TRPV1 is essential for cystitis-induced bladder mechanical hyperreactivity. Also, TRPV1 participates in development of visceral pain, as reflected by referred increased mechanosensitivity in peripheral tissues in the presence of visceral inflammation.     

Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells

Proliferation of activated hepatic stellate cells (HSC) is an important event in the development of hepatic fibrosis. Insulin-like growth factor-1 (IGF-1) has been shown to be mitogenic for HSC, but the intracellular signaling pathways involved have not been fully characterized. Thus, the aims of the current study were to examine the roles of the extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3-K) and p70-S6 kinase (p70-S6-K) signaling pathways in IGF-1- and platelet-derived growth factor (PDGF)-induced mitogenic signaling of HSC and to examine the potential crosstalk between these pathways. Both IGF-1 and PDGF increased ERK, PI3-K and p70-S6-K activity. When evaluating potential crosstalk between these signaling pathways, we observed that PI3-K is required for p70-S6-K activation by IGF-1 and PDGF, and is partially responsible for PDGF-induced ERK activation. PDGF and IGF-1 also increased the levels of cyclin D1 and phospho-glycogen synthase kinase-3beta. Coordinate activation of ERK, PI3-K and p70-S6-K is important for perpetuating the activated state of HSC during fibrogenesis.     

Inhibition of phosphorylase-a by fructose-1-phosphate, alpha-glycerophosphate and fructose-1,6-diphosphate: explanation for fructose-induced hypoglycaemia in hereditary fructose intolerance and fructose-1,6-diphosphatase deficiency

Abstract. The effect of fructose-1-phosphate, α-glycerophosphate and fruetose-1,6-diphosphate on the activity of liver phosphorylase-a has been investigated at different concentrations of inorganic phosphorus.—Inhibition of phosphorylase-a by fructose-1-phosphate is competitive, by α-glycerophosphate it is noncompetitive and by fructose-1,6-diphosphate uncompetitive. The K_i is 4 × 10^-3 M for fructose-1-phosphate and in the range of 5 to 6 × 10^-2 M for fructose-1,6-diphosphate and α-glycerophosphate. The activity of phosphorylase-a and its inhibition by fructose-1-phosphate depends very much on the concentration of inorganic phosphorus.—In fructose-1,6-diphosphatase deficiency glucose production from gluconeogenic precursors is impossible, whereas in hereditary fructose intolerance accumulated fructose-1-phosphate blocks the residual abnormal hepatic aldolase. Our findings explain fructose induced hypoglycaemia in hereditary fructose intolerance and fructose-1,6-diphosphatase deficiency by a block in glycogenolysis at the level of phosphorylase-a.