Increased Vδ1γδT cells predominantly contributed to IL-17 production in the development of adult human post-infectious irritable bowel syndrome.

BACKGROUND: γδT cells play an important role in the mucosa inflammation and immunity-associated disorders. Our previous study reported that γδ T cells producing IL-17 were involved in the pathogenesis of post-infectious irritable bowel syndrome (PI-IBS). However, their subset characteristic profile in this kind of disease remains unclear. Thus the current study's aim is to investigate the functionally predominant subset and its role in PI-IBS. METHODS: The total T cells were collected from the peripheral blood of patients with PI-IBS. The peripheral proportion of Vδ1 and Vδ2 subset was detected by FACS after stained with anti δ1-PE and anti δ2-APC. The local colonic proportion of this two subsets were measured under laser confocal fluorescence microscope. Vδ1 γδ T cells were enriched from the total peripheral T cells by minoantibody-immuno-microbeads (MACS) method and cultured, functionally evaluated by CCK-8 assay (proliferation), CD69/CD62L molecules expression assay (activation) and ELISA (IL-17 production) respectively. RESULTS: 1. Vδ1 γδ T cells significantly increased while Vδ2 γδ T cells remained unchanged in both the peripheral blood and local colonic tissue from PI-IBS patients (p < 0.05). 2. When cultured in vitro, the Vδ1 γδ T cells remarkably proliferated, activated and produced IL-17 (p < 0.05). CONCLUSIONS: Our results suggest that Vδ1 γδ T cells was the predominant γδ T cells subset in both peripheral and intestinal tissue, and was the major IL-17 producing γδ T cells in PI-IBS.

RMP promotes venous metastases of hepatocellular carcinoma through promoting IL-6 transcription.

Hepatocellular carcinoma (HCC) is believed to arise from tumor-initiating cells (T-ICs), which are responsible for tumor relapse and metastases. Portal vein tumor thrombus (PVTT) is raised from HCC and strongly correlated to a poor prognosis. However, the mechanism underling the formation of PVTT is largely unknown. Herein, we provide evidence that RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) was progressively upregulated in PVTT and overexpressed RMP appeared to increase T-ICs self-renewal. Moreover, RMP promoted metastases of PVTT cells and HCC cells in vitro and in vivo. Knockdown of RMP attenuated T-ICs self-renewal and reversed epithelial-mesenchymal transition (EMT) in HCC and PVTT cells. The neutralizing assays suggested that interleukin-6 (IL-6) had an indispensable role in RMP regulating metastases and self-renewal of HCC cells. Furthermore, the transcription of IL-6 was verified to be modulated by RMP via interaction with p65 and RPB5, through which expanding the T-IC/cancer stem cell populations, as well as inducing EMT was promoted. These results suggested that RMP may promote PVTT formation by promoting IL-6 transcription. Thus, RMP serves as a potent factor contributed to develop PVTT and a promising therapeutic target for HCC patients.

URI regulates tumorigenicity and chemotherapeutic resistance of multiple myeloma by modulating IL-6 transcription.

Unconventional prefoldin RPB5 interactor (URI), which acts as an oncoprotein in solid tumors, is associated with RNA polymerase II subunit 5. However, its impact on multiple myeloma (MM) has not been determined. We demonstrate here that URI is overexpressed in MM compared with plasma cells derived from healthy volunteers. Side population (SP) cells sorted from MM cells showed a much higher level of URI than non-SP cells. Using lentivirus-delivered shRNA, we established stable URI knockdown MM cell lines. URI inhibition significantly attenuated the proliferation of MM cells and decreased colony formation compared with the control cells. Tumor growth assays in NOD/SCID mice further confirmed the promotion role of URI during MM development in vivo. Furthermore, URI knockdown markedly reduced the abundance of SP in MM cell lines and enhanced the chemotherapeutic sensitivity of MM towards bortezomib. Mechanically, URI appears to be critically involved in modulating STAT3 activity through regulating interleukin (IL)-6 transcription via interaction with NFκBp65. In conclusion, URI may have an important role in the development of MM and chemotherapeutic resistance through activating the IL-6/STAT3 pathway.

Asparagine synthetase is an independent predictor of surgical survival and a potential therapeutic target in hepatocellular carcinoma.

BACKGROUND: Asparagine synthetase (ASNS) is associated with drug resistance in leukaemia, and the function of this enzyme in the context of hepatocellular carcinoma (HCC) is not clear. In this study, the relationship between ASNS expression and clinical outcomes after surgical resection was investigated, and the therapeutic value of ASNS was also evaluated. METHODS: The expression of ASNS was evaluated in HCC samples by real-time PCR and immunohistochemistry assays. The correlation between ASNS expression and clinicopathological features was investigated. Potential clinicopathological prognostic factors were examined by univariate and multivariate survival analysis. Asparagine synthetase was overexpressed and knocked down in HCC cell lines to assess the influence of the enzyme on cell proliferation, migration and tumourigenicity. L-asparaginase was used to treat HCC cells with high or low levels of ASNS in vitro and in vivo to examine the therapeutic efficacy. RESULTS: The expression of ASNS was higher in HCC tumour tissues and was closely correlated with the serum AFP level, tumour size, microscopic vascular invasion, tumour encapsulation, TNM stage and BCLC stage. Patients with low ASNS expression levels had a poor prognosis with respect to overall survival (OS). The multivariate survival analysis indicated that ASNS is an independent prognostic factor for OS. Furthermore, functional studies demonstrated that ASNS significantly inhibits the proliferation, migration and tumourigenicity of HCC cells. The knockdown of ASNS markedly increased sensitivity to L-asparaginase, indicating that cells with different ASNS protein levels have different sensitivities to L-asparaginase. CONCLUSION: The expression of ASNS is an independent factor affecting the survival of HCC patients, and low ASNS expression in HCC was correlated with worse surgical outcomes. The ASNS may be a promising therapeutic target for the treatment of HCC.

Impairment of the ryanodine-sensitive calcium release channels in the cardiac sarcoplasmic reticulum and its underlying mechanism during the hypodynamic phase of sepsis.

Changes in Ca2+-induced Ca2+ release in cardiac sarcoplasmic reticulum (SR) during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The 45Ca2+ release studies show that the amount of Ca2+ released from the passively and the actively loaded SR vesicles was unaffected during the early sepsis (9 h after CLP), but it was significantly decreased during the late phase (18 h after CLP) of sepsis. The [3H]ryanodine binding assays reveal that the Bmax for ryanodine binding was unaffected during the early phase, but was decreased by 32.1% during the late phase of sepsis. The affinity of ryanodine receptor for Ca2+ remained unchanged during sepsis. ATP, AMP-PCP, and caffeine stimulated binding, while MgCl2 and ruthenium red inhibited [3H]ryanodine binding in control, early sepsis, and late sepsis groups. The EC50 and IC50 values for these regulators were unaffected during the progression of sepsis. Digestion of control SR with phospholipase A2 decreased [3H]ryanodine binding and the decrease was reversible by the addition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS). Addition of PC, PE, or PS to the SR isolated from septic rats stimulated [3H]ryanodine binding. These data demonstrate that Ca2+-induced Ca2+ release from cardiac SR remained relatively unaffected during the early phase, but was significantly impaired during the late phase of sepsis. The sepsis-induced impairment in SR Ca2+ release is a result of a quantitative reduction in the number of Ca2+ release channels. Furthermore, the reduction is associated with a mechanism involving a modification of membrane lipid profile in response to certain stimuli such as activation of phospholipase A2.

Calcium uptake by sarcoplasmic reticulum is impaired during the hypodynamic phase of sepsis in the rat heart.

Alterations of the ATP-dependent Ca2+ uptake in the cardiac sarcoplasmic reticulum (SR) during the 2 hemodynamically distinct phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham-operated. The SR vesicles were isolated by sucrose gradient centrifugation. The results show that the rates of ATP-dependent Ca2+ uptake in the cardiac SR were unaffected during the early hyperdynamic phase, whereas they were decreased by 41-46% (P < 0.01) during the late hypodynamic phase of sepsis. Analysis of the kinetics of Ca2+ transport indicates that during the late phase of sepsis, the Vmax values of Ca2+ pump for ATP and Ca2+ were decreased, whereas the affinities of Ca2+ pump for ATP and Ca2+ were unaffected. Magnesium stimulated, whereas vanadate inhibited the ATP-dependent Ca2+ uptake, but the Mg2+-stimulated and the vanadate-inhibited Ca2+ uptake activities were significantly lower during the late sepsis. Phosphorylation of SR by the cAMP-dependent and the calmodulin-dependent protein kinases stimulated the ATP-dependent Ca2+ uptake in the control and the early septic experiments, whereas it failed to stimulate Ca2+ uptake in the late sepsis. The extent of the phosphorylation-stimulated Ca2+ uptake activities was reduced by 65-69% (P < 0.01) during the early sepsis, and they were completely abolished during the late sepsis. These data indicate that the ATP-dependent Ca2+ uptake in cardiac SR was impaired during the late hypodynamic phase of sepsis. The impaired Ca2+ uptake during late sepsis was associated with a defective phosphorylation of SR proteins. Because the ATP-dependent Ca2+ uptake by cardiac SR plays an important role in the regulation of contraction-relaxation coupling, our findings may contribute to the understanding of the pathogenesis of altered cardiac function during the progression of sepsis.

Biphasic redistribution of muscarinic receptor and the altered receptor phosphorylation and gene transcription are underlying mechanisms in the rat heart during sepsis.

OBJECTIVE: The purpose of this study was to investigate intracellular redistribution of muscarinic cholinergic receptor (m2AChR) and the roles of receptor phosphorylation and gene transcription as underlying mechanisms in the rat heart during different phases of sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). The density of m2AChR in the sarcolemmal and light vesicle fractions was studied using [3H]-quinuclidinyl benzilate ([3H]-QNB). Phosphorylation of m2AChR was studied by labeling of the myocardial ATP pool by perfusing isolated hearts with [32P]H3PO4 followed by identification of the phosphorylated m2AChR with SDS-PAGE. The steady-state level of m2AChR mRNA was determined by RT-PCR and Southern blot analysis. RESULTS: Septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP, early sepsis) and a subsequent hypocardiodynamic (18 h after CLP, late sepsis) state. During early sepsis, the Bmax for [3H]-QNB binding was increased in sarcolemma (+69%) but decreased in light vesicles (-22%), whereas during late sepsis, the Bmax was decreased in sarcolemma (-20%) but increased in light vesicles (+32%). The sum of Bmax for sarcolemmal and light vesicle fractions was increased during early sepsis (+43%) but decreased during late sepsis (-14%). The phosphorylation of m2AChR was decreased during early sepsis (-73%) but increased during late sepsis (+36% to +90%). The m2AChR mRNA abundance was increased during early sepsis (+52%) but decreased during late sepsis (-28%). CONCLUSIONS: The m2AChR in the rat heart was externalized from light vesicles to sarcolemma (overexpression) during early sepsis but internalized from surface membranes to intracellular sites (underexpression) during late sepsis. Furthermore, changes in the receptor phosphorylation and gene transcription are responsible for the biphasic redistribution and the altered expression of m2AChR in the rat heart during the progression of sepsis.

Transcriptional and posttranscriptional regulation of beta(2)-adrenergic receptor gene in rat liver during sepsis.

Changes in beta(2)-adrenergic receptor (beta(2)-AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic (9 h after CLP; early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [(3)H]dihydroalprenolol binding studies show that the density of beta(2)-AR was decreased by 12 and 35% during the early and late phases of sepsis, respectively. Western blot analyses depict that the beta(2)-AR protein level was reduced by 37 and 72% during early and late sepsis, respectively. The reverse transcription polymerase chain reaction and Southern blot analyses reveal that the steady-state level of beta(2)-AR mRNA was decreased by 37% during early phase and 77% during late phase of sepsis. Nuclear run-off assays show that the rate of transcription of beta(2)-AR mRNA was reduced by 36% during early sepsis and 64% during late sepsis. The stability assays indicate that the half-life of beta(2)-AR mRNA was shortened by 21 and 50% during the early and late phases of sepsis, respectively, indicating that the rate of degradation of beta(2)-AR mRNA was progressively enhanced during sepsis. These findings demonstrate that the beta(2)-AR gene was underexpressed in the liver during the progression of sepsis, and, furthermore, the underexpression of the beta(2)-AR gene was the result of a reduction in the rate of transcription coupled with an enhancement in the rate of degradation of beta(2)-AR gene transcripts. Thus our findings that the transcriptional and posttranscriptional regulation of beta(2)-AR gene associated with decreases in beta(2)-AR number and its protein expression may provide a molecular mechanistic explanation for the development of hypoglycemia during the late stage of sepsis.

Transcriptional regulation of alpha1-adrenoceptor gene in the rat liver during different phases of sepsis.

Changes in alpha1-adrenoceptor (alpha1AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic phase (9 h after CLP, early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [3H]prazosin binding studies show that the density of alpha1AR was increased by 30% during the early phase while it was decreased by 24% during the late phase of sepsis. Western blot analyses reveal that alpha1AR protein level was elevated by 48% during early sepsis but was decreased by 55% during late sepsis. Northern blot analyses depict that the steady-state level of alpha1bAR mRNA was enhanced by 21% during the early phase but was declined by 29% during the late phase of sepsis. Nuclear run-off assays show that the transcription rate of alpha1bAR gene transcript was increased by 76% during early sepsis while it was decreased by 29% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of alpha1bAR mRNA remained unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the increase in the rate of transcription of alpha1bAR gene paralleled with the elevations in the alpha1bAR mRNA abundance and alpha1AR protein level, while during the late phase of sepsis, the decrease in the rate of transcription of alpha1bAR gene coincided with the declines in the alpha1bAR mRNA abundance and the alpha1AR protein level in the rat liver. These observations indicate that the altered expression of alpha1AR genes in the rat liver during the progression of sepsis was regulated transcriptionally.

Alterations of G-protein and adenylate cyclase signaling in rat liver during the progression of sepsis.

Changes in the protein level of various subunits of G-protein and the activity of adenylate cyclase in rat liver plasma membranes during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that the protein levels of Galphai-2 and Galphai-3 were unchanged during the early hypermetabolic (hyperglycemic) phase (9 h after CLP), whereas Galphaai-2 and Galphaai-3 were increased by 32.4 and 59.1%, respectively, during the late hypometabolic (hypoglycemic) phase (18 h after CLP) of sepsis. The protein levels of Galphas and Gbeta remained unaltered during both the early and the late phases of sepsis. The activity of adenylate cyclase remained unchanged during the early phase, whereas it was decreased by 26% (p < .05) during the late phase of sepsis. Since the G-protein/adenylate cyclase signaling system mediates hormonal control of hepatic glucose metabolism, the observed increases in the Galphai-2 and Galphai-3 protein levels coupled with a decrease in the activity of adenylate cyclase may contribute to the development of the hypoglycemia during the late stage of sepsis.

[Alteration of ryanodine receptors in cardiac sarcoplasmic reticulum and nuclear envelope of rats during sepsis].

To investigate changes of ryanodine receptors in the sarcoplasmic reticulum (SR) and the nuclear envelope (NE) of rat cardiac myocytes during sepsis induced by cecal ligation and puncture (CLP), myocardial SR and NE were fractionated with density gradient centrifugation and the characteristic of ryanodine receptor was assayed with a method of radioreceptor binding assay. The result showed that Bmax of ryanodine receptors in cardiac SR was increased by 23% during early sepsis (9 h after CLP), but decreased by 38% during late sepsis (18 h after CLP). Bmax of ryanodine receptors in cardiac NE, on the other hand, was increased by 100% and 160% during early and late sepsis respectively. Kd of ryanodine binding to SR and NE remained unchanged during sepsis. These results demonstrated up-regulation of ryanodine receptors in SR occurred during early sepsis and down-regulation of these receptors in SR occurred during late sepsis, while up-regulation of ryanodine receptors in NE occurred during both the early and the late sepsis.

Role of endogenous carbon monoxide in the pathogenesis of hypotension during septic shock.

A sepsis model induced by cecal ligation and puncture was used to study the role of endogenous carbon monoxide in hypotension pathogenesis of rats during septic shock. After administration of zinc deuteroporphyrin 2,4-bisglycol (ZnDPBG),an inhibitor of heme oxygenase (HO),blood pressure (BP),HO activity and carbon monoxide (CO) release from vascular muscle tissue were measured. The results showed that BP of sepsis rats, including systolic and diastolic arterial BP, decreased significantly while HO activity and CO content were significantly increased. In contrast, after administration of ZnDPBG, BP of sepsis rats was significantly increased while the HO activity and CO production were significantly decreased. These findings suggest that HO activity and CO release within vascular musculature are increased during septic shock; inhibition of HO may elevate BP of rats during septic shock through a decrease of endogenous CO production. It is concluded that endogenous CO derived from vascular muscle cells plays an important role in regulating vascular tone, and the up-regulation of HO activity followed by subsequent CO production contributes to hypotension pathogenesis during septic shock.

Phospholipase A2 activities are decreased during early but increased during late phases of sepsis in rat heart.

BACKGROUND: Changes in the activities of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in the rat heart during early hyperdynamic and late hypodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of cardiac dysfunction during sepsis. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. PLA2 activity was measured based on the rate of hydrolysis of 1-palmitoyl-2-[1-(14)C]-oleoyl phosphatidylcholine. RESULTS: The results show that under physiological conditions, sPLA2 and cPLA2 activities were time and protein dependent. The optimal Ca2+ concentrations for sPLA2 and cPLA2 activities were 3 mM and 40 microM, respectively. During sepsis, sPLA2 activity was decreased by 25% (P < 0.01) during early phase while it was increased by 49% (P < 0.01) during late phase of sepsis. Similarly, cPLA2 activity was decreased by 23% (P < 0.01) during early sepsis while it was increased by 60% (P < 0.01) during late sepsis. CONCLUSIONS: Since PLA2 functions to maintain cell membrane integrity and function, a biphasic change in sPLA2 and cPLA2 activities may contribute to the development of the two cardiodynamically distinct phases during the progression of sepsis.

Phosphorylation of beta-adrenergic receptor leads to its redistribution in rat heart during sepsis.

The role of receptor phosphorylation on the redistribution of beta-adrenergic receptors (beta-ARs) in rat hearts during different phases of sepsis was investigated. Sepsis was induced by cecal ligation and puncture (CLP). Changes in the distribution of beta-ARs in the sarcolemmal and light vesicle fractions were studied using (-)-[4,6-propyl-3H]dihydroalprenolol ([3H]DHA). Phosphorylation of beta-ARs was studied by perfusing hearts with [32P]H3PO4 followed by identification of the phosphorylated beta-ARs with immunoprecipitation using anti-beta 1-AR antibody. The results show that septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP; early sepsis) and a subsequent hypocardiodynamic (18 h after CLP; late sepsis) state. [3H]DHA binding studies show that, during early sepsis, the maximum binding capacity (Bmax) was increased by 26% in sarcolemma but was decreased by 30% in light vesicles, whereas, during late sepsis, the Bmax was decreased by 39% in sarcolemma but increased by 31% in light vesicles. These data indicate that beta-ARs in the rat heart were externalized from light vesicles to sarcolemma during early sepsis but were internalized from surface membranes to intracellular sites during late sepsis. The immunoprecipitation studies reveal that the externalization of beta-ARs during early sepsis was coupled with a concomitant decrease (-28.5 to -30.6%, P < 0.01) in the receptor phosphorylation, whereas the internalization of beta-ARs during late sepsis was accompanied by a simultaneous increase (30.3 to 33.8%, P < 0.01) in the receptor phosphorylation. Because the phosphorylation/dephosphorylation of beta 1-ARs regulate their functional coupling and may reflect their subcellular distribution, it is suggested that the increase in receptor phosphorylation seen in late sepsis leads to the receptor internalization observed in late sepsis; similarly, externalization of (dephosphorylated) receptors in early sepsis may give rise to the apparent decrease in sarcolemmal receptor phosphorylation observed during this interval.

GTP-binding protein mediated phospholipase A2 activation in rat liver during the progression of sepsis.

Effects of GTP-binding proteins on the activation of secretory phospholipaseA2 (sPLA2) and cytosolic phospholipaseA2 (cPLA2) in rat liver during two different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The results show that in the absence of G-protein modulator, hepatic sPLA2 and cPLA2 activities were activated by 40.8-46 and 91.6-105.8%, respectively, during early and late phases of sepsis. GTPgammaS and fluoroaluminate (AlF4-) stimulated sPLA2 and cPLA2 activities within each experimental group, i.e., control, early sepsis, and late sepsis. The GTPgammaS and AlF4(-)-stimulated sPLA2 and cPLA2 activities remained significantly elevated during early phase (22.3-65.6% increase) and late phase (32.5-109.1% increase) of sepsis. Further analyses demonstrate that cholera toxin significantly stimulated sPLA2 and cPLA2 activities within each experimental group, and that the cholera toxin stimulated sPLA2 and cPLA2 activities remained significantly higher during early phase (23.5-37% increase) and late phase (56.7-70% increase) of sepsis. In contrast, pertussis toxin significantly inhibited sPLA2 and cPLA2 activities within each experimental group, and that the pertussis toxin-inhibited sPLA2 and cPLA2 activities remained significantly higher in early septic (57-68.5% increase) and late septic (34.6-45.5% increase) experiments. These data demonstrate that cholera toxin-sensitive G alpha s and pertussis toxin-sensitive G alpha i were both involved in the activation of sPLA2 and cPLA2 activities in rat liver during the progression of sepsis.

Role of endogenous carbon monoxide in hypertension pathogenesis of rats.

The present study investigated the contribution of endogenous heme oxygenase (HO)/carbon monoxide (CO) system to hypertension pathogenesis of rats. Zinc deuteroporphyrin 2,4-bisglycol (ZnDPBG), an inhibitor of heme oxygenase (HO), was used to inhibit HO activity in vivo. It was found that the blood pressure of rats with HO inhibition was significantly elevated, and plasma levels of adrenaline, noradrenaline, endothelin, nitrate and nitrite were significantly increased. HO activity and HbCO formation within vascular smooth muscle tissues were significantly inhibited after administration of ZnDPBG. Furthermore, administration of exogenous CO into HO inhibiting rats led to MABP decrease, but injection of HO substrate, heme-L-lysinate, had no effect on HO inhibition-induced hypertension. In spontaneously hypertensive rats, injection of exogenous CO resulted in a significant decrease of MABP, and heme-L-lysinate had a similar effect with exogenous CO. These data show that HO/CO system has an anti-hypertension biological action, suggesting that endogenous CO plays an important role in hypertension pathogenesis.

Group II phospholipase A2 gene expression is transcriptionally regulated in rat liver during sepsis.

Changes in group II phospholipase A2 (PLA2) gene expression in rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that PLA2 activity was increased by 41 and 92% during early hyperdynamic phase (9 h after CLP) and late hypodynamic phase (18 h after CLP) of sepsis, respectively. Western blot analysis reveals that group II PLA2 protein levels were elevated by 53 and 95% during early and late sepsis, respectively. Northern blot analysis depicts that the steady-state levels of group II PLA2 mRNA were enhanced by 39 and 114% during early and late sepsis, respectively. Nuclear run-off assay shows that the transcription rates of group II PLA2 gene transcript were stimulated by 36 and 74% during early and late sepsis, respectively. The actinomycin D pulse chase study indicates that the half-life of group II PLA2 mRNA remained unaffected during early and late phases of sepsis. These results demonstrate that group II PLA2 gene transcripts were overexpressed in rat liver during the progression of sepsis and that the overexpression was a result of the enhanced synthesis of group II PLA2 mRNA. Because PLA2 plays an important role in the maintenance of cell membrane integrity, our findings may contribute to the understanding of the pathogenesis of hepatic dysfunction during the progression of sepsis.

[Impairment in the phosphorylation of Ca(2+)-transport ATPase of rat liver endoplasmic reticulum during sepsis].

The phosphorylation of Ca(2+)-transport ATPase of rat liver endoplasmic reticulum (ER) during early and late septic shock induced by cecum ligation and puncture (CLP) was investigated by determining incorporation of [gamma-32P] ATP into Ca(2+)-ATP phosphoprotein intermediate. Hepatic endoplasmic reticulum was isolated by differential centrifugation with sucrose density gradient. The Ca(2+)-ATPase phosphoprotein intermediate was identified by SDS-PAGE. The results showed that the phosphorylation of Ca(2+)-ATPase (115 kD) was decreased respectively by 15-23% (P < 0.05) and 17-27% (P < 0.05) at 9 h (early sepsis) and 18 h (late sepsis), following the CLP in the rough, intermediate and smooth ER preparations. Kinetic analysis using rough ER showed that the Vmax for Ca2+ and for ATP for the phosphorylation of Ca(2+)-ATPase were decreased dramatically during early and late sepsis, but without changes in the K(m) values. These results demonstrate that the phosphorylation of the phosphoprotein intermediate of Ca(2+)-ATPase in rat liver was impaired during different phases of sepsis.

[Changes in the calcium channels in rat cardiac cells during sepsis].

Changes in the number of calcium channels in two subcellular fractions, the sarcolemma and the light vesicle, of rat cardic cells were studied during sepsis. Sepsis was induced by cecal ligation and puncture (CLP). The results showed that some of the calcium channels in the light vesicle translocated to the sarcolemma during the early sepsis (9 h after CLP) while during the late sepsis (18 h after CLP), some of these in the sarcolemma translocated to the light vesicle. The mechanisms of redistribution of the calcium channels in the sarcolemma and the light vesicle during sepsis was not associated to the phosphorylation of the calcium channels by cAMP dependent protein kinase (PKA), Ca2+/calmodulin dependent protein kinase (PKM) and protein kinase C (PKC). Since beta-adrenergic receptors, muscarinic cholinergic receptors and Na+/K(+)-ATPase were also redistributed during sepsis, it is suggested that the redistribution might be non-specific.

Effects of Arg-Gly-Asp-Ser on Ca2+ transport of myocardial sarcoplasmic reticulum in rat septic shock.

AIM: To study the effects of Arg-Gly-Asp-Ser (RGDS), a synthetic short peptide of fibrinogen degradation, on the Ca2+ transport function of cardiac sarcoplasmic reticulum in rat septic shock. METHODS: RGDS 5 mumol.kg-1 was injected i.v. at 4 h and 14 h after cecal ligation and puncture (CLP) operation on rats. Highly purified membrane of sarcoplasmic reticulum (SR) was prepared from rat hearts. Assays were made of ATP-dependent Ca2+ uptake by cardiac SR and [3H] ryanodine binding to SR. RESULTS: The initial rate and the capacity of SR Ca2+ uptake were increased by 104% (P < 0.01) and 12% (P < 0.05), respectively, paralleled by an increase in Ca(2+)-ATPase activity and a decrease in calcium accumulation of myo- cardium of septic rats, whereas the Bmax and Kd values of Ca2+ activated [3H]ryanodine binding to SR were unaffected after RGDS administration. CONCLUSIONS: The results indicated that RGDS have cardioprotective effects of maintaining Ca2+ homeostasis of cardiac myocytes by enhancing SR Ca2+ uptake in rat septic shock.