pharmacological research Trends in pharmacological research

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pharmacological research Editor Prof. Viktor BAUER, MD., DSc. Consulting Editors Michal DUBOVICKÝ, PhD. Assoc. Prof. Magda KOUŘILOVÁ, PhD. Mojmír MACH, PhD. Jana NAVAROVÁ, PhD. Prof. Radomír NOSÁĽ, MD., DSc. Ružena SOTNÍKOVÁ, PhD.

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BRATISLAVA 2008

Trends in Pharmacological Research ISBN 978–80–970003–7–0 Published by Institute of Experimental Pharmacology, SASc. Dúbravská cesta 9, SK-841 04 Bratislava, Slovak Republic fax: +421-2-59477 5928 • e-mail: [email protected] Printed in Slovak Republic Cover, Interior Design & Typesetting Mojmir Mach, PhD.

Copyright © 2008 Institute of Experimental Pharmacology All rights reserved. No part may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or ortherwise, without prior written permission from the Copyright owners.

pharmacological research Table of Contents

Editorial V. Bauer

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Brief history of the Institute of Experimental Pharmacology R. Nosáľ

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Trends in studies of drug metabolism and of related drug–drug interactions P. Anzenbacher, E. Anzenbacherová

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Smooth muscle tissues as models for study of drug action V. Bauer, R. Sotníková, V. Nosáľová, J. Navarová, Š. Mátyás, V. Pucovský, V. Rekalov, K. Szőcs, J. Nedelčevová, Z. Kyseľová, V. Dytrichová, J. Fatyková, M. Kollárová, L. Máleková, M. Srnová, Z. Stojkovičová, G. Tóthová 15 New ways of supplementary and combinatory therapy of rheumatoid arthritis (RA) by synthetic and natural substances with antioxidant properties: New perspectives for routinely administered drugs in RA K. Bauerová, S. Poništ, K. Valachová, D. Mihalová, L. Šoltés, D. Komendová, V. Tomeková, M. Štrosová, P. Gemeiner, G. Poli

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Effect of cytochrome P450 induction on drug disposition in isolated rat liver preparation Š. Bezek, M. Kukan, T. Trnovec

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Reflection on animal modeling of human cardiac diseases in preclinical pharmacology: From measurements of coronary blood flow and cardiac function to sophisticated approaches of protease-catalyzed soluble cardiac protein expression with experimental myocardial infarction J. Dřímal, V. Knezl, A. Babulová, L. Bacharová, F. Borovičová, M. Dravecká, P. Gibala, J. Gvozdjak, A. Gvozdjaková, J. Jakubovský, M. Kittová, D. Magna, A. Rybár, F.V. Selecký, R. Sotníková, S. Štolc, K. Strížová, J. Tokárová, R. Nosáľ, A. Sauberer, E. Nikšová, S. Markovič, J. Toroková, A. Puškárová, T. Kollár 41

pharmacological research New computational approach to mathematical modeling in pharmacological research M. Ďurišová, L. Dedík, M. Tvrdoňová

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Breeding and testing facility Dobrá Voda A. Gajdošík, A. Gajdošíková, E. Ujházy, D. Golhová, B. Kopecká, V. Krchnárová

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From comparative interspecies and ontogenetic pharmacokinetics up to the usage of microcamera-techniques for drug bioavailability studies (Historicizing comments on three decades of the existence of an experimental biopharmaceutical research in Hradec Králové, Czech Republic) J. Květina

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The use of electrochemical measurement for real-time monitoring of nitric oxide generation by macrophages in vitro A. Lojek, M. Pekarová, R. Nosáľ, J. Hrbáč

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H1-antihistamine Dithiaden® suppressed platelet aggregation and oxidative burst of neutrophils in vitro R. Nosáľ, K. Drábiková, V. Jančinová, T. Mačičková, J. Pečivová, M. Petríková, Z. Straková

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Research focuses of the pharmacology in Martin G. Nosáľová, S. Fraňová, A. Strapková, J. Mokrý, M. Šutovská, V. Sadloňová

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Trends in pharmacological research – contribution from studies of the membrane transport and cell signaling K. Ondriaš

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Vasoactive effects of Provinols™ in experimental hypertension O. Pecháňová, I. Bernátová

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Substituted pyridoindoles as antioxidants and aldose reductase inhibitors in prevention of diabetic complications: A preclinical study in vitro and in an animal model of experimental diabetes in vivo M. Štefek, P.O. Djoubissie, A. Gajdošík, A. Gajdošíková, M. Jusková, Ľ. Križanová, Z. Kyseľová, M. Májeková, L. Račková, V. Šnirc

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pharmacological research New pyridoindoles with antioxidant and neuroprotective actions S. Štolc, V. Šnirc, A. Gajdošíková, A. Gajdošík, Z. Gáspárová, O. Ondrejičková, R. Sotníková, Á. Viola, P. Rapta, P. Jariabka, I. Syneková, M. Vajdová, S. Zacharová, V. Nemček, V. Krchnárová

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Trends of research in pharmacology M. Tichý, P. Urban

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Trends in developmental toxicology: Protection of the developing organism – an ever topical issue E. Ujházy, M. Mach, J. Navarová, A. Gajdošíková, A. Gajdošík, J. Janšák, V. Dytrichová, M. Dubovický

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Angiogenesis A perspective target in cancer therapy L. Varinská, L. Mirossay, J. Mojžiš

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Cytokine-stimulatory effects of acyclic nucleotide analogues: extrapolation of immunopharmacological data from animal to human cells Z. Zídek, E. Kmoníčková, A. Holý

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AUTHORS INDEX

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Editorial Pharmacology is different from most other biological sciences because it does not ask how nature works, but rather how can we change nature? Answering this question requires the integrated effort of multiple techniques (molecular, biochemical, cellular and systems-based) to come to a total understanding of the action of a drug. K. Brune, Trends in Pharmacological Sciences 22(6), 323–324, 2001.

Pharmacological investigation has existed for as long as people have been taking drugs (natural and synthetic compounds). In its simplest sense, pharmacology means what drugs do to the body and vice versa. One of the most exciting aspects of pharmacology lies in the unique way it encourages interaction between different scientific approaches. This book is published on the occasion of the 60th anniversary of the Institute of Experimental Pharmacology of the Slovak Academy of Sciences. It embraces articles in experimental pharmacology and toxicology from the viewpoint of the basic scientists, the pharmacologist or the toxicologist. Our intention was to direct the attention of the medical community on pharmacological and toxicological aspects of drugs. Moreover, we are presenting recent developments in studies of drug action which were, are, and are intended to be involved in the scope of interests of pharmacological institutions in the Slovak and Czech Republic. In both countries, diverse goals were achieved during the last decades in basic pharmacological research (covering a range of sub-groups, such as neuro-, cardiovascular, gastrointestinal, respiratory, etc. pharmacology), pharmacokinetics, toxicology (drug toxicology, adverse and side effects, environmental toxicology, food toxicology), and clinical pharmacology. To characterize effects of biologically active compounds, besides classical and clinical pharmacology, drug metabolism, pharmacokinetics and analytical and clinical toxicology, different experimental approaches of various biomedical disciplines, like electrophysiology, biochemistry, molecular biology, pharmacogenetics, immunology, molecular toxicology, drug epidemiology, pharmacy and clinical pharmacy, among others, were employed on living matter such as cells, tissues or organs, both in animals and humans. Since some of the experimentators may not see the whole animal just sense details, and thus instead of grasping the whole, they dissect it to end up with just a foot, an ear or a piece of the tail (in other words, perhaps a few G proteins, kinases, lipases or phosphatases, etc.), their results call upon certain reservation in interpretation. Nevertheless, they opened up new perspectives and opportunities in drug design and development with or without direct relevance to therapeutics. Biomedical research in experimental and clinical pharmacology, targeting drug therapy and toxicology by exploiting the present knowledge on drug mechanisms of action, fate and toxicity is a rapidly progressing area. Our aim was to evidence that in our institutions not only carefully integrated hypotheses are generated but we also wish to stress the importance of maintaining a critical balance between the molecular understanding of drug targets, action and safety with their effects and toxicity in the whole animal in health and sickness.

Viktor Bauer

pharmacological research Brief history of the Institute of Experimental Pharmacology Radomír NOSÁĽ Director of the Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratilsava, Slovak Republic

The Institute of Experimental Pharmacology, Slovak Academy of Sciences, represents the most advanced research institution in basic and applied pharmacology in Slovakia. The Institute has played an important role in the development of the scientific fields “pharmacology” and “toxicology”. An integral part of the Institute is the Department of Toxicology and Animal Breeding located at Dobrá Voda near Trnava. It is the only center for toxicological research in breeding of laboratory animals in Slovakia. The basis of the contemporary Institute was founded in 1947 when, due to increasing demands on evaluation of the quality of new products in former Czechoslovakia, the Department of Biological Control of the Chemical and Pharmaceutical Works Inc. was established in Bratislava. In 1950, the Chemical and Pharmaceutical Works established the Research Institute for Pharmacy. Its Department of Experimental Medicine can be considered the actual germ of the present Institute of Experimental Pharmacology. The Department performed descriptive pharmacological analyses of a broad spectrum of new substances (e.g. follicular hormone, bee-venom, intravenous preparation of iron) as well as routine assessments of the toxicity of some biologically active substances. In 1951, due to lack of healthy and standardized animals for experiments, the Research Breeding Center was founded at Dobrá Voda near Trnava. The Breeding Centre has been supplying experimental animals to institutes of the SASc, universities and institutes of the Ministries of Health, Agriculture and Industry. In the same year, the Institute was incorporated into the Research Institute of Pharmacy and Biochemistry in Prague. Research activities were focused mainly on alkaloids, hormones, purine derivatives, optically active ephedrine and compounds derived from antimony for veterinary purposes. In 1953, the Institute was incorporated into the newly established Slovak Academy of Sciences within the Institute of Chemical Technology of Organic Compounds. In this period industrial projects were completed and research work became gradually oriented to basic science. Hypotensive alkaloids and cardioactive glycosides of wild-growing plants in Slovakia were isolated and studied. In 1963, the Central Laboratory of Pharmacology of the Institute of Organic Chemistry and Biochemistry of the Czechoslovak Academy of Sciences (CsASc) in Prague and the

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Department of Pharmacology of Natural Substances of the Institute of Chemistry of SASc in Bratislava including its Research Breeding Laboratory at Dobrá Voda, merged to establish the Institute of Pharmacology of the CsASc with Departments in Prague and Bratislava. Main interests of the newly formed Department in Bratislava were focused on mechanism of action of different endogenous and synthesized substances on synaptic transmission in the peripheral and central nervous system, peripheral and coronary blood-vessels, on myocardial contractility and side effects of antituberculotics. The year 1969 is important in the history of the Institute. The Slovak Departments of the Pharmacological Institute of the CsASc became independent and the Institute of Experimental Pharmacology of the SASc was created. From this time on the main interests of the Institute were concentrated on receptor specificity and the mechanism of action of alpha- and beta- adrenoceptor blocking drugs, on the studies of inhibitory and excitatory modulation of synaptic transmission by biogenic amines, theophylline, 5-hydroxytryptamine and the polyene antibiotic cyanein, on elucidation of the mechanism of action of papaverine in the therapy of some neurosomatic diseases, on pharmacokinetics of pyrazolidine and xanthine derivatives and beta-adrenoceptor blockers, and on the relationship between chemical structure and specific mechanism of action of adrenergic receptor antagonists and carbanilate local anesthetics. On studying the mechanism of action of carbidine, a new neuroleptic drug stobadine was synthetized, its membrane stabilizing, alpha-adrenergic receptor-blocking and antihypoxic properties were demonstrated. The research in pharmacodynamics was focused on several areas, particularly systemic pharmacology, like cardiovascular and neuropharmacology, smooth muscle, cellular and biochemical pharmacology. In applied pharmacology the Institute has a long tradition and is keeping in progress in teratology and pharmacological toxicology as documented also by the international symposia on toxicology biennially organized by the Institute. The two areas on basic teratology and toxicology yielded important results and provided many reports concerning studies on new drug registrations and production of drugs to be used therapeutically. A similar development holds true for the research in pharmacokinetics concerning studies in basic pharmacokinetics, particularly in the metabolism and fate of biologically active substances in the living organism. With the aim to find optimal therapeutical regimens, theoretical modeling of drug pharmacokinetics in the human and animal body has been introduced.

Over the last decade the following scientific issues have been investigated: • Study of receptor and non-receptor interactions between cationic amphiphilic substances and isolated cells at molecular and cellular levels (effect of beta-adrenoreceptor antagonists and antihistaminic drugs, stobadine and chloroquine on platelets, polymorphonuclear leukocytes and their interactions). • Preclinical study of the action of compounds affecting generation and/or action of reactive oxygen species in nervous tissue. Bauer et al. Trends in Pharmacological Research

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R. Nosáľ

• Study of intracellular signalization in the smooth muscles of vessels and in the heart tissues. • Effect of reactive oxygen species on smooth muscles of intestines, air passages and vessels. • Mechanisms of absorption, distribution and elimination of drugs, mathematical modeling of pharmacokinetic processes. • Creation of structural models of biomedical systems, detection of metabolic pathways of drugs and development of controlled systems of drug release. • Metabolic changes of xenobiotics; study of chemical and enzymatic mechanisms of molecular oxygen activation; toxic effects of reactive oxygen species and glucose in long-term hyperglycemia and protective effect of natural and synthetic antioxidants. • Study of molecular mechanisms of transport and antioxidative properties of selected antioxidants by means of QSAR method. • Possible negative effects of new drugs on pre- and postnatal development (embryotoxicity, teratogenicity and neurobehavioral development of offspring). • Acute and chronic toxicities. • Preclinical study of the original pyridoindole stobadine developed at the Institute (The Prize of the Slovak Academy of Scienceswas awarded for the year 2000). At present, the main interest of the Institute is focused on the study of pharmacological interventions in oxidative stress and proinflammatory reaction-induced injury of the organism. One of the main interests of the Institute is to develop new pharmacotherapeutic approaches to diseases associated with pro-inflammatory processes and oxidative stress. An integral part of R&D activities are studies in the field of biopharmaceutical (medicinal) chemistry focused on the design and synthesis of new pyridoindole derivatives with anti-inflammatory and anti-radical properties and research of the use of hyaluronan biopolymers. Pharmacodynamic, pharmacokinetic as well as toxicological mechanisms of action of biologically active substances are investigated at body, organ, cellular, membrane, receptor and molecular levels. Potential side toxic effects of drugs are studied using a battery of toxicity tests. The Department of Toxicology and Animal Breeding at Dobrá Voda possesses Accreditation for Breeding of Laboratory Animals and Experimentation on Laboratory Animals from the State Veterinary Administration SR No 7656/02-220, Statement of GLP Compliance No 23/2000 from Slovak National Accreditation Service (area of expertise: toxicity studies, carcinogenicity, care and housing of animals) and Statement of Entry in the Register of Forages SR No 8922. The Institute plays an importnat role not only in the field of research and development but also in education of young scientists. In cooperation with Comenius University and the Slovak Technical University, the Institute educates future pharmacologists, toxicologists and biochemists who become qualified specialists for biomedical research not only in Slovakia but also in research institutes abroad. Copyright © 2008

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pharmacological research Trends in studies of drug metabolism and of related drug–drug interactions Pavel ANZENBACHER 1, Eva ANZENBACHEROVÁ 2 1 Department of Pharmacology and 2 Department of Medical Chemistry and Biochemistry, Faculty of Medicine

and Dentistry, Palacky University at Olomouc, Hnevotinska 3, 775 15 Olomouc, Czech Republic

Key words: drug metabolism, drug interactions, cytochrome P450, conjugation enzymes, drug transport, nuclear complexes

Introduction Last twenty years in life sciences and hence also in pharmacology could be characterized by attempts to find molecular basis of function (as well as of dysfunction) of processes in living organisms. In medicine, an exponential increase of new findings and of experimental data has appeared. The amount of new information on the other hand has caused at least in significant number of colleagues an increasing feeling that either everything has been already found or that it is almost impossible to keep the pace with this progress. The reason for that may be a simple fact that “we cannot see the forest because of the trees”, in other words, that the intimate reason for the research in the particular field is not clearly stated or that it is not explained in a way acceptable even for rather learned part of the society. Let us hope that the need for an individualized medicine, and, hence, also for an individualized pharmacotherapy is generally accepted. The contribution of research in pharmacogenetic applications in pharmacokinetics, namely, in drug metabolism incl. its regulation and drug transport is expected here. The most general approach is to see the organism as a whole, incl. its ability to cope with metabolism of foreign compounds, with their transport and on the regulation of these processes. The trends in the field of studies on drug transport and metabolism are (i) to understand the mechanisms which determine the ability of individual (so many?) enzymes and proteins of drug transport and metabolism to pursue their function, (ii) to understand the differences in the ability of these proteins to exhibit their action due to genetically determined structural alterations and (iii) to understand the clinical consequences of the presence of these structurally altered (or even nonfunctional or missing) proteins. P. Anzenbacher & E. Anzenbacherová (2008) Trends in Pharmacological Research (Eds. V. Bauer et al.): 11–14.

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Drug metabolism enzymes Cytochromes P450 (CYP) The spatial structure of the most of the human liver microsomal cytochromes P450 (CYP) has been determined in last five years by X-ray crystallography, including the most important enzymes CYP3A4, CYP2C9, CYP2D6 which are responsible for metabolism of approximately three quarters of all drugs biotransformed in the man [1]. The understanding of the ability of these enzymes to metabolize the drugs and other xenobiotics is apparently determined not only by the spatial structure of the active site and the access/egress channels by which the compounds enter and leave the active site, but also by the flexibility of these parts of the molecule [2,3]. Deeper understanding of the function of cytochromes P450 and of the drug biotransformation however needs deeper insight into the differences in the structural properties of the genetically determined variants of the CYP enzymes which are present in significant part of population. For example, the CYP2C9 variant *3 is known to possess isoleucine instead of leucine in position 359 which leads to lowering of ability of this protein to metabolize warfarin down to one tenth of the activity of the protein coded by the normal, wild type allele (www.imm.ki.se/cypalleles). As the number of patients with this variant allele represents at least 10% of population, the detection of this genotype is contributing significantly to improvement of warfarin pharmacotherapy.

Enzymes of the 2nd phase of drug biotransformation In majority of cases, the drug is metabolized by enzymes of conjugation phase of drug metabolism. Here, the glucuronidation is the major pathway; and it is becoming clear that also these enzymes contribute to individual differences in drug metabolism and to the need of individualized treatment. The first known example was the increased hepatotoxicity of paracetamol in individuals with Gilbert’s syndrome, which is caused by a lower activity of uridine diphosphate glucuronosyltransferase form 1 (UGT1A1 or UGT1.6) due to mutations in exons 2 and 5 [4]. However, little is known about the links with this and other genetic variants of the UGT enzymes with defects in drug metabolism.

Regulation of levels of drug metabolism enzymes, nuclear receptors The most important mechanism deciding on the levels of the enzymes of the first two phases of drug (xenobiotic) metabolism is the regulation of transcription of their corresponding genes by nuclear receptors and their complementary responsive elements in the promoter sequences of the corresponding genes. The immediate step leading to the activation of a receptor is the binding of a regulatory molecule (e.g. a polycyclic hydrocarbon molecule) to the corresponding receptor. There is a family of these receptors in the cells (mostly in the liver) which regulate expression of enzymes of both phases of drug metabolism as well as of the transporting pumps (as the P-glycoprotein Copyright © 2008

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of the multidrug resistance complex, MDR1 or ABCB1). There is a considerable body of evidence that these receptors are involved in regulation of the enzymes mentioned in the preceding paragraph; for example, the genetic variants in the UGT enzymes may well reflect the variants of the regulatory molecules as of the receptors (pregnane X receptor PXR, constitutive androstane receptor CAR, peroxisome proliferator activated receptor PPAR) [5].

Systems of active drug transport (ABC pumps) As it has been introduced in the preceding paragraph, the presence of protein membrane-bound efflux transporters in human cell membranes is one of the factors which may decide on the available level of a drug in the target tissue, in other words, on the efficacy of the drug applied. Hence, genetic polymorphisms connected with an absence or dysfunction of a particular system may be reflected in an ineffective treatment or with a significant decrease in drug efficacy. Whereas the connection of the polymorphisms of the genes of these proteins with development of many serious diseases is known and accepted [6,7], the effect of these polymorphisms on the pharmacokinetics of drugs is known in several cases only. For example, it has been shown that the common polymorphic variants of the MDR1 protein were associated with higher digoxin serum concentrations [8], or, that there is an association of the MDR1 gene polymorphisms and the efficacy and safety of the simvastatin treatment [9].

Conclusion In other words, the individualization of pharmacotherapy seems to be one of the most important trends in medicine of the 21st century with tools offered by recent developments in pharmacology, molecular biology, biochemistry and analytical chemistry. The need for cooperation of specialists in these fields is only stressed by complexity of the life sciences, however, as it has been written in the introduction, the main aim should be kept in mind, namely, a significant improvement of the drug efficacy and safety. Drug metabolism studies aimed at the pharmacogenetics of drug metabolizing enzymes, on the regulation of these enzymes as well as on the pharmacogenetics of drug membrane-bound efflux transporting systems will certainly bring many information of key importance.

Acknowledgment The financial support from the Grant Agency of the Academy of Sciences of the Czech Republic KAN200200651 is gratefully acknowledged.

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REFERENCES [1] Anzenbacher P, Anzenbacherová E: Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 2001; 58, 737–747. [2] Skopalík J, Anzenbacher P, Otyepka M: Flexibility of human cytochromes P450: molecular dynamics reveals differences between CYPs 3A4, 2C9, and 2A6, which correlate with their substrate preferences. J Phys Chem B 2008; 112: 8165–8173. [3] Anzenbacher P, Anzenbacherová E, Lange R, Skopalík J, Otyepka M: Active sites of cytochromes P450: What are they like? Acta Chim Slov 2008; 55: 63–66. [4] Parkinson A: Biotransformation of xenobiotics. In: Casarett and Doull´s Toxicology, the Basic Science of Poisons, Chapter 6. Editor: Klaassen CD. Mc Graw Hill, 2001, p. 133–224. [5] Zhou J, Zhang J, Xie W: Xenobiotic nuclear receptor-mediated regulation of UDP-glucuronosyltransferases. Curr Drug Metabol 2005; 6:289–298. [6] Kimura Y, Morita SY, Matsuo M, Ueda K: Mechanism of multidrug recognition by MDR1/ABCB1. Cancer Sci 2007; 98: 1303–1310. [7] Turgut S, Yaren A, Kursunluoglu R, Turgut G: MDR1 C3435T polymorphism in patients with breast cancer. Arch Med Res 2007; 38: 539–544. [8] Aarnoudse AJ, Dieleman JP, Visser LE, Arp PP, van der Heiden IP, van Schaik RH, Molokhia M, Hofman A,Uitterlinden AG, Stricker BH: Common ATP-binding cassette B1 variants are associated with increased digoxin serum concentration. Pharmacogenet Genomics 2008; 18: 299–305. [9] Fiegenbaum M, da Silveira FR, Van der Sand CR, Van der Sand LC, Ferreira ME, Pires RC, Hutz MH: The role of common variants of ABCB1, CYP3A4, and CYP3A5 genes in lipid-lowering eficacy and safety of simvastatin treatment. Clin Pharmacol Ther 2005; 78, 551–558.

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pharmacological research Smooth muscle tissues as models for study of drug action Viktor BAUER, Ružena SOTNÍKOVÁ, Viera NOSÁĽOVÁ, Jana NAVAROVÁ, Štefan MÁTYÁS, Vladimír PUCOVSKÝ, Vladimír REKALOV, Katalyn SZŐCS, Jana NEDELČEVOVÁ, Zuzana KYSEĽOVÁ, Viera DYTRICHOVÁ, Jozefína FATYKOVÁ†, Mária KOLLÁROVÁ, Lubica MÁLEKOVÁ, Monika SRNOVÁ, Zuzana STOJKOVIČOVÁ, Gizella TÓTHOVÁ Department of Smooth Muscle Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic, E-MAIL: [email protected]

Key words: smooth muscle, autonomic nerves, epithelium, endothelium, drug actio

Introduction Direct action of drugs in smooth muscles (SM) or via their innervation, endothelium or epithelium affects SM tone and/or contractility. Properties common for all types of SM and special properties of the particular ones, cellular and subcellular organization, innervation, role of endothelium and epithelium make them suitable to discover fundamental physiological, pathophysiological processes and characterize features of drug action [1]. Although the complexity of the SM preparations calls upon certain reservation in interpretation of results obtained in vitro and in vivo, with some precaution these are applicable also for other systems involving similar mechanisms as SMs.

Methods Our department deals mainly with effects of drugs on autonomous (ANS, i.e. cholinergic, adrenergic, non-adrenergic, non-cholinergic – NANC) and sensoric nerves; processes linked to epithelium, endothelium and to their biologically active mediators and modulators; membrane and subcellular receptors and receptor coupled processes; ion channels; enzymes and availability of Ca2+. Introduction of sophisticated electrophysiological, pharmacological, biochemical, isotope and morphological methods have provided the possibility to elucidate causality and targets of drug action in diseases and pathological conditions, such as: asthma, gastric ulcer, colitis, ischemia/reperfusion (I/R), diabetes, oxidative stress, etc. Details of the methods used in our studies are described in papers [1–19].

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Results and discussion 1. Calcium and smooth muscle activities Ca2+ has an extremely important role in regulation of SM activity, because there is a greater gradient between concentrations of the free extracellular ([Ca2+]o) and intracellular ([Ca2+]i) calcium than for other ions. Elevation of [Ca2+]i above 0.1 μmol/l and its binding with calmoduline (CaM) activates not only myosine light chain kinase (MLCK) resulting in SM contraction but also further enzymes affecting SM activity. Ca2+ homeostasis is maintained by its influx via selective voltage (VOC, Figure 1A) and receptor (ROC) operated Ca2+ channels and less selective cationic channels, by chemically operated Ca2+ release from intracellular stores and by its active transport out of the cell and to intracellular stores, by Ca2+ pumps and exchange mechanisms. Some SMs (phasic ones, like intestine or portal vein) generate inward Ca2+ current on their entry to the cell, tetrodotoxine (TTX) insensitive action potentials and SM contraction, which are inhibited by Ca2+ channel blockers, indicating the essential role of Ca2+ in these processes [2,3]. Other SMs (tonic ones, like vessels or trachea) generate action potentials only under specific conditions (e.g. inhibition of membrane conductance for K+ by tetraethylammonium – TEA) and their contraction develops without or during pro-

Figure 1. Patch clamp recording of the effect of nifedipine on inward Ca2+ current (A), hypoxia on spontaneous transient outward K+ currents (STOCs) and whole cell current (B) and H2O2 on single K+ channel activity evoked by current RAMPs from +20 to -40 mV (C) (channel conductivity and reversal potential are indicated) of guinea pig (GP) taenia coli (TC) SM cells. Copyright © 2008

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longed low amplitude membrane depolarization. There is, however, some evidence of SM contractile proteins activation without participation of an increase in [Ca2+]i [3,4]. The following membrane elements participate in maintaining the SM membrane potential and alterations of its conductivity: the VOC (L-type, T-type, CRAC-type); ROC; chemical agonists activated nonselective cationic channels (fast and slow) permeable for Na+ and Ca2+ (responsible for excitatory junction potentials) or permeable for Na+ and K+ (responsible for prolonged depolarization); voltage dependent Na+ channels (myocardial and neuronal type); voltage dependent K+ channels (transient type, responsible for A current); TEA sensitive and insensitive outward and inward rectifiers; Ca2+ and voltage dependent maxi- (responsible for transient outward current) and low- conductance (responsible for oscillations of the membrane potential and STOCs) K+ channels (Figure 1B, C); membrane potential independent K+ channels (receptor operated N type; ATP and Ca2+ sensitive; ATP sensitive and Ca2+ insensitive responsible for resting membrane potential, M current and S current); second messenger activated voltage and Ca2+ dependent channel (conductive for anions, mainly Cl–); the Na+ pump; Ca2+ pump; Na+ - Ca2+ exchanger; Na+ - H+ exchanger and K+ - Na+ - Cl– exchanger [1,5]. The effects of Cai2+ may be modified by: its binding (e.g. by chelators); modulation of CaM and its interaction with Ca2+ (e.g. by phenothiazines); influence of MLCK and myosine phosphatase (e.g. by substance A-3) or myosine and actine (e.g. by H2O2). Mechanisms coupled to intracellular Ca2+ stores participate as well in maintaining free [Ca2+]i. From one of these stores (S) Ca2+ is released by IP3 sensitive (IICR) and ryanodine sensitive (CICR) channels, while from the other (Sß) only through IICR channels. IP3 receptor activation released Ca2+ evokes additional Ca2+ releases (Ca2+ induced Ca2+ release) by means of CICR channels. Free Cai2+ activates proteinkinase C and by Cai2+/ CaM interaction proteinkinaseII, which phosphorylates VOC and IICR channels and releases further Ca2+. Transport of Ca2+ back to its intracellular stores is materialized by a tapsigargin sensitive Ca2+ pump. If in Sß the concentration of Ca2+ becomes low, calcium influx factor (CIF) is produced. CIF activates Ca2+ release activated channels (CRAC) in plasma membrane and mediates the influx of Ca2+ to the SM cell.

2. Interaction among smooth muscle layers SMs are composed of mechanically and electrically coupled cells in close vicinity with the surrounding connective tissues. Myosine possesses a more substantial role in adjustment of SM tone than actine. Their structural organization grants transfer of contractile protein generated force along the whole tissue. It is believed that the longitudinal muscle (LM) of guinea pig ileum (GPI), used for more than a hundred years as SM model, is liable for isotonic shortening or elongation and isometric raise or loss of SM tone [6]. The role of the circular muscle (CM) and of the so-called “microcosmos” of ANS is neglected, though there are significant variations between reactivities of LM and CM layers. To evoke contraction of GPI longitudinal muscle strips (LSt) single pulse stimulation of intramural nerves (ES) is sufficient; while for activation of circular strips Bauer et al. Trends in Pharmacological Research

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(CSt) tetanic stimulation is needed. The responses of CSt have features of rebound contractions (RC) rather than of primary ones. Dose response curves (DRC) of isoprenaline (Iso), acetylcholine (Ach) and histamine (Hi) have higher amplitudes on LSt than on CSt, while the contrary applies in the case of carbachol (Crb) and KCl. The sensitivity of LM, holding the intramural myenteric plexus (MP), to ES, Iso, Ach, Hi, H2O2, Crb and KCl did not differ from that of LSt. In contrast, isolated CM, poor in MP, loses reactivity to Crb and KCl. Mild reduction of difference between responses of CM and CSt by hexamethonium suggests that ganglionic transmission does not participate in communication between the muscle layers. Peristaltic activity and rhythmic longitudinal shortenings evoked by Ach, Crb and KCl are in the case of Crb and KCl transient and succeeded by elongation of the GPI. Elongation results most likely from the presence of transversally oriented muscle fibers in CM and the less regular organization of actine and myosine which endows greater contraction capability compared with LM (parallel longitudinal arrangement of cells converts to transversal direction on shortening and slewing around the longitudinal axis)[7]. Yet the possible participation of Cajal cells in the different reactions of muscle layers can not be excluded.

3. Adrenergic transmission Using α- and β-adrenoceptor agonists and antagonists, we found that postsynaptic α1-drenergic receptors dominate in terminal, while α2-receptors in intermediate and proximal parts of the GPI. The mainly inhibitory β-adrenoceptors are homogenously distributed. The cholinergic and NANC nerve terminals possess modulatory α2- inhibitory receptors [8,9]. Isolation of SM cells under which α1-adrenoceptors do not lose their function was developed using antioxidants (dithiothreitol or taurine), high concentration of bovine serum albumin and the relatively specific enzyme, collagenase Type XI (Sigma). Phenylephrine (PhE) induced α1-adrenergic receptor mediated membrane hyperpolarization in TC and LM of GPI. It enhanced the amplitude of inward Ca2+ current, the frequency and amplitude of voltage and temperature dependent STOCs, elicited low amplitude sustained outward current, reduced the inward and enhanced the outward component of the whole cell current. These results are in favor of the assumption that SM relaxation and membrane hyperpolarization in GPI and TC are realized at least in part as a consequence of activation of Ca2+ dependent K+ conductance [9].

4. NANC transmission The SM tone and its spontaneous activity are modulated beyond the ion channels and transport mechanisms, also as a result of receptor and enzyme activation. Besides circulating hormones (e.g. steroids, catecholamines) the receptors and enzymes are affected also by neurotransmitters such as Ach, noradrenaline (NA), substance P (SP), nitric oxide (NO), vasoactive intestinal polypeptide (VIP), etc., released from the ANS and sensoric nerves and by mediators such as endothelium-derived relaxing (EDRF/NO), Copyright © 2008

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contracting (EDCF), and hyperpolarizing (EDHF) factor, eicosanoids, Hi, bradykinine (BK), angiotensin, serotonin (5-HT), endothelin, reactive oxygen species (ROS), etc. released from nerves, epithelium, or endothelium. Receptors on SM membrane are coupled to ion channels directly (e.g. chemically activated cation channels), by enzymes (e.g. tyrosine kinase, phospholipase A2), or through G-proteins and enzymes (e.g. adenylate cyclase, guanylate cyclase, phospholipase C). The produced second messengers (e.g. cAMP, cGMP, prostaglandins, IP3, DAG) subsequently affecting intracellular receptors (e.g. IICR) or enzymes (e.g. proteinkinases) activate myosinkinase (phosphorylates myosine resuling in SM contraction), or phosphatase (dephosphorylates myosine resulting in SM relaxation). In the presence of atropine and guanethidine, stimulation of NANC nerves evokes in GPI LM relaxation-contraction with RC, while in GPI CM, TC, GP and cat (C) airways (AW) relaxation with RC [10,11]. Using microelectrodes and sucrose-gap methods, TTX and Mg2+ sensitive NANC excitatory (e.j.p) and inhibitory (i.j.p.) junction potentials were recorded from GPI LM and NANC i.j.p. from GPI CM, TC and CAW. Rebound depolarization was recorded on both layers. Frequency profiles demonstrated that NANC responses arose at higher frequencies than cholinergic ones. Thus GPI LM possesses in addition to cholinergic and adrenergic, both excitatory and inhibitory NANC innervation. The NANC excitation is denser in the terminal, while the NANC inhibition in the proximal parts of GPI. In contrast the GPI CM and TC possess homogenous NANC inhibitory innervation. To unveil the nature of NANC transmission we used ATP, ADP, apamin, TEA, VIP, SP and its derivatives, capsaicine, BK, calcitonin gene-related peptide, catecholamines, 6-hydroxydopamine, reserpine, Hi, 5-HT, GABA, indomethacine (Indo), carbamate local anesthetics, 3,4-diaminopyridine, opioids, dipyridamole, ROS, inhibitors of NO synthase (INOS) and the method of cross desensitization. Our results suggest that in the GPI, TC, GPAW and CAW ATP, adenosine and prostaglandins do not contribute to the generation of NANC response. SP is probably the excitatory and VIP and NO are the inhibitory NANC transmitters [10,11].

5. Effects of ROS ROS produced in cell membrane lipid bilayers, in the electron transport system of mitochondria, in cell organelles, like peroxisomes, lysosomes, endoplasmic reticulum, as well as in the cytoplasm by enzymatic and non-enzymatic reactions are essential for physiological function, metabolism and defense of the majority of cells and tissues [12]. In GPI LM, TC, GPAW, CAW and rat aorta (RA) ROS evoked contraction, relaxation or biphasic response. Their amplitudes depended on basal tone (spontaneous or elevated by Hi, 5-HT or KCl). INOS ameliorated the ROS induced contractions. The ROS effects were dependent on intact endothelium and mucosa. H2O2 and superoxide (O2•–) in GPI LM were more effective than hydroxyl radical (•OH) and singlet oxygen (1O2). During relaxation, which follows the initial phasic contraction the responses of GPI LM elicited by Ach and ES were suppressed. The NANC excitation was more sensitive to ROS action Bauer et al. Trends in Pharmacological Research

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than is NANC inhibition. While native neutrophils did not affect the muscle tone, the fMLP activated ones (ANT) induced contraction, relaxation or a biphasic response of RA precontracted by PhE, NA or KCl, due to production of O2•– and its transformation to other ROS. The muscle tone of SMs changed as the result of direct (altered membrane activity, e.g. Figure 1B, C) or indirect effects of ROS (elimination of the protective role of the endothelium, interaction with NO or its production, or with cyclooxygenase and/ or lipoxygenase pathways) [13,14]. CAW possesses diethyldithiocarbamic acid sensitive superoxide dismutase activity. The multiple actions of O2•– generating systems appear to result from the presence and simultaneous action of at least two different ROS (O2•– and H2O2). While O2•– inhibited cholinergic and NANC transmission and participated at least in part in the evoked relaxation, H2O2 seems to be responsible for elevation of muscle tone and augmentation of cholinergic contractions and e.j.p.s, resulting from increase of [Ca2+]i, with subsequent augmentation of stimulation of evoked contractions, as well as of the Ca2+ and voltage sensitive K+ conductance[15].

6. Pathologic conditions Introduction of the above mentioned electrophysiological, biochemical, isotope and morphological methods have provided the possibility to elucidate causality and targets of drug action in numerous pathological conditions.

a) Ischemia/Reperfusion (I/R) Effects of I/R on endothelial function (tested in PhE precontracted rings using Ach) [13] and production of ROS (detected by luminol enhanced chemiluminiscence) was evaluated in Wistar rats. Ischemia was induced by clamping of the superior mesenteric artery (SMA) for 60 min followed by reperfusion (for 5 or 30 min). Ischemia or ischemia followed by 5 min-reperfusion did not change SMA reactivity and ROS production. Prolongation of reperfusion to 30 min increased the spontaneous production of ROS in SMA, which correlated with functional injury, i.e. impairment of endothelium-dependent relaxation (EDR) and decrease in EC50 values of Ach. Inhibition of prostaglandin and NO synthesis by Indo and NG-nitro-L-arginine methyl ester (L-NAME) attenuated EDR from sham-operated and I/R groups, suggesting that I/R damages both systems, the EDRF and EDHF (Figure 2).

b) Experimental diabetes Circulatory and gastrointestinal disorders have often been reported in diabetes. In diabetic macroangiopathies hyperglycemia facilitates deterioration of EDR through increased production of ROS. Ten weeks of streptozotocine (STZ)-induced diabetes resulted in diminished EDR of RA, increased endothelemia, elevated systolic blood pressure and increased concentration of ROS in RA and blood. Incubation of aortic rings in solution with high glucose concentration led to impairment of EDR. Reduced EDR Copyright © 2008

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Figure 2. Effect of mesenteric ischemia (60 min) followed by reperfusion (30 min) on EDR of the SMA before (A) and after INOS with 100 μmol/l L-NAME (B). Relaxation is expressed as % of contraction induced by 1 μmol/l PhE. Data are means ± SEM of 8-12 measurements. *p60 sec to those with SMS 60 sec relative to those with SMS