Acetazolamide Inhibits Electrogenic Sodium Bicarbonate Flux through kNBC1. Molecular Mechanisms and Computer Simulations.
GROSS E
Department of Physics and Astronomy, University of Arkansas, Fayetteville, AR, 72703, USA.
Background: The HCO3- : Na+ cotransport stoichiometry of the electrogenic sodium bicarbonate cotransporter kNBC1 determines the reversal potential (E(rev)) and thus the net direction of transport of these ions through the cotransporter and thus across the cell membrane (i.e. efflux or influx). Phosphorylation of kNBC1-Ser(982) in the carboxy-terminus of kNBC1, by cAMP-protein kinase A (PKA), shifts the stoichiometry from 3 : 1 to 2 : 1. Downstream of Ser(982) in kNBC1 is a D986NDD motif. A homologous motif (D887ADD) in the carboxy-terminus of the anion exchanger AE1 binds to carbonic anhydrase II (CAII). We thus studied the binding of kNBC1 to CAII and the role of the D986NDD motif in this protein-protein interaction.
Methods: We used isothermal titration calorimetry to measure the binding constant of CAII to kNB1 and Ussing chamber electrophysiology apparatus to measure the electogenic flux of sodium and biacrboanet through the cotransporter.
Results: In isothermal titration calorimetry experiments, CAII was found to bind to wt kNBC1-Ct with a K(D) of 160 +/- 10 nM. Acetazolamide inhibited the short-circuit current through the cotransporter by 65 +/- 6 % when the latter operated in the 3 : 1 mode, but had no effect on the current in the 2 : 1 mode.
Conclusions: We propose a model in which CAII, when bound to kNBC1, builds a high local concentration of bicarbonate in the vicinity of the cotransporter’s anionic binding site. Phosphorylation of kNBC1 by PKA removes CAII and as a result lowers local bicarbonate concentration and shifts the stoichiometry to 2:1. This model is also supported by computer simulations with a six-state transport binding scheme and electric field modulated binding constants and membrane translocation steps.
|
Infusion Monitoring of Anesthetic Drugs: Propofol in Respiratory Gas
GROSSHERR1, HENGSTENBERG2, VARADARAJAN B2, DIBBELT3, SCHMUCKER1, GEHRING1
1Dept. of Anaesthesiology and 3 Central Laboratory, Department of Clinical Chemistry; UK S-H, Campus Luebeck, Luebeck, 2 Research Unit, Draegerwerk Drägerwerk AG Co. KGaA, Germany
Background: The continuous monitoring of propofol concentration in breathing gas by an electrochemical sensor (ELCH) allows a determination of changes in plasma concentration. The following animal study should test the hypothesis whether a bolus of propofol induces time related changes in breathing gas, that can be compared to the changes in calculated plasma and effect site propofol concentration.
Methods: After the approval of the regional authority 8 pigs in a healthy condition were investigated. After propofol free induction propofol was applied as a bolus (4 mg/ kg body weight) at 0 and 30 minutes and with a continuous infusion of propofol (9,6 mg/ kg body weight x h) simultaneously. The time to peak after the propofol bolus was determined for the concentration of propofol in breathing gas by ELCH and in plasma and effect site (MARSH –model). The non-parametric Mann – Whitney U-test was used to compare them (*: p <5%).
Results: In 4 of the 8 animals the first and in all 8 animals the second bolus was detected as a peak of the propofol concentration in breathing gas measured by ELCH (see table 1).
Treatment
|
Number of animals
|
t (breathing gas)
|
t (plasma)
|
t (effect site)
|
|
(1)
|
(s)
|
(s)
|
(s)
|
Bolus 0 min
|
4 of 8
|
321
|
25
|
115
|
Bolus 30 min
|
8 of 8
|
354 (*)
|
20
|
110
|
The ELCH resolved a bolus as a peak in breathing gas concentration after propofol has been given already for 30 min as a continuous infusion. At the beginning of a propofol anaesthesia a bolus was indicated as a concentration change and not as a peak in 50% of the animals.
Conclusions: Differences in time to peak in breathing gas compared to effect site and plasma illustrate how fast propofol is distributed to different compartments. Ongoing investigations using other methods for real time measurement of propofol in breathing gas will help to further separate possible influences of the measured appearance of propofol in breathing gas due to equipment from influences due to physiology.
Authors’ disclosure statement:
There are financial obligations between the authors and the Drägerwerk AG Co. KGaA, Germany
|
Increased repolarization reserve as a new anti-arrhythmic principle
GRUNNET M, DINESS TG, OLESEN SP, HANSEN RS
NeuroSearch A/S and Department of Biomedical Sciences, University of Copenhagen, Denmark
Background: A healthy human heart will beat approximately 3.000.000.000 times during a normal lifespan without any disturbances. Any electrical deviation from this regular pathern is termed an arrhythmia. Arrhythmias can result in anything from minor palpations to sudden cardiac death. A number of arrhythmias are due to malfunction of a cardiac ion channel named HERG1. This channel is essential for appropriate repolarisation of the cardiac action potential. It is well known that unintended inhibition of the HERG channel is pro-arrhythmic. We have therefore developed a new concept of HERG channel activation an investigated the anti-arrhythmic properties of such activators.
Methods: The experimental approach is translational. Patch-clamp experiments have been conducted applying native cardiomyocytes or by using heterologous expression systems in oocytes and mammalian cells of the HERG channel. In addition, ex vivo Langendorff experiments and in vivo studies in both conscious and anaestezied animals have been conducted.
Results: A number of anti-arrhythmic properties was demonstrated for the HERG channel activators. In native cardiomyocytes action potential was abbreviated and post-repolarisation refractory period was increased significantly. Further HERG channel activation rendered the cardiomyocytes more resitance towards early-afterdepolarisations (EAD’s). In intact hearts investigated in Langendorff set-up extrasystolis could be prevented by HERG channel activation and a tendency towards less dispersion of the length of action potentials was observed. In in vivo studies HERG channel activation could prevent drug induced prolongation of the QT interval and significantly reduce the incidence of extrasystolis and ventricular fibrillations.
Conclusions: In conclusion we believe it is demonstrated that under certain circumstances, activation of the cardiac HERG channel can be a new antyi.-arrhythmic principle
|
Treatment of ovarian cancer cells with drug combinations targeting ErbB receptor tyrosine kinases and fatty acid synthase
GRUNT TW1,3, WAGNER R1, SHABBIR W1, BERGER W2, MARIAN B2, GRUSCH M2, ZIELINSKI C1,3, LUPU R4
1Div. Oncology and 2Inst. Cancer Res., Dept. Med. I, Med. Univ. Vienna, 3Ludwig Boltzmann Cluster Oncology, Vienna, Austria; 4Dept. Med., Evanston Northwestern Healthcare Res. Inst., Evanston, IL, USA.
Treatment of ovarian cancer (OC) is still suboptimal necessitating the search for novel therapies. In normal tissue, the key lipogenic enzyme fatty acid synthase (FASN) converts dietary carbohydrates to triglycerides, whereas in cancer, FASN represents a metabolic oncogene and produces phospholipids for membrane microdomains (lipid rafts) that accommodate clusters of receptor tyrosine kinases including Epidermal Growth Factor Receptor (EGFR, ErbB1) and ErbB2 (HER-2/neu) thus setting the stage for signal initiation. Importantly, both FASN and ErbBs are overexpressed in tumors including OC and represent drugable targets. Recent data suggest a link between FAS and ErbB2 in breast cancer. In OC, the relationship between FAS and ErbB is still elusive. Therefore, we examined the effect of FAS and ErbB inhibition on A2780 ovarian cancer cells (OCC). A FASN inhibitor (C75) and 2 irreversible ErbB inhibitors (EKB-569, Wyeth; CI-1033, Pfizer) inhibit growth of OCC (MTT assay - IC50: C75=22 µM; EKB-569=5,1 µM; CI-1033=3,7 µM). Interestingly, C75 synergizes with EKB-569 or CI-1033 in cell growth inhibition (p<0.01) suggesting cooperation between FAS and ErbB pathways during OCC growth. RT-PCR, real-time analysis and Western blotting revealed that C75 slowly and concordantly reduces EGFR mRNA, protein and activity in OCC. Thus, C75 silences EGFR gene expression at transcriptional levels without directly affecting EGFR signaling. C75 caused deprivation of overall and phosphorylated ErbB2 protein, but failed to diminish ErbB2 mRNA. Although C75 post-transcriptionally represses ErbB2, it does not directly disrupt ErbB2 activity. C75 also caused shut-down of FAS mRNA and protein. On the other hand, EKB-569 abolishes EGFR and ErbB2 protein expression and phosphorylation, but only weakly depresses mRNA levels. Strikingly, EKB-569 also represses FAS mRNA and protein. CI-1033 also failed to affect EGFR and ErbB2 transcript levels, but compromised EGFR activity (but not EGFR protein expression) and ErbB2 protein expression and function. Generally, CI-1033 reduced ErbB function rather than ErbB protein expression. Moreover, CI-1033 correspondingly down-regulated FAS mRNA and protein. Our data indicate that ErbB and FAS pathways mutually interact with each other in OCC. Thus, interference with the FAS and the ErbB systems effectively abrogates their oncogenic activities and may be exploited for OCC treatment. Supported by ‚Initiative Krebsforschung’, Vienna, Austria.
|
The PI3K/AKT pathway determines EGFR/HER/ErbB drug efficacy in breast cancer cells.
SHABBIR W1, BRÜNNER-KUBATH C1, GRUSCH M2, BERGER W2, MARIAN B2, WAGNER R1, LÖTSCH D2, ZIELINSKI C1,3, GRUNT TW1,3
1Div. Oncology and 2Inst. Cancer Res., Dept. Med. I, Med. Univ. Vienna, 3Ludwig Boltzmann Cluster Oncology, Vienna, Austria
ErbB transmembrane proteins belong to the family of tyrosine kinase receptors. Four members have been described: ErbB1 (EGFR), ErbB2, ErbB3, and ErbB4. ErbB1 and 2 are overexpressed/hyperactivated in many tumors, including ovarian and breast cancer. They stimulate carcinogenesis and malignant progression, and confer unfavorable prognosis. Clinical success has recently been obtained by targeting ErbB2 in ErbB2+ breast cancer. However, only 30% of ErbB2+ breast cancers respond to targeted ErbB2 blockade and most of the responders develop secondary resistance. The situation is even worse, when ovarian cancer is considered. Unfortunately, predictive markers for assessing ErbB inhibitor sensitivity/resistance are still widely lacking. Using MTT assay and Western blotting we examined the effects of the novel irreversible ErbB inhibitor pelitinib (EKB-569, Wyeth) on the growth activity and on ErbB-triggered signaling in 11 human breast and 11 human ovarian cancer cell lines. SKBR3 and T47D were identified as most sensitive and most resistant breast cancer cell lines, respectively. In contrast, the sensitivity of the ovarian cancer cell lines did not vary as much. Interestingly, the antiproliferative activity of the drug did not correlate with EGFR and ErbB2 protein levels. Moreover, drug-dependent inhibition of EGFR, of ErbB2 and of ERK1/2 phosphorylation was seen in both pelitinib-sensitive and pelitinib-resistant cells indicating that inhibition of ERK1/2 downstream signaling is not sufficient for drug-dependent growth arrest. In contrast, phosphorylation of ErbB3 at Tyr1289, of AKT at Ser473 and at Thr308, and of GSK3beta at Ser9 was blocked only in the sensitive, but not in the resistant cells. Moreover, ectopic expression of constitutively active AKT induced resistance to pelitinib in SKBR-3 cells. Conversely, pelitinib rapidly induced phosphorylation of PTEN at Ser380 in sensitive, but not in resistant cells. Taken together, our data suggest that ErbB3/PI3K/AKT, but not ERK1/2 signaling plays crucial roles in determining sensitivity/resistance of the cells against the irreversible dual EGFR/ErbB2 inhibitor pelitinib. Therefore, we propose that drug-mediated downregulation of phospho-AKT and phospho-ErbB3 levels might be useful surrogate markers for ErbB drug efficacy in breast cancer. Supported by ‚Initiative Krebsforschung’, Vienna, Austria.
|
Expression of Fas on human T lymphocytes under stimulation with Borrelia burgdorferi sensu lato.
GRYGORCZUK S1, OSADA J2, ŚWIERZBIŃSKA R1, CHMIELEWSKI T3, ZAJKOWSKA J1, KONDRUSIK M1, PANCEWICZ S1, DĄBROWSKA M2, TYLEWSKA-WIERZBANOWSKA S3
1Department of Infectious Diseases and Neuroinfections, Medical University in Białystok, Poland; 2Department of Haematologic Diagnostics, Medical University in Białystok, Poland; 3Department of Rickettsiae, Chlamydiae and Zoonotic Spirochetes, National Institute of Hygiene, Warsaw, Poland
Background: Pathogenesis of late Lyme disease (LD) remains controversial, and the possibility of 1) autoimmune complications after elimination of Borrelia burgdorferi sensu lato (B.b.) infection and 2) impaired immunity preventing pathogen elimination, has been proposed. As apoptosis of activated T lymphocytes, on the Fas receptor pathway, is essential in control of inflammatory/immune response, its abnormalities in contact with B.b. could lead to both of these conditions.
Methods: We measured Fas expression on peripheral blood CD3+ and CD4+ lymphocytes under stimulation with B. burgdorferi s.l. Peripheral blood mononuclear cells were derived from 23 patients with late LD (18 with Lyme arthritis - LA and 5 with neuroborreliosis - NB) and 13 healthy persons (controls, C). Cells were incubated for 48 hours without stimulation (neg.) or with the suspension of inactivated Borrelia burgdorferi spirochetes: B.afzelii VS 46110, B.garinii 20047 or B.burgdorferi sensu stricte B-31, with bacteria to PBMC ratio 10:1, as antigenic stimulation. Fas expression on CD3+ (for all studied subjects) and CD4+ (for 8 subjects in LA, 4 in NB and 8 in C group) cells was measured by flow cytometry with FITC-labeled anti-Fas monoclonal antibody.
Results: Expression of Fas on CD3+ and CD4+ cells increased with age. When corrected for age, there was no difference between between LD and C groups. In LD patients, Fas expression did not depend on clinical form and duration of the disease. Median Fas expression increased significantly (p < 0,05) under stimulation with any of the B.b. strains: on CD3+ cells from 34% to 41-42% and on CD4+ from 23% to 24-26%. For CD3+ the increase was comparable in LA, NB and C groups, while for CD4+ cells it was signifiacant only in LA group.
Conclusions: Exposition to B.b causes moderate, but significant, increase of Fas death receptor on CD3+ and CD4+ peripheral blood limfocytes, which may render these cells more susceptible to apoptosis. Possible role of this phenomenon in pathogenesis of LD requires further study.
|
Dopamine transporter as the target and carrier of illicit and therapeutic drugs – PK/PD approaches to develop MAGIC BULLETS for cocaine abuse
GU H
The Ohio State University College of Medicine, Columbus, OH, USA
Background: Cocaine is a powerful psychostimulant and an addictive drug of abuse. There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies support the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to expectations, DAT knockout mice (DAT-KO), and SERT or NET knockout mice still display the rewarding property of cocaine. These studies indicate that none of these transporters are required for the cocaine effects and led to the re-evaluation of the DA hypothesis and the proposal of redundant reward pathways. However, the knockout mice have very significant adaptive changes during development to compensate for the complete absence of a critical protein, which might have altered how cocaine produces its effects in these mice.
Methods: To study the role of DAT in cocaine reward, we have engineered a functional but cocaine-insensitive mutant of DAT and generated a knock-in mouse line carrying this DAT mutant (DAT-CI mice). Normal doses of cocaine still block SERT and NET but have little effect on DAT in these mice which provide a unique tool to study the role of DAT in mediating cocaine effects. We also used an Adenyl Associate Virus (AAV) vector to re-introduce the wild type DAT back into specific brain regions of DAT-CI mice to study whether cocaine responses can be restored and what brain regions are critical for which cocaine responses.
Results: In DAT-CI mice, cocaine did not elevate extracellular DA in the nucleus accumbens (NAc), cocaine did not stimulate locomotor activity but suppressed it, and cocaine failed to produce reward as measured by conditioned place preference and by drug self administration. In contrast, amphetamine, another psychostimulant, was able to stimulate locomotor activity and produce reward, indicating that the reward system functioned well in these mice. In addition, re-introducing wild type DAT back into the brains of fully developed DAT-CI mice restored the ability of cocaine to stimulate locomotor activity and to produce conditioned place preference.
Conclusions: Our results indicate that cocaine blockade of DAT is required for the stimulating and rewarding effects of cocaine in mice with a functional DAT. While cocaine can produce reward in mice without DAT but it is through a mechanism different from that in normal mice. Therefore, under some abnormal conditions, possibly when the DA system is defective, cocaine may produce reward by interacting with targets other than DAT. Furthermore, our results suggest that drugs that prevent cocaine binding to DAT should reduce the stimulating and rewarding effects of cocaine and thus may be effective in treating cocaine addiction.
MAGIC BULLETS development: We are now collaborating with other investigators to screen large chemical libraries for MAGIC compounds that prevent cocaine binding while still allow transport. We are also working on the natural esterases responsible for cocaine degradation to develop a MAGIC enzyme that significantly reduce the bioavailability of cocaine.
|
Drugs Legeslation and Regulation in Pakistan
GUL R, IFTIKHAR ALAM KHATTAK
Department of Human Nutrition, Faculty of Nutrition Sciences, NWFP Agricultural University, Peshawar, NWFP-Pakistan, Email: romanticadil@yahoo.com
Objectives: The present study focused on data regarding the current situation of drugs regulation in Pakistan, its developmental history and other related issues.
Introduction: Pakistan is committed to the goal of health for all, inspired by the principle of social equity. To achieve this, the government is taking all possible measures in general, and drugs in, particular. National drugs policy emphasizes to ensure the availability, efficacy, safety and quality of the essential drugs in affordable Prices. Essential drugs are those which help in combating diseases, maintaining and improving the health status of the population. Government is also emphasizing to ensure the availability and acceptability of drugs in the country, to protect the public from hazards of substantial and unsafe drugs. To achieve this goal they are trying to develop skillful persons in the drugs manufacture fields, so they developed an operational and applied research in the field of pharmaceuticals.
Materials: Data about the drugs regulation in Pakistan was collected using the internet database and other published materials. Wherever needed, personal interviews with the concerned personals or other communication means were used for data collection. The information collected were arranged and compiled in a proper sequence.
Results & Discussion: Pakistan has a fairly modern legislation namely the Drugs Act 1976. Under this act rules have been formed on the various aspect of drugs control. It provides a system of licensing of all manufacturing houses and registration of the finished drugs to ensure efficacy, safety and quality of the marketing drugs. A board has been established for the export, import and quality control on federal and provincial bases. The quality is controlled by inspectors and laboratory services. The laws have been considered to be fairly modern in favor of public safety. From production and marketing view point, the government extends full support to the drug producers and drugs dealers. Incentives are provided to the hospitals and the local industry. About 80% of the drugs are locally prepared by 285 companies including 25 multinational and trying for self sufficiency. To ensure the quality, 8 inspectors on federal whereas 81 inspectors are working in provincial setup.
Conclusion & Recommendations: The traditional system of medicines is not properly regulated and hence efforts are made to regulate it through law with a view to their rationalization, to improve standard and for the protection of the public from hazards. Comprehensive public information should be launched to enhance under standing and acceptance of the essential drugs concept by the health professionals. For the selection of essential drugs, the ease in availability of essential and genuine drugs should be ensured by the government.
|
Dostları ilə paylaş: |
