Decrease In Intracellular Concentration Research Articles

Peptide Sodium Channels Modulator Mu-Agatoxin-Aa1a Prevents Ischemia-Reperfusion Injury of Cells

Ischemia-reperfusion injury (IRI) is an irreversible functional and structural injury. Restoration of normal oxygen concentration exacerbates the emergence and development of deadly cells. One of the possible moments of reperfusion damage to cells is an increase in the intracellular concentration of sodium ions. In this article, we study the mu-agatoxin-Aa1a, a modulator of sodium channels, on the processes of IRI cells damage. The toxin was synthesized using an automatic peptide synthesizer. Hypoxia was induced by reducing the content of serum and oxygen in the CHO-K1 culture. The influence of the toxin on the level of apoptosis; intracellular concentration of sodium, calcium, and potassium ions; intracellular pH; totality of reactive oxygen species (ROS), nitric oxide (NO), and ATP; and changes in the mitochondrial potential were studied. The experiments performed show that mu-agatoxin-Aa1a effectively prevents IRI of cells. Toxin reduces the level of apoptosis and prevents a decrease in the intracellular concentration of sodium and calcium ions during IRI. Mu-agatoxin-Aa1a contributes to the maintenance of elevated intracellular pH, reduces the intracellular concentration of ROS, and prevents the decrease in intracellular NO concentration and mitochondrial potential under conditions of reoxygenation/reperfusion. An analysis of experimental data shows that the mu-agatoxin-Aa1a peptide has adaptogenic properties. In the future, this peptide can be used to prevent ischemia/reperfusion tissue damage different genesis.

Study of the Effect of Proton Pump Inhibitors on the Development of Hypomagnesemia in Patients with Paroxysmal Atrial Fibrillation on the Background of Autonomic Sinus Node Dysfunction

Aim. To study the effect of proton pump inhibitors (PPIs) on the development of hypomagnesemia in patients with paroxysmal atrial fibrillation (AF) on the background of autonomic sinus node dysfunction (ASND).Material and methods. A prospective cohort study included 32 patients with an established diagnosis of ASND and often recurrent paroxysmal AF. The main group consisted of 17 patients who took pantoprazole at a dose of 20 mg 2 times a day as treatment of diagnosed duodenal erosions within 6 weeks from the time of the initial visit to an arrhythmologist. The remaining 15 patients did not take PPIs and were included in the control group. The determination of magnesium in whole blood, plasma and formed elements was carried out twice in all patients – at the initial visit and after 6 weeks. In addition, in the experiment, complex-forming activity of solutions of various PPIs (esomeprazole, pantoprazole, rabeprazole and omeprazole) with respect to magnesium ions was studied. The author’s method was used, based on turbidimitric determination of the light transmission during the heterogeneous reaction of the formation of magnesium phosphates in the presence of the analyzed drug or without it.Results. There were no significant differences in the magnesium content in the blood of patients of the main and control groups before taking PPIs. After taking of pantoprazole by the patients of the main group there were significant differences between main and control groups in magnesium in whole blood (0.48 [0.44-0.51] mmol/l vs 0.55 [0.5-0.61] mmol/l, p=0.01) and its values in formed elements (0.52 [0.45-0.67] mmol/l vs 0.75 [0.65-1.2] mmol/l, p=0.009). Analysis of magnesium content in patients of the main group before and after taking of pantoprazole also showed a significant decrease in intracellular concentrations of the element (0.6 [0.51-1.0] mmol/l vs 0.52 [0.45-0.67] mmol/l, p=0.002), as well as decrease in its total content in whole blood (0.51 [0.45-0.59] mmol/l vs 0.48 [0.44-0.51] mmol/l, p=0.04). To substantiate the observed effects it was experimentally proved the possibility of formation of strong complex compounds between the ions of magnesium and PPIs: the highest activity was demonstrated by rabeprazole and pantoprazole (coefficients of complex formation per unit of total organic carbon – 1.5 and 0.72, respectively) and the lowest – omeprazole and esomeprazole (0.04 and 0.09, respectively).Conclusion. A decrease of magnesium content in whole blood and formed elements in patients with paroxysmal AF on the background of ADSN was mediated by a six-week intake of pantoprazole in a dose of 20 mg 2 times a day for the treatment of concomitant erosion of the duodenum. When choosing PPIs for arrhythmological patients with concomitant gastro-duodenal pathology, it is advisable to be guided by the complexing activity of drugs with respect to magnesium ions: the most pronounced metal-ligand interaction with magnesium was shown by rabeprazole and pantoprazole, and the least – omeprazole and esomeprazole.

LRRC8A potentiates temozolomide sensitivity in glioma cells via activating mitochondria-dependent apoptotic pathway.

Chloride (Cl-), a primary anion in the extracellular fluid, plays an important role in a variety of physiological and pathological processes, such as cell apoptosis and proliferation. However, the information about Cl- in cancer cell apoptosis and chemoresistance is poorly understood. In the present study, we found that temozolomide (TMZ) treatment led to a decrease in intracellular concentration of Cl- ([Cl-]i) in both U87 and TMZ-resistant U87/R glioma cells. The decrease in [Cl-]i was more noticeable in U87 cells than in U87/R cells. Moreover, the expression of LRRC8A was reduced in U87/R cells compared with U87 cells. LRRC8A downregulation inhibited TMZ, induced the decrease in [Cl-]i and abolished the difference of [Cl-]i between U87 cells and U87/R cells. Knockdown of LRRC8A using small interfering RNA attenuated TMZ-induced U87 cell growth inhibition and apoptosis, while overexpression of LRRC8A by adenoviral infection enhanced the effect of TMZ on U87 and U87/R cell viability and apoptosis. Furthermore, LRRC8A downregulation inhibited TMZ-induced mitochondria-dependent apoptosis, including elevated Bcl-2 expression, reduced Bax expression, cytochrome c release, and caspase nine and caspase three activation. On the contrary, upregulation of LRRC8A augmented the activation of mitochondria-dependent apoptotic pathway in U87 and U87/R cells. In conclusion, this study demonstrates that LRRC8A potentiates TMZ-induced glioma cell apoptosis via promoting mitochondria-dependent apoptosis, suggesting that LRRC8A can be represented as a novel target for drug resistance treatment in glioma cells.

Ageing reduces the reproductive performance of male white shrimp Litopenaeus vannamei by altering sperm intracellular Ca2+ concentrations and interfering with sperm apoptosis

Although the effects of age on the reproductive performance of various invertebrates, including white shrimp Litopenaeus vannamei are increasingly well documented, the mechanisms manifesting these impacts remain poorly understood. To ascertain the mechanisms of age on reproductive performance, the sperm quality, intracellular contents of Ca2+, insemination and hatching rates, and status of sperm apoptosis in terms of the expression of key regulatory genes were investigated in 11 and 16 month old male L. vannamei. The aged male individuals (16 months) had lesser reproductive performance in terms of fertilization and hatching rates. In addition, fewer and less viable sperm were detected in aged shrimp, which may be due to the altered expression of apoptosis-related genes. Furthermore, the aged males had lesser intracellular contents of Ca2+ in the sperm which may decrease the capacity of these gametes to undergo a complete acrosome reaction. In general, due to the decrease in intracellular contents of Ca2+ and alterations in the process of apoptosis, aged L. vannamei have relatively lesser quality sperm and may, therefore, have lesser reproductive performance.

Loss of SLCO1B3 drives taxane resistance in prostate cancer.

Background:Both taxanes, docetaxel and cabazitaxel, are effective treatments for metastatic castration-resistant prostate cancer (mCRPC). However, resistance to taxanes is common. Our objective was to investigate mechanisms of taxane resistance in prostate cancer.Methods:Two docetaxel-resistant patient-derived xenografts (PDXs) of CRPC were established (PC339-DOC and PC346C-DOC) in male athymic nude mice by frequent intraperitoneal administrations of docetaxel. Next-generation sequencing was performed on PDX tissue pre- and post-docetaxel resistance and gene expression profiles were compared. [14C]-docetaxel and [14C]-cabazitaxel uptake assays in vitro and cytotoxicity assays were performed to validate direct involvement of transporter genes in taxane sensitivity.Results:Organic anion-transporting polypeptide (SLCO1B3), an influx transporter of docetaxel, was significantly downregulated in PC346C-DOC tumours. In accordance with this finding, intratumoural concentrations of docetaxel and cabazitaxel were significantly decreased in PC346C-DOC as compared with levels in chemotherapy-naive PC346C tumours. In addition, silencing of SLCO1B3 in chemo-naive PC346C resulted in a two-fold decrease in intracellular concentrations of both taxanes. Overexpression of SLCO1B3 showed higher sensitivity to docetaxel and cabazitaxel.Conclusions:The SLCO1B3 determines intracellular concentrations of docetaxel and cabazitaxel and consequently influences taxane efficacy. Loss of the drug transporter SLCO1B3 may drive taxane resistance in prostate cancer.

Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

Islet transplantation has become a promising treatment for type 1 diabetes. However, immunosuppressive drugs used today cause islet deterioration and modification strategies are necessary. But little is known about pharmacokinetics interactions and intracellular concentrations of immunosuppressive drugs in human islets. We determined the pharmacokinetics of tacrolimus and sirolimus in islets by measuring intracellular concentration after exposure alone or in combination at two different doses up to 48h. A quantification technique established in our laboratory using a Micromass Quattro micro API MS/MS-instrument with electrospray ionization was used. Islets function was measured by oxygen consumption rates. Presence of drug transporters OATP1B1 and ABCB1 and metabolizing enzyme CYP3A4 in islets were quantified using real-time quantitative PCR. Islets incubated with tacrolimus and sirolimus had a significant decrease in intracellular concentration of sirolimus compared to sirolimus alone. Reduced intracellular sirolimus concentration was followed by increased p70S6k phosphorylation suggesting preservation of the mTOR-signaling pathway. Drug transporters OATP1B1 and ABCB1 and enzyme CYP3A4 were expressed in human islets, but were not involved in the reduced sirolimus concentration by tacrolimus. These findings provide new knowledge of the drug interaction between tacrolimus and sirolimus, suggesting that tacrolimus has an inhibitory effect on the intracellular concentration of sirolimus in human islets.

P01.17 * ROLE OF PHOSPHATYDILCHOLINE-SPECIFIC PHOSPHOLIPASE C IN MODULATION OF CXCL12/CXCR4 AXIS IN A HUMAN GLIOMA CELL LINE

BACKGROUND: The CXCR4 chemokine receptor and its ligand CXCL12 are one of the most well studied chemokine systems in tumor biology. In gliomas CXCL12/CXCR4 axis regulates multiple mechanisms that sustain tumor growth as migration, proliferation, angiogenesis and is emerging as a novel biologically relevant target for brain tumor therapy. Previous studies, in our laboratory highlights a role of the phosphatydilcholine-specific phospholipase C (PC-PLC) in the regulation and expression of growth factor membrane receptors, suggesting a relationship between the metabolism of phosphatydilcholine (PC) and cell receptor status. AIM: Investigate the crosstalk between the chemokine receptor CXCR4 and PC-PLC and its implications in glioma cells growth. MATERIALS AND METHODS: U87MG, a glioblastoma cell line was treated with a conventional CXCR4 antagonist, AMD3100 (10 µM), or with a PC-PLC inhibitor, D609 (Xantate) (100 µM). Confocal Laser Scanning Microscopy (CLSM) and Western Blotting (WB) experiments were performed on cells to investigate protein expression and intracellular localization. Cell metabolism on both treated or untreated U87MG cells was evaluated by in vitro Nuclear Magnetic Resonance Spectroscopy (MRS). RESULTS: CLSM analyses highlighted for the first time an extensive co-localization of CXCR4 and PC-PLC on the plasma membrane of U87MG cells. Furthermore we observed a down-modulation of CXCR4 from cell membrane in response to both inhibitors, AMD3100 and D609, however D609 only induced a decrease in CXCR4 total protein content. Moreover, D609 treatment reduced PC-PLC expression, induced a reduction of pAKT and exerted a significative anti-proliferative effect on U87MG cells (49% inhibition). MRS analyses revealed alterations on U87MG metabolism, showing in D609-treated cells an increase of choline-containing metabolites (Glycerophosphocholine, GPCho; Choline, Cho) (increase of 77%) that could be correlated with activation of lipases and phospholipases that can induce an accumulation of neutral lipids associated with the inhibition of cell proliferation. D609 treatment also induced a significative decrease of intracellular concentration of Lactate (38%), and preliminary analyses on lactate deydrogenase (LDH) showed a decrement of the enzyme activity (30%) suggesting that inactivation of PC-PLC could impaired the glycolysis pathway in U87MG. CONCLUSIONS: In this study we identified for the first time an interaction between the chemokine receptor CXCR4 and the enzyme PC-PLC on a glioma cell line. The specific inhibition of PC-PLC modulates CXCR4 receptor localization on cellular membrane and protein expression. Our findings suggest that the inhibition of PC-PLC could represent a new molecular approach in CXCR4-overexpressing glioma cells.

Steatotic Rat Hepatocytes in Primary Culture Are More Susceptible to the Acute Toxic Effect of Acetaminophen

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in humans. Non-alcoholic fatty liver disease is the most frequent chronic liver disease in developed countries. The aim of our work was to compare the effect of APAP on intact rat hepatocytes and hepatocytes isolated from steatotic liver in primary cultures. Male Wistar rats were fed with standard diet (10 % energy from fat) and high-fat diet (71 % energy from fat) for 6 weeks and then hepatocytes were isolated. After cell attachment, APAP (1; 2.5; 3.75 and 5 mM) was added to culture media (William's E medium) and hepatocytes were cultured for up to 24 hours. APAP caused more severe dose-dependent damage of steatotic hepatocytes as documented by increased release of lactate dehydrogenase (LDH) and LDH leakage, decreased activity of cellular dehydrogenases (WST-1 test) and reduced albumin production. Intact steatotic hepatocytes contained lower amount of reduced glutathione (GSH). Treatment with APAP (1 and 2.5 mmol/l) caused more pronounced decrease in GSH in steatotic hepatocytes. ROS (reactive oxygen species) formation after 24-hour incubation was significantly higher in fatty hepatocytes using APAP at concentration of 3.75 and 5 mmol/l. Interleukin 6 (IL-6) production was elevated in 2.5 mM APAP-treated nonsteatotic and steatotic hepatocyte cultures at 8 hours, compared to appropriate controls. In conclusions, our results indicate that steatotic hepatocytes exert higher sensitivity to the toxic action of APAP. This sensitivity may be caused by lower content of GSH in intact steatotic hepatocytes and by more pronounced APAP-induced decrease in intracellular concentration of GSH.

Effect of vitrifying solutions, ethylene glycol and 1, 2-propandiol, on intracellular K+ and lactate dehydrogenase (LDH) of hepatocytes in primary culture

Shortage of donor liver tissue for the isolation of hepatocytes requires the development of improved cryopreservation techniques for long-term storage of these cells for more efficient use. The aim of this study is to compare two cryoprotectants for preserving living cells in culture by vitrification. The viability of hepatocytes after cryopreservation was determined by the intracellular concentration of potassium (K+) and lactate dehydrogenase (LDH). Cryomicroscopy was used to check the state of vitrification and morphology of these cells subjected to the process of freezing and thawing. This experiment made it possible to observe an early vitrification with 40% of Ethylene glycol (EG) or 1,2-propanedio (1, 2-PD) to reach the final vitrified state with 60%. The treatment of these cells in culture with solutions containing EG or 1, 2-PD revealed a failure in functional properties of the plasma membrane. Thus, a decrease of intracellular concentration of LDH and K+ was noticeable. This decrease was more significant when it came with 60% EG or 1, 2 - PD. The use of a cryopreservation solution additionally containing NaCl, KCl, Ca2+ and sucrose and the treatment of hepatocytes in culture at +4°C greatly minimized the functional impairment of the plasma membrane. These results suggest that the osmo-diffusional mechanism is at the basis of membrane damage letting out LDH and K+ into the extracellular compartment. However, this mechanism can be controlled by a selection of treatment solutionparameters such as temperature, osmolarity and electrolytes concentration while taking the cryoprotectants coefficient of permeability into consideration. Key words: Hepatocyte, cryopreservation, cryoprotectant, vitrified state, vitrification.

Creatine Phosphate: Pharmacological and Clinical Perspectives

Since the 1970s, extensive experimental and clinical research has demonstrated that relevant reductions of creatine phosphate (CrP) or phosphocreatine availability occur in a wide spectrum of pathophysiological situations. A decrease in intracellular concentrations of creatine (Cr) and CrP results in a hypodynamic state of cardiac and skeletal muscle pathology. Many experimental and clinical studies have evaluated the possibility to improve cardiac and skeletal muscle performance by exogenous administration of CrP. Furthermore, many experimental studies have shown that CrP may play two important roles in the regulation of muscle energetics and work. First, CrP maintains local adenosine triphosphate pools and stabilizes cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic hearts, protects the sarcolemma of cardiac cells against ischemic damage, decreases the frequency of arrhythmias, and increases post-ischemic recovery of contractile function. Recent research on CrP has demonstrated positive therapeutic results in various clinical applications. These benefits have been applied in several pathological conditions, such as heart failure, acute myocardial ischemia, chronic ischemic heart disease, cardiac surgery, skeletal muscle hypotonotrophy, and cerebral ischemia. This review describes the CrP shuttle, pathophysiological basis of the supplementation of CrP, and its therapeutic effects in multiple clinical conditions. The major aim is to summarize results of the intense research carried out over 40 years to provide evidence to support the adjunctive use of CrP in many pathological conditions that may target cellular energy impairment; thus, increasing energy metabolism.

Models to Predict Unbound Intracellular Drug Concentrations in the Presence of Transporters

Knowledge of free drug intracellular concentration is necessary to predict the impacts of drugs on intracellular targets. The goal of this study was to develop models to predict free intracellular drug concentrations in the presence of apical efflux transporters. The apical efflux transporter P-glycoprotein (P-gp), encoded by human gene multidrug resistance 1 (MDR1), was studied. Apparent permeabilities for 10 compounds in Madin-Darby canine kidney (MDCK) and MDR1-MDCK cell monolayers were obtained experimentally. Six of these compounds were evaluated additionally in the presence of the P-gp inhibitor cyclosporine A. A three-compartment model was developed, and passive and apical efflux clearances (CL(d) and CL(ae), respectively) were estimated. Endogenous canine transporters also were delineated. The three-compartment model was unable to simulate experimentally observed lag times and exhibited systematic bias across the simulations. Next, a five-compartment model with explicit membrane compartments was developed. This model resulted in lower systematic errors and simulated the lag time observed experimentally. Apical efflux was modeled out of the cell or out of the membrane. The five-compartment model with apical efflux out of the membrane predicted marked differences in unbound intracellular concentrations between the apical-to-basolateral and the basolateral-to-apical directions. Upon apical drug addition, large decreases in intracellular concentrations were observed with the efflux transporter. No such difference was predicted upon basolateral drug addition. This is consistent with experimental differences in the impact of P-gp on hepatic and brain distribution and supports the hypothesis that apical efflux occurs out of the apical membrane.

Pseudomonas aeruginosa PAO1 Preferentially Grows as Aggregates in Liquid Batch Cultures and Disperses upon Starvation

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.

Na–Picotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 micromol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P(i)) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P(i) cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P(i) cotransporter expressed in these primary cell cultures could be identified as Na-P(i) type I. Although the exact physiological role of Na-P(i) type I still is unresolved, these experiments demonstrate that apical Na-P(i) type I activity is increased at the onset of ATP depletion in MTAL cells.

Decrease in Intracellular Concentration Causes the Shift in Km Value of Efflux Pump Substrates

Passive permeability and active efflux are parallel processes in transcellular flux. Therefore, the observed kinetics of a transporter substrate depends on both of these factors. The transporter expression has been shown to affect both the apparent K(m) and V(max) values. Kinetic parameters can be obtained from various experimental settings, but these do not necessarily reflect the situation in transcellular flux. Kinetic absorption models need reliable estimates of saturable kinetics when accurate in silico predictions are to be made. The effect of increasing P-glycoprotein expression on apparent transport kinetics was studied using quinidine and digoxin as model compounds. The intracellular concentrations of drugs during the transport process were also measured. A dynamic simulation model was constructed to study the observed data. The apparent K(m) and V(max) values increased as the P-glycoprotein expression increased. Simulations reproduced the shift in both kinetic parameters as a function of efflux pump expression. In addition, the apparent K(m) value showed a strong inverse relationship to the passive permeability. In contrast, the apparent V(max) value reached a maximum at intermediate passive permeability and declined above and below this passive permeability. The true V(max) and K(m) values were never reached. The shift in K(m) was assigned to a decrease in intracellular concentration at the P-glycoprotein interaction site with both experimental and simulation data. In conclusion, the apparent kinetic parameters in transcellular permeability assays depend on passive permeability and efflux pump activity. Therefore, parameters that are obtained from in vitro assays should be cautiously applied to in vivo predictions.

Bradykinin activates ADP-ribosyl cyclase in neuroblastoma cells: Intracellular concentration decrease in NAD and increase in cyclic ADP-ribose

ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastoma × glioma NGPM1-27 hybrid cells was measured by monitoring [ 3H] cyclic ADP-ribose (cADPR) formation from [ 3H] NAD +. Bradykinin (BK) at 100 nM increased ADP-ribosyl cyclase activity by about 2.5-fold. Application of 300 nM BK to living NGPM1-27 cells decreased NAD + to 78% of the prestimulation level at 30 s. In contrast, intracellular cADPR concentrations were increased by 2–3-fold during the period from 30 to 120 s after the same treatment. Our results suggest that cADPR is one of the second messengers downstream of B 2 BK receptors.

Antisense oligodeoxynucleotides directed against a novel angiotensinogen mRNA-stabilizing protein reduce blood pressure in spontaneously hypertensive rats

We have previously reported that hypertension in the young spontaneously hypertensive rat (SHR) is associated with an elevation in tissue angiotensinogen and a novel polysomal protein known to stabilize angiotensinogen mRNA. In our current study we determined the role of the mRNA-stabilizing protein in the regulation of tissue angiotensinogen expression and mean arterial pressure (MAP) in the SHR utilizing antisense oligodeoxynucleotide (AON) inhibition. Three AONs (RNASTAAS1, position 31-50; RNASTAAS2, position 21-40; RNASTAAS3, position 143-162 of the cDNA coding for the polysomal protein) were administered intravenously (dose 450, 900, and 1,800 microg/kg; 1 dosage/day over 3 days) in conscious, chronically instrumented male SHRs at the age of 7 wk. Control SHRs received corresponding scrambled oligodeoxynucleotide sequences (SCR1, SCR2, SCR3). Each animal received the increasing dose schedule. RNASTAAS2 resulted in a reduced expression of the polysomal protein to 21% (liver), 12% (brain), 27% (heart), 18% (renal cortex), and 22% (renal medulla) of control. Angiotensinogen expression was inhibited to 54% (liver), 41% (brain), 68% (heart), 52% (renal cortex), and 74% (renal medulla) compared with control SHRs. Decreases in plasma concentrations of angiotensinogen and plasma renin activities were associated with a significant decrease in MAP from 147 +/- 6 mmHg (after SCR2) to 106 +/- 4 mmHg after RNASTAAS2. The effects of the two other AONs on MAP were less (RNASTAAS1, -31 mmHg; RNASTAAS3, -16 mmHg) with corresponding decreases in mRNAs coding for angiotensinogen and the polysomal protein. A significant decrease in intracellular concentrations of the polysomal protein accompanied AON inhibition. The magnitude of effects (-15 to -41 mmHg) was comparable to the effects of captopril (100 mg x kg(-1) x day(-1) for 3 days: -32 mmHg) and an AT(1) receptor antagonist (L-158809, 1.5 mg x kg(-1) x day(-1) for 3 days: -36 mmHg). These data suggest an important role of the mRNA-stabilizing protein for hepatic and extrahepatic angiotensinogen expression and MAP in the SHR.

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H 2O 2 and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.

Apoptosis induced by hydrogen peroxide is mediated by decreased superoxide anion concentration and reduction of intracellular milieu

Hydrogen peroxide (H 2O 2) is considered to be a mediator of apoptotic cell death but the mechanism by which it induces apoptosis is unclear. Here, we show that cells undergoing apoptosis from exposure to H 2O 2 display a significant decrease in intracellular concentration of superoxide (O − 2) which is associated with a reduction of the intracellular milieu, as measured by an increase in the GSH/GSSG ratio and a decrease in intracellular pH. The notion that a decrease in intracellular O − 2 concentration triggers apoptosis is supported by the observation that H 2O 2-mediated apoptosis could be retarded in cells in which the intracellular O − 2 concentration is maintained at or above the cellular baseline level by inhibition of the major O − 2 scavenger superoxide dismutase (Cu/Zn SOD). Taken together, our observations indicate that a decrease in the intracellular O − 2 concentration, reduction and acidification of the intracellular milieu constitute a signal for H 2O 2-mediated apoptosis, thereby inducing a reductive as opposed to an oxidative stress.

Modulation of albumin gene expression by amino acid supply in rat liver is mediated through intracellular concentration of pyridoxal 5′-phosphate

Rats were nourished by infusion of total parenteral nutrition solutions containing 0% or 3.3% amino acids for 7 days. The level of albumin mRNA in the liver of amino acid-infused rats was found to be several fold higher than that in the liver of amino acid-depleted rats. Expression of albumin gene is known to be regulated by tissue-specific transcription factors such as HNF-1 and C EBP . We determined the binding activities of liver nuclear extracts to the HNF-1- and C EBP -binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of amino acid-infused rats were greater than those of amino acid-depleted rats. In view of our recent finding that vitamin B 6 modulates albumin gene expression through inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate, we determined the intracellular concentrations of vitamin B 6 derivatives. We found that the concentration of pyridoxal phosphate in the liver of amino acid-infused rats was decreased to almost half of that of amino acid-depleted rats, whereas the concentration of pyridoxamine phosphate was increased in the opposite direction. These observations suggest that an increase in albumin mRNA level in the liver of amino acid-infused rats may be caused by a decrease in intracellular concentration of pyridoxal phosphate, which in turn relieves inactivation of tissue-specific transcription factors.

Agonists and Antagonists for Metabotropic Glutamate Receptors

&lt;p&gt;Glutamate (Glu) is a prominent excitatory amino acid (EAA) neurotransmitter in the central nervous system, and it exerts its actions at a number of different receptor subclasses. lonotropic Glu receptors, such as the N-methyl-D-aspartate (NMDA), 2- amino-3-(5-methyl-3-hydroxyisoxazol-4-yl)propanoic acid (AMPA), and kainic acid subtypes, are coupled to ion channels, and transduce signals by gating the flow of calcium and sodium ions into the cell. Metabotropic EAA receptors (mGluRs) are coupled through G-proteins to intracellular enzymes such as phospholipase C (PLC) and adenylate cyclase (AC). Agonist activation of mG!uRs coupled to PLC produces an increase in the intracellular concentrations of inositol triphosphate (IP3) and diacylglycerol (DAG), while agonist activation of mG!uRs coupled to AC produces a decrease in intracellular concentrations of cyclic­ adenosine monophosphate (cAMP). At least eight distinct metabotropic receptor proteins (mGluRl-8) have been identified, and these can be divided into three groups. Group 1 mG!uRs consist of mG!uRl and mG!uR5, and are PLC-coupled receptors. Group 2 mG!uRs include mG!uR2 and mG!uR3, and are negatively coupled to AC. &lt;/p&gt;&lt;p&gt; Group 3 mG!uRs consist of mGluR4, mG!uR6, mGluR 7and mGluR8, and they are also negatively coupled to AC. Many studies are currently underway to identify novel agonists and antagonists for these receptors. The discovery of such compounds will be an important step toward understanding the physiological and pathophysiological consequences of mG!uR activation in vivo, and will ultimately be essential in defining the therapeutic potential of selective mGluR agents. Novel aspects of the medicinal chemistry and pharmacology of metabotropic receptors are discussed in this review.