TITLE: Method for evaluating the effects of smoke on bronchioles

FIELD OF INVENTION The invention relates to a method to evaluate the effects of the lipid soluble part obtained from smoke on bronchioles as well as identifying bioactive agents, which is capable of reducing the effects of smoke on smooth muscle cells in blood vessels or bronchioles.

BACKGROUND OF THE INVENTION

Cigarette smoking has since long been recognized as a strong risk factor that is linked with high incidence of atherosclerosis, acting in concert with hyperlipidemia and hypertension. Stroke, myocardial infarction and claudication are common among cigarette smokers (Wyngaarden & & Smith. Cecil Textbook of Medicine, 1-2404 (W.B. Saunders company, Philadelphia, 1988). The direct medical cost of cardiovascular disease is enormous in the Western world and smoking has been found to cause 5 million premature deaths per year worldwide, the majority of these are interestingly due to cardiovascular events (Lightwood. The economics of smoking and cardiovascular disease, Prog Cardiovasc Dis 46, 39-78 (2003). It is considered that smoking particles cause damage to the arterial wall with localized dysfunction of the endothelium and enhanced plaque formation (Holden et al., Endothelium-dependent effects of cigarette smoke components on tone of porcine intrapulmonary bronchioles in vitro {Toxicol Appl Pharmacol 104, 191-9 (1990)). However, the molecular mechanisms to explain how smoking can participate in and exacerbate the atherosclerotic process are poorly understood. Therefore, there is a need of developing a method in which the effects of tobacco smoke can be evaluated at the bronchial level. By the development of such a method it will be possible to evaluate the effects of existing tobacco as well as that of new tobacco products, which may have a lower effect or no effect at all on the arterial walls and airways and hopefully being less dangerous to the human being consuming the tobacco. The method can also be used for the screening for agents that can prevent the changes that tobacco products induce in smooth muscle cells of blood vessels or airways as well as using the method to determine the expression of receptor regulation, which may be changed due to smoking, i.e., it is possible to use the method as a diagnostic method

SUMMARY OF THE INVENTION

The invention relates to a method of evaluating the effects of the lipid soluble part obtained from smoke on dissected bronchioles, such as arterial or

airway smooth muscle cells as well as identifying bioactive agents, which are capable of reducing the effects of smoke on vessels or the bronchioles. Thereby it is for the first time possible to compare different smoke such as different smoke from different tobaccos or tobacco products to each other and identify tobacco which is less harmful to the human being utilising said tobacco as well as using the method in finding new tobacco which do not or to a low extent affect the expression of all receptors which are influenced by smoke and are present within the smooth muscle cells in blood vessels or the bronchioles, for example the endotelin receptors in the arterial walls or airways and thereby reduce constriction of the smooth muscle cells in blood vessels or the bronchioles.

According to a first aspect the invention relates to a method of evaluating the effects of the lipid soluble tobacco smoke particles on the smooth muscle cells in blood vessels or the bronchioles comprising the steps of; providing dissected bronchioles or vessels from an animal, exposing said dissected bronchioles or vessels to lipid soluble smoke particles, inducing upregulation of one or more receptors mediating tone prior to dissecting said bronchioles or vessels, immersing said dissected bronchioles or vessels in a medium and applying a tension on said bronchioles or vessels, determining the contractile response of said dissected bronchioles or vessels and evaluating the effects of smoke on smooth muscle cells in blood vessels or bronchioles.

In a second aspect the invention relates to a method of evaluating the effects of tobacco smoke on the smooth muscle cells in blood vessels or the bronchioles comprising the steps of; providing dissected bronchioles or vessels from an animal, exposing said dissected bronchioles or vessels to lipid soluble smoke particles, inducing upregulation of one or more receptors mediating tone prior to dissecting said bronchioles or vessels, immersing said dissected bronchioles or vessels in a medium and applying a tension on said bronchioles or vessels, adding a bioactive agent, determining the contractile response of said dissected bronchioles or vessels and evaluating the effects of smoke on smooth muscle cells in blood vessels or bronchioles and obtaining a bioactive agent.

In a third aspect the invention relates to a bioactive agent, which has been obtained by the above mentioned method as well as the use of said bioactive agent for the treatment of a disorder involving constriction of vessels or bronchioles such as after chronic obstructive lung disease or asthma bronchioles.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1. Effects of nicotine in comparison with DSP on ACh-induced vasodilatation. Concentration 0.11 mg/L of nicotine that is nicotine concentration in DSP was used.

Since nicotine and smoke particles were dissolved in DMSO, the same volume of DMSO was added in the culture medium and served as controls. The arteries treated with nicotine or DSP for 6 (A) and 12 hrs (B). Data are shown as mean ± S.E.M. n = 8. *p < 0.05 and **p < 0.01 vs. control.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the context of the present application and invention the following definitions apply: The term "lipid soluble smoke particles" are intended to mean the fraction obtained in example 1.

The term "bioactive agent" is intended to mean an agent being capable of acting as a protein kinase inhibitor being capable of normalising vascular tone by decreasing vasoconstriction or increasing vasodilatation of blood vessels, such as in arteries after ischemic brain injury.

The term "medium" is intended to mean a solution, in which receptors of dissected arteries or bronchioles may be upregulating. Examples of receptors are endothelin, such A ₁ and A ₂ , angiotensin, such as AT ₁ and AT ₂ , serotonin, such as 5HT _1B ^an( i bradykinin, such as B ₁ and B _2. Examples of physiological solutions will be obvious for a person skilled in the art. Examples are modified KREB 's, DMEM, fysiological NaCl, phosphate buffer etc

The term "treatment" according to this invention means that by inhibiting the signal resulting in transcription and translation, the receptor upregulation will be prevented and, hence, e.g. the sequel leading to penumbral zone damage and neuronal loss will be revoked.

The term "upregulation" is intended to mean that receptors in the blood vessels and in the airways are changed in phenotype and/or increased in number, which will lead to deleterious effects on circulation and pulmonary function.

The term "bronchioles" is intended to mean the airways after trachea.

Method of the invention

The present invention relates to the area of evaluating the effects of smoke on the smooth muscle cells in blood vessels or the bronchioles and thereby obtain knowledge about which of the particles in smoke are responsible for certain effects. The lipid soluble smoke particles may be from different tobacco products, such as conventional cigarettes or cigars. Particles which then may be removed from the tobacco and thereby a healthier tobacco may be obtained, which has less negative effects on the human being consuming the tobacco. It has surprisingly been found that tobacco smoke upregulate at least the endothelin receptors in arterial and

bronchial smooth muscle cells (SMC). The method may also be used to identify new bioactive agents, which can be use to treat disorders involving the bronchioles or the vessels as well as to determine expression of receptors, which becomes upregulated by lipid soluble smoke particles and thereby use the method to determine the degree of the disorder or disease caused by the lipid soluble smoke particles.

To be able to identify such tobacco lipid soluble smoke particles we have developed a method in which constriction and/or dilatation may be induced in parallel or in sequence with upregulation of one or more receptors such as the endothelin receptors. The inventors have surprisingly found that blood vessels of tissue subjected to ischemia both in vivo and in vitro, such as the basilar artery and the middle cerebral artery, undergoes phenotypic changes so that the vasoconstrictor receptor population is upregulated, which result in excessive arterial constriction, and that for example the endothelin, the 5-hydroxytryptamine and angiotensin receptors are among those becoming upregulated. The same effects, albeit much stronger are seen by the use of tobacco lipid soluble smoke particles. The method can be used to understand the molecular mechanism involved in the upregulation of e.g., endothelin receptors; this process is involved in vascular and pulmonary disease. From this it is then possible to identify bioactive agents, which can be used to treat smoke induced diseases. The bioactive agent may act as a protein kinase inhibitor.

The arterial or airway SMCs can be obtained from an animal such as rat, mouse, guinea-pig, dog, cat, horse or a synthetic version thereof. The SMCs may be obtained by the use of the technique disclosed by Adner et al., Plasticity of contractile endothelin-B receptors in human arteries after organ culture. Br J Pharmacol 119, 1159-66 (1996) and airway such as in Granstrδm et al., Upregulation of endothelin receptor function of mRNA expression in airway smooth muscle cells following sephadex-induced airway inflammation. Basic&Clinical Pharmacol Toxicol. 2004;95:43-48. The isolated arteries or bronchioles are then exposed to tobacco smoke or tobacco lipid soluble smoke particles, which have been extracted from said smoke, such as water soluble particles or lipid soluble particles. One way to exatrct said lipid soluble smoke particles are Example 1.

The bronchioles may be dissected into smaller segments or discs either prior to be exposed to the tobacco lipid soluble smoke particles or after.

The bronchioles will during the exposure to the lipid soluble smoke particles either upregulate the endothelin receptors or the endothelin receptors may remain more or less active. However, so far all tobacco, which has been evaluate have activated the endothelin receptors and the conclusion is that if the particles which upregulate the endothelin receptors are removed from the tobacco a healthier tobacco may be obtained or the tissues will not be prone to diseases. Therefore the

invented method is important and should be a useful tool in the development of new tobacco. The method can also be used for the screening for agents that can prevent the changes that tobacco products induce in smooth muscle cells of blood vessels in airways as well as using the method to determine the expression of receptor regulation, which may be changed due to smoking, i.e., it is possible to use the method as a diagnostic method

The examples below are included to further illustrate the invention and the invention should not be limited thereto.

EXAMPLE 1

Extraction of DSP.

Three cigarettes (0.8 mg nicotine per cigarette) were "smoked" by a water aspirator and the smoke was directed through a cotton wool filter. The retained smoke particles in the filter were dissolved in 1 ml DMSO. The DMSO-soluble smoke particle (DSP) preparations were analyzed by gas chromatograph-flame ionisation detection (GC-FID, Agilent 6890N, USA) with a 0.23 mm x 15 mm x 0.25 m DB-5MS capillary column (Agilent, USA). The GC-FID temperatures were programmed from 5O ⁰ C, increased with 5°C/min to 280 ⁰ C and remained for 3 min. The concentration of nicotine in DSP was calculated according to standard nicotine peak value and area. After DSP preparations have been analyzed by the gas chromatograph, they were diluted by DMSO to standard nicotine content (0.11 mg/L) and used for organ culture experiments.

EXAMPLE 2

Tissue preparation

Sprague-Dawley rats (250-300 g, male or female) were euthanized with CO2, the head cut off and exsanguinated. The superior mesenteric artery was removed gently, immersed in cold oxygenated buffer solution (in mM: NaCl 119, NaHCO3 15, KCl 4.6, MgC12 1.2, NaH2PO4 1.2, CaC12 1.5, glucose 5.5, pH 7.4) and dissected free of adhering tissue under a light microscope. Human middle cerebral arteries (MCA) were obtained from 3 subjects from the Department of Forensic Medicine of Xian Jiaotong University (China). The arteries were collected from three men and used for organ culture between 4 to 6 hrs after death due to a traffic accident. The experimental protocol for using rat and human arteries was approved by local Ethic's Committee of Shaanxi province (China).

EXAMPLE 3

Organ culture.

We have developed an in vitro method of inducing phenotypic changes of GPCR in vascular cells through culture of arterial segments for a period of time (Adner et al., Plasticity of contractile endothelin-B receptors in human bronchioles after organ culture. Br J Pharmacol 119, 1159-66 (1996)). One mm long ring segments are incubated (endothelium and adventitia removed) are incubated in serum free DMEM at 37 ⁰ C in humidified atmospheric air enriched with 5% CO ₂ for 24 hrs with or without 0.2 μl/ml of DSP.

EXAMPLE 4

In vitro pharmacology.

For contractile experiments, the bronchioles are mounted on two wires (Myograph, J.P. Trading, Denmark) submerged in a temperature controlled buffer solution (37°C) of the following composition (mM) NaCl 119, NaHCO ₃ 15, KCl 4.6, MgCl ₂ 1.2, NaH ₂ PO ₄ 1.2, CaCl ₂ 1.5 and glucose 5.5. The buffer is continuously aerated with oxygen enriched with 5% CO ₂ resulting in a pH of 7.4. Measurements are recorded via a PowerLab unit (AD Instruments, Hastings, England) for continuous recording on a personal computer and analyzed with the software program Chart ^® (Adner et al., Plasticity of contractile endothelin-B receptors in human bronchioles after organ culture. Br J Pharmacol 119, 1159-66 (1996)).

EXAMPLE 4

Immunohistochemistry.

Vessel segments, three samples in each group, were placed on Tissue TEK (Gibco), frozen and subsequently sectioned into 8 μM thick slices. The primary antibodies used were rabbit antihuman ET _B (IBL, 16207), diluted 1 :400, goat anti human ET _A (Santa Cruz Biotechnologies, sc-2 1194), diluted 1 : 100, mouse anti rat smooth muscle actin (Serotec, MCA 1 905T) diluted 1 :100 and mouse anti rat CD3 1 (Serotec, MCA1746), diluted 1 :200. All dilutions were done in PBS with 10% fetal calf serum. The secondary antibodies used were donkey-anti-mouse Cy™5 conjugated (Jackson ImmunoResearch, 715- 175-150) 1 :100, donkey-anti-rabbit Cy™3 conjugated (Jacksonl mmunoResearch, 711-165-152) 1 :100 in PBS with 10% fetal calf serum. The antibodies were then detected at the appropriate wavelength in a confocal microscope (Zeiss, USA). As control only secondary antibodies were used. The absolute fluorescence were investigated with Image J

(http://rsb.info.nih.gov/ij) and background fluorescence subtracted. The measurement was done where there was positive staining for smooth muscle actin, hence in the muscle portion of the vessel. The values obtained were mean fluorescence in the area selected.

EXAMPLE 5

Intracellular signal transduction studies.

After organ culture, vessels were collected and placed on ice, homogenized in lysis- buffer with protease- and phosphatase inhibitors (10 mM Tris pH 7.4, 50 mM β- Glycerophosphate, 100 μM Na ₃ VO ₄ , 0.5% Deoxycholate, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na ₄ P ₂ O ₇ , 1% Triton X-IOO, 1 mM DTT, 20 μM Pepstatin, 20 μM Leupeptin, 0.1 U/ml Aprotinin, 1 nM Calyculin and 1 mM PMSF). Total protein concentration was determined using a BioRad DC kit (Hercules, CA, USA) and a Genesys 6 spectrophotometer (Thermo, Waltham, MA, U.S.A.). 45 μg total protein was loaded per lane on a 10% SDS-PAGE gel and blotted onto a Hybond PVDF membrane for 30 min at 1.5 mA/cm ² . Subsequently the membrane was blocked in 5% non-fat milk for 1 hour at room temperature (RT) and incubated with primary antibody over-night at 4 ⁰ C and with secondary antibody (Pierce, Rockford, IL, U.S.A.) for 1 hour at RT. The membranes were developed using the Supersignal Dura kit (Pierce, Rockford, IL, U.S.A.) and visualized using a Fujifilm LAS-1000 Luminiscent Image Analyzer (Stamford, CT, U.S.A.). pAkts _ER.473 , pJNK, phospho-p38, (Cell Signaling Technology, Beverly, MA, U.S.A.), pELKl, ρPKCδ _SE R645, ρPKCε _SE R729 (Biosource, Camarillo, CA, U.S.A.), ET _A and ET _B (Chemicon, Temecula, CA, U.S.A.) were used at 1 :1000. pERK (Promega, Madison, WI, U.S.A.) was used at 1:2500.

EXAMPLE 6

Pharmacology in vitro

Circular segments including two cartilage segments were cut from trachea and bronchi. Each segment was denuded of its epithelium with a metal tread of 1 mm in diameter and a surface as a fmegraded file to avoid confounding dilatory effects (Hadj-Kaddour et al, 1996).

They were then mounted on two L-shaped metal prongs. One prong was connected to a force displacement transducer attached to a computer for continuous registration of isometric tension and the other to a displacement device. The

segments were then immersed into small (2.5 ml) temperature-controlled (37 ⁰ C) tissue baths containing the same buffer solution. The solution was equilibrated with 5 % CO ₂ in O ₂ , resulting in a pH of 7.4. Initially a tension of 1 mN was applied to each segment, which was then allowed to stabilise at this level of tension for 60 min. The contractile ability of each segment was first examined by exposure to a potassium rich (60 mM) buffer solution (for composition, see below). Only after two strong (>lmN) reproducible contractions (variation <10 %) had been elicited were the individual segments used for further studies. The contraction induced by K ⁺ -solution (60 mM) was used as the contractility reference. Comparing experiments between epithelium-denuded and epithelium-intact segments proved that the ability to contract was not influenced or harmed by denudation .

Concentration-response experiments were conducted in three parts: First, the endothelin receptors in trachea where characterised using endothelin-1 and S6c in the absence or the presence of the endothelin ET _A -antagonist FR 139317 (lOμM), the endothelin ET _B -antagonist BQ 788 (lOμM), or in the presence of a combination of the two antagonists or the combined ET receptor antagonist bosentan (lOμM). Second, after the eosinofilic inflammation had been established (24h), the effect of endothelin-1 and S6 was studied at two levels of the rat airways, trachea and bronchus. Acetylcholine was added (1 mM) when maximum contraction was reached by the endothelin agonists. All segments could be used in this study, i.e. no segment was damaged through removal of epithelium or from the Sephadex induced inflammation. The airway epithelial cells were removed mechanically by wodden stick and the result controlled by histological investigation after the experiments (haematoxylin -eosin staining).

Buffer solutions and drugs

Standard buffer solution (mM): NaCl 119, NaHCO ₃ 15, KCl 4.6, MgC12 1.2,

NaH ₂ PO ₄ 1.2, CaCl ₂ 1.5, glucose 5.5. Analytical grade chemicals and double- distilled water were used for preparing all solutions. S6c and ET-I (Auspeptide,

Aus) were dissolved in water with bovine serum albumin (Kabi, Sweden) (0.1% w/v). All MAPK inhibitors were dissolved in DMSO and diluted in water.

PD98059, SB239063, SB408039, SB386023b and SB203580 were purchased from

Sigma, St. Louis, USA.

Calculations and statistics

Data are expressed as mean values ± S.E.M. Contractile responses in each segment are expressed as a percentage of the K ⁺ -induced contraction. E _max represents the

maximal contraction induced by an agonist, and given either in absolute values or expressed as a percentage of the K ⁺ -induced response for each vessel segment. The PEC ₅₀ value was calculated from the line between the concentrations above and below the midpoint of the concentration-response curve. Kruskal-Wallis nonparametric test with Dunn's post-hoc test was used for all statistical analyses. Differences were considered significant at P < 0.05.

EXAMPLE 7

PCR Real-time

Samples of the lung were snap-frozen in liquid nitrogen immediately after acquisition and total cellular RNA was extracted using the TRIzol reagent (Gibco BRL) following the supplier's instructions. The resulting RNA pellet was finally washed with 70% ice-cold ethanol air-dried and redissolved in 10 μl diethyl- pyrocarbonate (DEPC)-treated water. The amount of RNA was determined by use of spectrophotometry (DU64 spectrophotometer; Beckman, Fullerton, CA, USA) considering a ratio of OD _260:280 > 1.6 as pure. RT-PCR was carried out using the GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA, USA) on a DNA Thermal cycler (Perkin-Elmer). Specific primers for the rat endothelin ET _A - (generating a 264 basepair product) and endothelin ET _B receptors (generating a 560 basepair product) were designed as follows: Endothelin ET _A -receptor forward: 5 '-TACAAGGGCGAGCAGCACAGGA-3 ' reverse: 5 ^' -CACAGGGCGAAGATGACAACCAA-S '

Endothelin ET _B -receptor forward: 5 '- TGACGCCACCCACTAAGAC-3 ' reverse: 5 '-GACAGCCAGAACCACAGAGA-3 '

The reverse transcription of total RNA to cDNA and subsequent PCR were carried out with the GeneAmp RNA PCR kit (Perkin Elmer, Foster City, USA) in a Perkin Elmer DNA Thermal Cycler. First strand cDNA was synthesised from 1 μg total RNA in a 20- μl reaction volume using random hexamers as primers. The reaction mixture was incubated at 25 ⁰ C for 10 min, 42 ⁰ C for 15 min, heated to 99 ⁰ C for 5 min, and chilled to 5 ⁰ C for 5 min. For each primer pair a 9-μl portion from the resultant cDNA was amplified by PCR in a final volume of 50 μl. The PCR was carried out with the following profile: 5 min at 95 ⁰ C for 1 cycle, followed by 30 cycles of 1 min at 95 ⁰ C, 1 min at 57 ⁰ C and 30 sec at 72 ⁰ C; final extension was

done for 7 min at 72 ⁰ C. A blank (water) was included in all experiments. Control experiments showed that 30 cycles were within the exponential phase of the PCR (not shown).

The identity of the PCR products was verified by restriction analysis as follows: The endothelin ET _A products were digested with SpH I (Boehringer Mannheim, Germany), generating three fragments of 27, 78 and 159 basepairs, respectively. The endothelin ETβ-products were cleaved with Eco RV (Boehringer), generating two fragments of 106 and 454 basepairs respectively. The lung tissue was snap frozen in -8O ⁰ C after removal. Total cellular RNA was extracted using the FastRNA™ Kit-GREEN and FastPrep® FP 120 Cell Disrupter (BIOlOl, Qbiogene, Carlsbad CA, USA) following the suppliers' instructions. The resulting pellet was finally redissolved in 10 μL diethyl-pyrocarbonate (DEPC)- treated water. Reverse transcription of total RNA to cDNA was carried out using the GeneAmp RNA PCR kit (PE Applied Biosystems, Foster City, CA, USA) in a Perkin-Elmer DNA Thermal cycler. First strand cDNA was synthesized from 1 μg total RNA in a 20 μL reaction volume using random hexamers as primers. The reaction mixture was incubated at 25°C for 10 min, 42°C for 15 min, heated to 99°C for 5 min and chilled to 5 °C for 5 min. Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System (Perkin-Elmer, Applied Biosystems, Foster City, CA, USA) using the GeneAmp S YBR ^® Green kit (Perkin-Elmer, Applied Biosystems Foster City, CA, USA) with the cDNA synthesized above as template in a 50 μL reaction volume. A no template control was included in all experiments. The GeneAmp 5700 Sequence Detection System monitors the growth of DNA in real-time using an optic and imaging system, via the binding of a fluorescent dye to double-stranded DNA. Specific primers for the rat ET _A and ET _B receptors were designed as follows:

ET _A receptor forward; 5 '-ATTGCCCTCAGCGAACAC-3 ' reverse; 5 '-CAACCAAGCAGAAAGACGGTC-S '

ET _B receptor forward; 5 '-GATACGACAACTTCCGCTCCA-3 ' reverse; 5 '-GTCCACGATGAGGACAATGAG-S '

Elongation factor- 1 (EF-I) mRNA was used as a reference, since it is the product of a housekeeping gene, continuously expressed to a constant amount in cells. The EF-

1 primers were designed as follows:

EF- 1 forward; 5 '-GCAAGCCCATGTGTGTTGAA-3 ' reverse; 5 '-TGATGACACCCACAGCAACTG-3 '

The real-time PCR was carried out with the following profile: 50°C for 2 min, 95°C for 10 min, 95°C for 15 sec times 40 and 60°C for 1 min. To prove that the cDNA of EF-I and the ET receptors were amplified with the same efficacy during real-time PCR, a standard curve were made where the C _τ -values were plotted against cDNA concentration based on the equation: C _T = (Ig (1 + E)) ^"1 lg(concentration), where E is the amplification efficiency with the optimal value of 1. To prove that each primer- pair generates only one PCR product, an agarose gel electrophoresis with the PCR products was run.

Drugs

PCR: Oligonucleotides and reagents for the PCR assay were purchased from Perkin- Elmer, Applied Biosystems Foster City, CA, USA.

Calculations

PCR: PCR experiments were performed on n sephadex treated rats and n saline treated rats. The amount of ET _A and ET _B receptor mRNA was calculated as relative to the amount of EF-I mRNA in the same sample by the formula: Xo/Ro = 2 ^ctR'ctx , where X ₀ = original amount of ET receptor mRNA, R ₀ = original amount of EF-I mRNA, CtR = C _τ -value for EF-I and CtX = C _τ -value for the ET receptor. Statistical analysis was performed, where p < 0.05 was considered significant.

EXAMPLE 8

Molecular biology of other receptors

Data suggest that other receptors _. Examples of physiological solutions will AT ₁ 5- HT ₁₅ and B ₁ , respectively, also are upregulated. There might be still other not yet defined receptors that are changed or over expressed in situations of lung disease, which also could contribute to the excessive vasoconstriction in asthma. Incidentally, not only are membrane bound receptors contributing to regulation of lung flow, as their coupling with the activity of different types of ion channels are also relevant. Taken together, an effective method to identify potentially beneficial bioactive agents would require the ability to keep track of different mechanisms.

Extraction of total RNA: Total cellular RNA was extracted using the FastRNA™ Kit-GREEN (BIO 101, Carlsbad CA, USA) following the suppliers' instructions. The resulting pellet was finally washed with 75% ethanol, air-dried and redissolved in 40 μL diethyl-pyrocarbonate (DEPC)-treated water. Reverse transcription of total RNA to cDNA was carried out using the GeneAmp RNA PCR kit (PE Applied Biosystems, Foster City, CA, USA) in a Perkin-Elmer DNA Thermal cycler. First strand cDNA was synthesized from total RNA in a 40 μL reaction volume using random hexamers as primers. The reaction mixture was incubated at 25 ⁰ C for 10 min, heated to 42 ⁰ C for 15 min, further heated to 99 ⁰ C for 5 min and chilled to 5°C for 5 min. Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System using the GeneAmp SYBR ^® Green kit (Perkin- Elmer, Applied Biosystems Foster City, CA, USA) with the cDNA synthesized above as template in a 50 μL reaction volume. A no template control was included in all experiments. The GeneAmp 5700 Sequence Detection System monitored the growth of DNA in real-time using an optic and imaging system, via the binding of a fluorescent dye to double-stranded DNA. Specific primers for the ATi and AT ₂ receptors, ACE and NF -KB were designed as follows:

AT ₁ receptor forward: 5 ^' -GGATGGTTCTCAGAGAGAGTACAT-S ' reverse: 5'-CCTGCCCTCTTGTACCTGTTG -3 ' AT ₂ receptor forward: 5 ^' -TCTGTTAGTGGGATGCATGTAATCA-S ' reverse: 5 '-TGTGGGCCTCCAAACCATT-S ' ACE forward: 5'-CCCGGAAATACGAAGAATTGC-S' reverse: 5 '-GGCTCTCCCCACCTTGTCTC-3 ' NF-KB forward: 5 '-GAGAGCCAGTAGCACGCATG-3 ' reverse: 5 '-CCTGGGTTCGTGGAATGAGT-S '

Elongation factor- 1 (EF-I) and glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) mRNA were used as endogenous standards, and the primers were designed as follows:

EF-I forward: 5 ^' -GCAAGCCCATGTGTGTTGAA-S ' reverse: 5 '-TGATGACACCCACAGCAACTG-3 ' GAPDH

forward: 5 '-GGCCTTCCGTGTTCCTACC-3 ' reverse: 5 '-CGGCATGTCAGATCCACAAC-3 '

The real-time PCR was carried out with the following profile: 50 ⁰ C for 2 min, followed by 40 cycles with 95°C for 15 sec and 60 ⁰ C for 1 min.

Calculations

PCR: PCR experiments were performed on 5-6 rats per tested primer (each sample in duplicate). The amount of mRNA was calculated as relative to the amount of EF- 1 and GAPDH mRNA in the same sample by the formula: X ₀ ZR ₀ = 2 ^ctR"CtX , where X ₀ = original amount of target mRNA, R ₀ = original amount of EF-1/GAPDH mRNA, CtR = C _τ -value for EF-1/GAPDH and C _T X = C _τ -value for the target. By C _T - values we refer to the number of PCR cycles performed per gene product in one sample at a specific point of time. Data are expressed as mean values + SEM. Statistical analyses were performed with unpaired Students' t-test and P < 0.05 was considered significant.

EXAMPLE 9

Western blot

Western blot was used to confirm the presence of activated (phosphorylated) MAPKs in the bronchioles. Segments of bronchioles to be used for Western blots were cultured for 0.5, 1, 2, 3 or 24 h, snap frozen in liquid nitrogen and subsequently stored at -8O ⁰ C until use.

The tissue used for Western blots was incubated initially in 500 DL Laemmli solution and homogenized with a polytron (IKA labortechnik, Germany). The lysates were centrifuged at 5000 X g for 5 minutes at 4° C and the supernatant transferred to new tubes. Further Laemmli solution was added to obtain a concentration of approximately 75 mg tissue/mL. The lysates were stored at -20° C until use.

The proteins were heated to 97-100° C for 3-5 minutes to denature most proteins. Equal amounts of the boiled supernatant fraction were loaded onto a 10 % polyacrylamid gel, separated by electrophoresis (Mini-protean 3, Bio-Rad, CA) and then transferred to semi-blot PVDF membranes (Bio-Rad) by semidry blotting (Trans-blot SD, Bio-Rad). The Western blots were blocked with a solution of 5 % skim milk in TBS-T (0.1 % Tween-20 in TBS) and incubated overnight at 4° C in TBS-T containing skim milk and an appropriate dilution of

polyclonal antibodies. The membranes were washed in TBS-T, incubated for 90 minutes with anti-rabbit IgG horseradish peroxidase-labelled secondary antibody (1:50 000). After further washing, the PVDF membranes were subjected to SuperSignal West and the chemiluminescence was detected by a Luminiscent Image Analyzer (Fujifilm Science Imaging systems, Japan). In the p38 and Akt-protein kinase gels, the cell extract from the rat bronchioles were applied to the gel as a positive control. For further details on the methodology see (Frodin et al., 2002).

Goat polyclonal antibody was used to detect ERK1/2 (Santa Cruz Biotechnology, USA), while a polyclonal rabbit antibody (Promega, USA) was used to detect phospho-ERKl/2 kinase. Rabbit polyclonal antibodies (Cell Signaling Technology, USA) were used to detect p38 MAP kinase, phospho-p38 MAP kinase (Thrl80/Tyrl82), Akt protein kinase, phospho-Akt protein kinase (Thr308) and phospho-Akt (Ser473). Peroxidase-conjugated anti-goat (DAKO, Denmark) and anti-rabbit (Amersham Pharmacia Biotech, UK) immunoglobulins were used as secondary antibodies. For development we used SuperSignal West Dura Extended Duration Substrate (Pierce, Perbio, USA).

EXAMPLE 10

Comparison of DSP with nicotine

DSP contains 0.11 mg/L of nicotine and thus this concentration was used to examine if nicotine was the key substance in the DSP-induced reduction of vasodilatation. Compared to nicotine, DSP had a significantly stronger effect in reducing the ACh-induced dilatation. However, in comparison with DMSO (control) nicotine did not produce any significant reduction of vasodilatation (Fig.