Browsing by Subject "nikotiini"
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Aivojen kolinerginen järjestelmä ja histamiinireseptorit Parkinsonin taudin 6-OHDA-jyrsijämallissa (2015)Parkinson's disease is a neurodegenerative disease where the nigrostriatal dopaminergic cells die gradually causing severe motor symptoms. Current treatment of the disease relieves the symptoms but does not affect the progression of the disease, nor does it have a neuroprotective effect. The most important drug for the treatment of Parkinson's disease is L-dopa, the precursor of dopamine. With long-term use, L-dopa loses its efficacy and patients start to get adverse effects. The most significant adverse effects are abnormal involuntary movements called dyskinesias. In the literature review of this thesis Parkinson's disease and its treatment is briefly described. Review focuses on the description of the brain cholinergic and histaminergic systems and their receptors along with the available studies about cholinergic and histaminergic neurotransmission in Parkinson's disease 6-hydroxydopamine (6-OHDA) rodent model. The experimental part of this thesis consisted of two different set of experiments and in both of these the dopamine neurons were destroyed unilaterally by injecting 6-OHDA into the striatum. The aim of the first experiment was to examine histamine H3-receptor antagonist JNJ-39220675 and α7-nicotinic receptor agonist PHA-543613, and their combination therapy effects on motor function and the concentrations of striatal neurotransmitters in hemiparkinsonian mice. Effects on motor function were studied two and four weeks after the 6-OHDA injection with cylinder test, the D-amphetamine-induced rotations, and the inverted grid test. After behavioral tests, mice were sacrificed and striatal neurotransmitter concentrations were determinated by HPLC. The aim of the second experiment was to examine if nicotine can relieve L-dopa-induced dyskinesias. In this experiment 6-OHDA was injected at two sites into the striatum, which was intended to produce more extensive destruction of dopaminergic neurons than in the first experiment. The extent of the lesion by 6-OHDA was verified before starting chronic L-dopa treatments with cylinder test. One month after the 6-OHDA injection, five mice were sacrificed and their striatum and substantia nigra sections were measured for destruction of dopaminergic neurons by immunohistochemical TH-staining. Chronic L-dopa treatment with benserazide was started 49‚àí63 days after the 6-OHDA injection. At the same time, mice were divided into two groups. Half of them got normal drinking water and half got nicotine water. During the chronic L-dopa treatment, development of dyskinesias was observed once a week by video tracking. The cylinder test was also done once again after starting the L-dopa treatment. In the first experiment, H3-receptor antagonist JNJ-39220675 showed promising results in improving motor function. Mice used the impaired (contralateral) paw more in the cylinder test and rotated less to the ipsilateral side in the D-amphetamine-induced rotation test than control animals two weeks after the 6-OHDA injection. Combination therapy also reduced the ipsilateral rotations but in the cylinder test it had no effect two weeks after 6-OHDA injection. Because the asymmetry in behavioural tests were caused by destroying dopaminergic neurons, balancing of the motor skills can result from decreased levels of dopamine in the intact side or from increased dopamine levels or stronger dopaminergic postsynaptic transmission in the lesion side. The results four weeks after 6-OHDA injection are not reliable because the striatal samples showed that dopamine concentrations in the lesion side were very close to that of the intact side indicating recovery from the lesion. In the second experiment, mice developed dyskinesias which were decreased with nicotine treatment. Mice also used the contralateral side paw less indicative of loss of dopamine neurons. In agreement, TH-immunostaining confirmed significant loss of TH-positive neurons. Based on these findings, the 6-OHDA injection site, the selected drug doses, and the experimental design seem to fit the evaluation of dyskinesias. The occurrence of dyskinesias and nicotine's effect on them was seen strongest in the body movements. Dyskinesias in forelimbs were minor, but the nicotine treatment decreased them also.
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(2012)Kolinergiset α6*-nikotiinireseptorit ovat kiinnostavia, koska ne liittyvät mahdollisesti Parkinsonin tautiin ja nikotiiniriippuvuuteen. Ionikanavina toimivat nikotiinireseptorit ovat muodostuneet viidestä alayksiköstä, jotka esiintyvät erilaisina yhdistelminä. α6-alayksikköä sisältävät nikotiinireseptorit sijaitsevat presynaptisesti ja säätelevät dopamiinin vapautumista dopaminergisessä hermopäätteessä. α6*-nikotiinireseptorit ovat keskittyneet vain tietyille aivoalueille ja niitä esiintyy runsaasti dopaminergisissä hermosoluissa. α6*-nikotiinireseptoreita on erityisesti mesolimbisen ja nigrostriataalisen hermoradan dopaminergisissä hermosoluissa. Lisäksi niitä on paljon näkemiseen liittyvillä aivoalueilla. Nikotiini toimii asetyylikoliinin tavoin aktivoimalla α6*-nikotiinireseptoreita, mikä johtaa dopamiinin vapautumiseen hermopäätteessä. α6*-nikotiinireseptoreiden sijainnin, määrän ja toiminnan tutkimisessa on käytetty apuna muun muassa niille selektiivisiä antagonisteja, saalistavista merietanoista peräisin olevia α-konotoksiineja, erityisesti α-konotoksiini MII:ta. Nigrostriataalisella hermoradalla, joka ulottuu substantia nigrasta striatumiin, α6*-nikotiinireseptorit voivat vaikuttaa liikkeen säätelyyn. Nikotiini vapauttaa dopamiinia nigrostriataalisen hermoradan päätepisteessä, striatumissa, mikä voi lisätä liikeaktiivisuutta. Nikotiinilla on havaittu olevan hyödyllisiä vaikutuksia Parkinsonin taudin eläinmalleissa, mutta Parkinsonin tautipotilailla nikotiinihoidosta saadut tutkimustulokset ovat ristiriitaisia ja puutteellisia. α6*-nikotiinireseptoreille voitaisiin kehittää selektiivisiä agonisteja, joiden avulla lääkehoito voitaisiin kohdentaa paremmin ja vältyttäisiin mahdollisilta haittavaikutuksilta. Tupakanvieroitukseen tarvittaisiin lisää uusia selektiivisiä lääkehoitoja, joilla olisi hyvä hoitomyöntyvyys ja mahdollisimman vähän haittavaikutuksia. Mesolimbinen hermorata, joka ulottuu ventraaliselta tegmentaalialueelta nucleus accumbensiin, liittyy riippuvuuden syntyyn. Nikotiinin vaikutukset välittyvät VTA:n kautta nucleus accumbensiin, jossa vapautuu dopamiinia. Osa nikotiinin vaikutuksista välittyy myös presynaptisten α6*-nikotiinireseptorien kautta. Selektiivisistä α6*-nikotiinireseptoreiden antagonisteista voisi olla hyötyä nikotiiniriippuvuuden hoidossa, sillä niiden vaikutus vastaisi osittaisagonistin vaikutusta. α-konotoksiini PIA:n vaikutuksia nikotiinin aiheuttamaan dopamiinin vapautumiseen tutkittiin in vivo mikrodialyysimenetelmällä. α-konotoksiini PIA saalistavasta merietanasta eristetty selektiivinen α6*-nikotiinireseptoreiden antagonisti. Tutkimuksessa käytettiin vapaana liikkuvia urospuoleisia Wistar-rottia. Tutkimuksen kohteena olevat aivoalueet olivat striatum ja nucleus accumbens. Rotille asennettiin anestesiassa ohjauskanyyli joko striatumiin tai nucleus accum-bensiin stereotaktisen laitteen avulla. Mikrodialyysikokeessa koetinten tasapainotuksen jälkeen kerättiin perustason näytteet ja pistettiin saliini tai nomifensiini tai vaihdettiin Ringer-ruiskun tilalle α-konotoksiini PIA-ruisku. Puolen tunnin päästä pistettiin saliini tai nikotiini ja vaihdettiin Ringer-ruisku takaisin. Näytteitä kerättiin 15 minuutin välein yhteensä 5,5 tuntia. Lopuksi aivot otettiin talteen ja niistä tehtyjen aivoleikkeiden avulla tarkastettiin koetinten paikat. Mikrodialyysinäytteistä määritettiin HPLC-menetelmällä dopamiinin ja sen metaboliittien DO-PAC:n ja homovaniliinihapon sekä koejärjestelyssä oletettavasti muuttumattomana pysyvän 5-HIAA:n pitoisuudet. Koejärjestelyssä päätettiin käyttää tutkittavien aivoalueiden dopamiinipitoisuuden nostamiseen nomifensiinia, joka estää dopamiinin takaisinottoa hermopäätteissä. Käsittelyryhminä olivat saliini-saliini (n=striatum ja nucleus accumbens, 8+7), saliini-nomifensiini (n=8+4), saliini-nikotiini (n=3+4), nomifensiini-nikotiini (n=10+13) ja nomifensiini-nikotiini-α-konotoksiini PIA (n=8+5). Rottia jouduttiin hylkäämään eri syistä joko ennen mikrodialyysia, mikrodialyysin aikana tai sen jälkeen. Rottia hylättiin yhteensä 70 kpl. Tilastollisessa analyysissä tutkittujen käsittelyiden tai aivoalueiden välille ei saatu merkitseviä eroja, koska eläinten välinen hajonta oli liian suurta. Silmämääräisesti nomifensiini-nikotiinikäsittely nosti striatumin ja nucleus accumbensin dopamiinipitoisuuksia. α-konotoksiini PIA näytti estävän dopamiinin vapautumista striatumissa ja nucleus accumbensissa, mutta erot nomifensiini-nikotiinikäsittelyyn eivät olleet tilastollisesti merkitseviä. AUC-arvolla mitattuna α-konotoksiini PIA esti dopamiinin vapautumista striatumissa 39,6 % ja nucleus accumbensissa 31,3 %. Aivoalueiden välillä ei ollut tilastollisesti merkitseviä eroja.
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(2013)Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channel receptors which are widely distributed in human brain. nAChRs are often expressed pre-synaptically and they modulate the release of other neurotransmitters. nAChRs consist of five subunits: nine different subunits have been identified so far, forming multiple different nAChR subtypes with different pharmacological properties. nAChRs participate extensively in physiological functions and pathophysiological conditions. nAChRs mediate the effects of endogenous agonist, acetylcholine, as well as commonly used substance of abuse, nicotine. Addictive drugs such as nicotine and opioids cause adaptive changes in central nervous system. In addition to binding site of acetylcholine, various allosteric binding sites have been identified in nAChRs. Allosteric ligands are able to modulate the effect of agonist by binding to allosteric binding site. The aim of the experimental part of the pro gradu was to study in vitro interactions of nicotine and three different opioids, codeine, oxycodone and tramadol in SH-SY5Y cells. SH-SY5Y cells express endogenously α3* and α7-nAChRs. Binding assays were performed with radioactive ligand [3H]-epibatidine. Functional interactions of nicotine and the opioids were studied with 86Rb+- efflux assay. Codeine, oxycodone and tramadol exhibited receptor level interactions with nicotine in SH-SY5Y cells. Observed interactions were mediated by nAChRs. The opioids inhibited nAChR activation caused by nicotine without binding to the [3H]-epibatide binding site. Codeine, oxycodone and tramadol appear to affect as weak non-competitive antagonists of nAChRs. These results give further information of nicotine-opioid interactions at receptor level. There are indications that nicotine and opioids also have interactions in vivo, which may be partly explained with these receptor level interactions.
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(2012)Smoking is one of the major causes for premature deaths worldwide. Tobacco smoke contains nicotine, which activates the nicotinic acetylcholine receptors (nAChR) expressed by the human body. nAChRs are part of the cholinergic system and its endogenous neurotransmitter is acetylcholine. The nAChRs are excitatory and the often regulate the release of other neurotransmitters. Nicotine is one of the most addicting compounds known. The rewarding effects of nicotine are mediated through the activation of the mesolimbic dopamine pathway. The mesolimbic pathway is triggered also by the compounds activating the endogenous opioid system thus mediating the rewarding effects and opioid addiction. The nicotine - opioid interactions have been widely studied. It is observed that majority of opioid abusers and patients receiving opioid replacement therapy are smokers. It has been also detected that nicotine releases endogenous opioid peptides in vivo in the brain regions mediating both addiction and analgesia. In addition, the rewarding effect of nicotine attenuates in opioid receptor knock-out rodents. Furthermore, it has been observed that nicotine's rewarding effects can be reduced with opioid receptor antagonists. In order to prevent smoking's negative effects the use opioid antagonists for smoking cessation has been clinically researched with poor results. Many of the opioids in clinical use have diverse and direct interaction with the nAChRs in vitro. E.g. it has been observed that methadone and morphine have an effect on the function of the nAChRs. This may explain partially the smoking behaviour of replacement therapy patients. Opioids are prescribed mainly for the treatment of moderate to intense pain. Nicotine is too found to be analgesic in vivo but in humans its analgesic effect has been questionable. In the experimental part of thesis binding and functional interactions with human's α4β2-nAChR expressed by SH-EP1-hα4β2 cell line was researched with clinically commonly used opioids codeine, oxycodone and tramadol. Competitive binding was studied using [3H]-epibatidine binding assay and the functional effects were studied using 86Rb+-efflux assay. The results suggest that oxycodone and tramadol act as weak competitive antagonists of α4β2-nAChR in vitro in concentrations that are clinically irrelevant. According to the results, however, codeine acts as positive allosteric modulator of α4β2-nAChR potentiating the effects of nicotine in micromolar concentrations. The effect is similar to galantamine, used in treatment of Alzheimer's disease. The clinical relevance of codeine's potentiating nicotine's effect on the function of α4β2-nAChR cannot be estimated according to the results from these studies. Therefore, in order to confirm the results experiments with codeine need to be done in vivo using e.g. α4- and β2-knock-out mice in order to clarify α4β2-nAChR's role in the analgesic and rewarding effects of codeine. However, the results from the experimental part provide valuable information on the interactions of nicotine and opioids. Results from studies conducted with α4β2-nAChRs have not been published enough to determine the importance of the phenomenon in i.a. drug addiction and analgesia.
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