Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

Aim: Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity. Patients and Methods: Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry. Results: Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027). Conclusion: CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis

Estimativa das contribuições dos sistemas anaeróbio lático e alático durante exercícios de cargas constantes em intensidades abaixo do VO2max

O objetivo do estudo foi estimar as contribuições do metabolismo anaeróbio lático (MAL) e alático (MAA) em intensidades abaixo do consumo máximo de oxigênio (VO2max). Dez homens (23 ± 4 anos, 176,4 ± 6,8 cm, 72,4 ± 8,2 kg, 12,0 ± 4,5 % de gordura corporal) realizaram um teste progressivo até a exaustão voluntária para identificação do VO2max, da potência correspondente ao VO2max (WVO2max) e do segundo limiar ventilatório (LV2). Na segunda e na terceira visita foram realizados seis testes de cargas constantes (três testes por sessão) com intensidades abaixo do VO2max. Houve uma predominância do MAL sobre o MAA durante os exercícios submáximos a partir da intensidade correspondente ao LV2, sendo significativamente maior em 90% VO2max (p < 0,05). Dessa forma, esses resultados podem auxiliar treinadores a aplicarem cargas de treinamento adequadas aos seus atletas, de acordo com a exigência metabólica da competição

Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases

Effects of epidermal growth factor and calcium omission on cholecystokinin-stimulated Cl⁻ conductance in rat pancreatic zymogen granules

Evidence suggests that cholecystokinin-octapeptide (CCK-8)-induced activation of a Cl− conductance in the membrane of zymogen granules (ZG) is closely related to pancreatic enzyme secretion [1,2,3]. Following stimulation of isolated pancreatic acinar cells with increasing concentrations of CCK-8, the Cl− conductance in the ZG from these acini increased, reached a maximum of 40±7 % above basal Cl− conductance at 10−12 M CCK-8, and then decreased at CCK-8 concentrations higher than 10−9 M to a level comparable to the basal Cl− conductance. We had interpreted the inhibitory action of high CCK-8 concentrations to be due to the generation of high concentrations of diacylglycerol and/or its metabolites by an “overstimulation” of phospholipase C at supramaximal CCK-8 concentrations [1]. We now show that EGF abolishes the downstroke of the dose response curve for CCK-8-induced ZG Cl− conductance and shifts the stimulatory response to higher CCK-8 concentrations. Similarly in a nominally “Ca2+-free buffer” (free [Ca2+] ∼0.2 nM), stimulated Cl− conductance at 10−12 M CCK-8 is nearly abolished and the decreased Cl− conductance at 10−8 M CCK-8 is increased to the level of maximal stimulation at 10−12 M CCK-8. We conclude that both EGF and low [Ca2+] affect CCK-8-induced ZG Cl− conductance by decreasing phospholipase C activity

PGE₂ regulates cholecystokinin-octapeptide (CCK-8)-stimulated Cl⁻ conductance in isolated zymogen granules from rat pancreas

In this study we have examined the effects of prostaglandin E2 (PGE2), the cyclooxygenase inhibitor, indomethacin, and a protein kinase A inhibitor (PKA‐I) on the Cl− conductance in isolated zymogen granules (ZG) from cholecystokinin octapeptide (CCK‐8) pre‐stimulated pancreatic acini. The Cl− conductance in isolated ZG from CCK‐8 pre‐stimulated rat pancreatic acini increases with increasing CCK‐8 concentrations and decreases at supramaximal CCK‐8 concentrations. The basal and CCK‐8‐stimulated Cl− conductance in ZG is inhibited by pretreatment of acini with PGE2 (10−6 M). This PGE2‐induced inhibition is abolished in the presence of PKA‐I (20 U/ml). Furthermore, pretreatment of acini with indomethacin (10−5 M) or PKA‐I (20 U/ml) abolishes the decrease in the Cl− conductance at supramaximal CCK‐8 concentrations (10−9 M). We conclude that the inhibition of the Cl− conductance in isolated ZG at high CCK‐8 concentrations is mediated by an enhanced production of PGE2, and that PGE2 operates by stimulating adenylate cyclase (AC) with a consequent rise in cAMP and activation of PKA

Effects of cholecystokinin, cholecystokinin JMV-180 and GTP analogs on enzyme secretion from permeabilized acini and chloride conductance in isolated zymogen granules of the rat pancreas

Previous studies have shown that hormonal activation of the Cl− conductance in pancreatic zymogen granules (ZG) is closely related to enzyme secretion from acinar cells. We have now examined the role of guanine nucleotides in stimulated and unstimulated protein secretion from isolated digitonin‐permeabilized pancreatic acini and in the Cl− conductance of isolated ZG. Protein secretion from permeabilized isolated acini, measured at 0.1 mM Ca2+, increased with increasing cholecystokinin octapeptide (CCK‐8) concentrations and decreased at high CCK‐8 concentrations. The maximum secretion, approximately twice the control level, was reached at 1 nM CCK‐8. The CCK analog, CCK JMV‐180, which supposedly acts as an agonist on high‐affinity CCK receptors and as an antagonist on low‐affinity CCK receptors, stimulated maximum enzyme secretion at a CCK JMV‐180 concentration of 0.1 μM and no decrease in secretion was observed at higher CCK JMV‐180 concentrations. 0.1 mM guanosine 5′‐[γ‐thio]triphosphate (GTP [S]) also increased the protein release by approximately twice that of the control and shifted the CCK‐8 concentration causing maximum stimulation from 1 nM to 0.01 nM. GTP[S] concentrations greater than 0.1 mM inhibited protein release evoked by an optimal concentration of 1 nM CCK‐8. 0.1 mM GTP[S] had no pronounced effect on the protein secretion stimulated by low concentrations of CCK JMV‐180, but inhibited protein secretion evoked by CCK JMV‐180 concentrations greater than 0.1 μM. This indicates that guanosine‐nucleotide‐binding proteins [G protein(s) coupling to CCK receptors also mediate both CCK‐induced increases and CCK‐induced decreases of enzyme secretion at low and high CCK concentration, respectively. ZG were prepared on a Percoll gradient from CCK‐8‐stimulated or CCK‐JMV‐180‐stimulated and unstimulated acini. Their Cl− conductances were estimated in the absence of Ca2+ and in the presence of 1 mM EGTA from the rate of decrease in absorbance following addition of the K+ ionophore valinomycin as a measure of ZG osmotic lysis. The Cl− conductance in ZG from CCK‐8‐stimulated and CCK‐JMV‐180‐stimulated acini was maximally activated at 1 pM and 10 nM respectively. At higher agonist concentrations, Cl− conductance was decreased. Direct addition of 10 μM GTP[S] to isolated ZG from unstimulated acini increased the rate of lysis by approximately 40% of the control value. This effect was approximately additive to that of CCK‐8 or of CCK JMV‐180 prestimulation. In the presence of Ca2+ concentrations greater than 1 μM, the ZG lysis rate was increased by approximately 30% of the control value and this effect was additive to the effect of CCK‐8 prestimulation, but not additive to GTP[S]‐stimulated ZG lysis. We conclude from our data that G proteins in the plasma membrane Gp and in the ZG membrane GE are involved in stimulated enzyme secretion. In the presence of GTP, activated GE couples to a Cl− channel in the ZG membrane, which leads to an increase in Cl− conductance, an influx of Cl−, cations and H2O into the ZG and flushing of stored enzymes over the ZG membrane which is fused to the luminal cell membrane. Inhibition of enzyme secretion by supramaximal CCK concentrations is reflected by a decrease in Cl− conductance. The mechanism of this inhibition is not known. It probably involves messengers or metabolites generated at the plasma membrane by CCK‐induced signal transduction, rather than an inhibitory G protein in the ZG membrane

Secretagogue and second messenger-activated Cl⁻ permeabilities in isolated pancreatic zymogen granules

Previous studies of enzyme secretion from isolated pancreatic acinar cells and of isolated zymogen granules (ZG) have reported that both a Cl− and a K+ permeability are present on the ZG membrane. It has been suggested that ion influx via these permeability pathways, followed by water movement is required for granular swelling which appears to be intimately related to exocytosis. However, little is known about the regulation of these pathways by secretagogues. Evidence suggests that cAMP-protein kinase A and diacylglycerol-protein kinase C are second messengers in stimulation of exocytosis. In the present study we have examined ion permeability pathways in ZG isolated from control cells and from cells pretreated with the acetylcholine analog carbachol (Cch), with the peptide hormone cholecystokinin (CCK) and with second messengers of hormone action such as cAMP and the diacylglycerol analog 12-O-tetradecanoyl phorbol-13-acetate (TPA). Ion and water influx rates in ZG and consequent swelling and lysis of granules was monitored by measuring changes in optical densities of ZG suspensions at 540 nm following additions of the electrogenic or electroneutral ionophores valinomycin and nigericin, respectively. The data show that both a Cl− conductance and an anion exchange pathway are present in the granule membrane. Both pathways are activated by pretreatment of isolated cells with CCK or of isolated permeabilised cells with cAMP, whereas only the Cl− conductance is increased by pretreatment with Cch or with TPA. The anion transport inhibitor DIDS abolishes Cl− conductance but has no effect on the anion exchanger. Granular lysis is also inhibited by buffers of high osmotic strength. The direct application of the catalytic subunit of protein kinase A (PKA) and ATP to isolated ZG inhibits both the Cl− conductance and the anion exchange pathway probably mediated by phosphorylation. This result does not seem to be consistent with the observation that in isolated ZG from cAMP prestimulated cells both Cl− conductance and the Cl−/anion exchanger are activated. We conclude that stimulation of pancreatic acinar cells by secretagogues results in activation of Cl− permeability pathways in the ZG membrane mediated by cAMP-protein kinase A and DG-protein kinase C mediated phosphorylation. It is possible that in addition to “on” reactions also “off” reactions exist which close the Cl− channel, e.g. by dephosphorylation and that in activation of Cl− permeability pathways different sites for cAMP-protein kinase A action or different protein kinases A are involved

Epidermal growth factor inhibits both cholecystokinin octapeptide-induced inositol 1,4,5-trisphosphate production and [Ca²⁺]_i increase in rat pancreatic acinar cells

We have studied the effects of epidermal growth factor (EGF) on both cholecystokinin octapeptide (CCK-OP)-induced inositol-1,4,5 trisphosphate (IP) production and on cytosolic free calcium concentrations [Ca2+]i by fluorescence measurements in fura-2-loaded pancreatic acini. Our data show that EGF inhibits CCK-OP induced IP3 production by 40 ± 9 % and decreases CCK-OP induced rise in cytosolic Ca2+ by 41 ± 9 %. These data indicate that activation of EGF receptors leads to inhibition of CCK-OP induced stimulation of phospholipase C (PLC)