Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: Cerca de 52% dos homens na faixa etÃria entre 40 e 70 anos sofrem de disfunÃÃo erÃtil (DE). A ioimbina (IOI), um inibidor adrenÃrgico &#945;2, vem sendo usada no tratamento da DE hà dÃcadas, e ainda tem as suas indicaÃÃes, porÃm seu mecanismo de aÃÃo ainda permanece obscuro. A maior parte dos estudos com IOI foi realizada em corpos cavernosos e aorta de ratos e coelhos, devido à dificuldade de se obter tecido humano. O objetivo deste estudo à definir o mecanismo de aÃÃo nÃo-adrenÃrgico nÃo-colinÃgico da IOI, avaliando a via nitrÃrgica e a via dos canais iÃnicos no mÃsculo liso de corpo cavernoso humano. MÃtodos: Os corpos cavernosos foram retirados de 13 doadores cadÃveres masculinos (idade 18-53 anos), durante cirurgia pra retirada de ÃrgÃos para transplante, seguindo os protocolos de doaÃÃo de tecidos. As tiras de mÃsculo cavernoso humano foram montadas verticalmente em paralelo, em banho de registro isomÃtrico em soluÃÃo de KHS (pH 7,4, a 37ÂC), constantemente aerada (O2-95% e CO2-5%). Foram realizadas curvas dose-resposta com IOI (10-12 â10-4 M) em tiras de corpos cavernosos de humanos prÃ-contraÃdas com fenilefrina (10 ÂM) e com soluÃÃo despolarizante rica em potÃssio (60 mM de K+), em associaÃÃo com inibidores da via nitrÃrgica e da via de canais iÃnicos (Na+, K+ e Cl-), para estudo das vias NANC. Resultados: A IOI obteve relaxamento importante das tiras de corpo cavernoso humano in vitro na dose 10-4 M. ApÃs prÃ-contraÃÃo com fenilefrina (10 ÂM), as tiras submetidas à IOI nesta dose, relaxaram 95,8%, e quando a prÃ-contraÃÃo foi realizada com soluÃÃo despolarizante rica em K+ (60 mM de K+), Ca++ (2 mM), contendo 10 ÂM guanetidina (simpatolÃtico quÃmico) e 10 ÂM fentolamina (bloqueador &#945;-adrenÃrgico), o relaxamento foi de 69,5% (p<0,05). A IOI (10-4M), apÃs prÃ-contraÃÃo com fenilefrina (10 ÂM), e na presenÃa de 7- NI (10ÂM) e do L-NAME (100ÂM) (bloqueadores das enzimas Ãxido nÃtrico sintetases constitutivas (nNOS e eNOS, respectivamente)) e do ODQ (inibidor da enzima guanilato ciclase solÃvel - 10ÂM) proporcionou relaxamento do mÃsculo cavernoso humano de 57,4%; 55,5%; 62,99%; respectivamente (p<0,05). Em associaÃÃo com TTX (Tetrodotoxina - bloqueador do canal de sÃdio neuronal - 100ÂM), TEA (tetraetilamonio - bloqueador de canais de potÃssio voltagem-dependentes ativados por cÃlcio - 100ÂM), e com apamina + charybdotoxina (inibidores dos canais de potÃssio ativados por cÃlcio de baixa, mÃdia e alta condutÃncia -0,1ÂM + 1ÂM), obteu-se relaxamento de 56,4%; 100%; 100% respectivamente (p>0,05). Jà com a glibenclamida (inibidor dos canais de KATP e de Cl- - 10ÂM), proporcionou relaxamento de 71,1% (p<0,05). ConclusÃo: Os resultados dos estudos farmacolÃgicos sugerem que a IOI relaxa o corpo cavernoso de humano por mecanismo outro que nÃo o seu bloqueio adrenÃrgico, possivelmente ativando a via nitrÃrgica, e via canais de KATP e de CL- (NO â GUANILATO CICLASE â GMPc â canais de KATP e de cloreto). A IOI nÃo age via canais de Na+, e nem atua via canais de potÃssio voltagem-dependentes ativados por cÃlcio, como tambÃm nÃo age nos canais de potÃssio ativados por cÃlcio de baixa, mÃdia e alta condutÃncia. Palavras-chave: ioimbina, corpo cavernoso, disfunÃÃo erÃtil, canais de K+. / Introduction: About 52% of men aged between 40 and 70 years old suffer with erectile dysfunction (ED). Yohimbine (YOH), an &#945;2-adrenergic blocker, has been used for ED treatment for several decades, and it still has its indications. However its mechanism of action still remains obscure. Most of the studies with YOH were made with corpus cavernosum and aorta of rats and rabbits, due to the difficulty of getting human tissue. The aim of this study is to define the non-cholinergic non-adrenergic mechanism of action of the YOH, evaluating the nitrergic pathway and the role of the ionic channels in the human cavernosum smooth muscle. Methods: The corpus cavernosum were removed from 13 male cadavers donors (18-53 years old), during surgery for removing of organs for transplant, following the national protocols for organs donation. The strips of human cavernosum muscle were vertically settled in parallel, in isometric register bath in KHS solution (pH 7,4, at 37ÂC), constantly gassed with (O2-95% and CO2-5%). Curves dose-reply were performed with IOI (10-12 - 10-4 M) in strips of human corpus cavernosum beings pre-contracted with phenilefrine (10 ÂM) and with rich depolarizing solution in potassium (60 mM of K+), in association with nitrergic and ionic channels inhibitors (Na+, K+ and Cl-), for study of NANC ways. Results: The YOH provided an important relaxation for the strips of in vitro human corpus cavernosum under the dose of 10-4 M. After pre-contraction with phenilefrine (10 ÂM), the strips submitted to the YOH in this dose, got relaxed 95.8%, and when the pre-contraction was made with rich depolarizing K+ solution (60 mM of K+), Ca++ (2 mM), containing 10 ÂM guanethidine (chemical simpatolitic) e 10ÂM phentolamine (&#945;-adrenergic blocker), the relaxation was of 69,5% (p<0,05). The YOH (10-4M), after pre-contraction with fenilefrina (10 ÂM), under the presence of 7-NI (10 ÂM) and of L-NAME (100 ÂM) (nitric oxide synthases inhibitors (nNOS and eNOS, respectively)), and of the ODQ (soluble guanylate cyclase enzyme inhibitor â 10 ÂM) provided relaxation of the human cavernosum muscle of 57,4%; 55.5%; 62.99%; respectively (p<0,05). In association with TTX (Tetrodotoxina - neuronal sodium channel inhibitor â 100 ÂM), TEA (tetraetilamonio - potassium channels voltage-dependents activated by calcium blocker â 100 ÂM), and with apamina + charybdotoxina (potassium channels activated by calcium blocker of low and high conductance - 0,1 ÂM + 1 ÂM), we got a relaxation of 56,4%; 100%; 100% respectively (p>0,05). With the glibenclamida (KATP and Cl- channels blocker â 10 ÂM), we got a 71,1% relaxation (p<0,05). Conclusion: The pharmacologic studies results suggest that the YOH relaxes the human corpus cavernosum by another mechanism different of his adrenergic blockade, possibly activating the nitrergic pathway, and through Cl- and KATP channels (NO - GUANILATO CICLASE - GMPc - chloride and KATP channels). The YOH doesnât act through Na+ channels, and doesnât act through potassium channels voltage-dependents activated by calcium, as well as it doesnât act in the K+ channels activated by calcium of low and high conductance.

Initiation of smoking mainly occurs during adolescence. Adolescents experience more stressful life events; therefore, stress may be a factor that contributes to this high risk of smoking initiation. The current study examines the effects of three different stressors (yohimbine, intermittent footshock and social defeat) on nicotine self-administration (NSA) in adolescent and adult rats. The effects of yohimbine and footshock were examined after the establishment of NSA behavior, while the effect of social defeat was tested on the initiation of NSA behavior. Yohimbine increased NSA, but the other two stressors did not. The increase in NSA induced by yohimbine tended to be higher in adults than in adolescents. No marked age differences in response to the other two stressors were observed. These results suggest that stress increases NSA in a stressor-specific manner, and that adolescents do not show enhanced vulnerability to the effect of stress on NSA.

Initiation of smoking mainly occurs during adolescence. Adolescents experience more stressful life events; therefore, stress may be a factor that contributes to this high risk of smoking initiation. The current study examines the effects of three different stressors (yohimbine, intermittent footshock and social defeat) on nicotine self-administration (NSA) in adolescent and adult rats. The effects of yohimbine and footshock were examined after the establishment of NSA behavior, while the effect of social defeat was tested on the initiation of NSA behavior. Yohimbine increased NSA, but the other two stressors did not. The increase in NSA induced by yohimbine tended to be higher in adults than in adolescents. No marked age differences in response to the other two stressors were observed. These results suggest that stress increases NSA in a stressor-specific manner, and that adolescents do not show enhanced vulnerability to the effect of stress on NSA.

It has been well established that the stress-related neurochemical systems corticotropin-releasing factor (CRF), noradrenaline (NA), and dopamine (DA) mediate stress-induced reinstatement of drug seeking. The three series of experiments presented in this dissertation constitute a further exploration of the role these neurochemical circuits play in reinstatement by providing the first direct exploration of whether central CRF and catecholamine (NA and DA) systems interact to influence reinstatement of cocaine seeking. The primary objective of the first series of experiments was to determine whether NA and CRF systems interact to mediate reinstatement of cocaine seeking and, if so, to determine the direction of this interaction. Results showed that central administration of NA induced reinstatement and up-regulated the expression of c-fos mRNA, a marker of neuronal activation, in brain regions involved in footshock-induced reinstatement. Pretreatment with a CRF antagonist blocked NA-induced reinstatement. In contrast, pretreatment with the α2-adrenoceptor agonist, clonidine, failed to block CRF-induced reinstatement. Taken together, these findings suggest a functional interaction between NA and CRF systems in mediating stress-induced reinstatement of cocaine seeking, whereby activation of CRF receptors occurs subsequent to, and downstream of, the sites of action of NA. A second series of experiments examined the role of D1- and D2-like receptors in CRF-induced reinstatement. Pretreatment with the D1- or D2-like receptor antagonists, SCH23390 and raclopride, respectively, dose-dependently blocked CRF-induced reinstatement of cocaine seeking. Taken together with previous findings, these results suggest that CRF-induced reinstatement of cocaine seeking likely involves DAergic signaling via D1- and D2-like receptors, subsequent to activation of CRF receptors. The final series of experiments investigated the neuropharmacology of yohimbine-induced reinstatement, focusing on the roles of α2-adrenoceptors, D1- and D2-like receptors. These experiments were prompted by an unexpected finding in the first series of experiments, in which a CRF antagonist failed to interfere in yohimbine-induced reinstatement of cocaine seeking. Results showed that pretreatment with the α2-adrenoceptor agonist, clonidine, or raclopride, prior to tests for yohimbine-induced reinstatement failed to influence responding. In contrast, pretreatment with SCH23390 blocked yohimbine-induced reinstatement. Taken together, these findings suggest that yohimbine may act through system(s) other than NA to have its effects.

It has been well established that the stress-related neurochemical systems corticotropin-releasing factor (CRF), noradrenaline (NA), and dopamine (DA) mediate stress-induced reinstatement of drug seeking. The three series of experiments presented in this dissertation constitute a further exploration of the role these neurochemical circuits play in reinstatement by providing the first direct exploration of whether central CRF and catecholamine (NA and DA) systems interact to influence reinstatement of cocaine seeking. The primary objective of the first series of experiments was to determine whether NA and CRF systems interact to mediate reinstatement of cocaine seeking and, if so, to determine the direction of this interaction. Results showed that central administration of NA induced reinstatement and up-regulated the expression of c-fos mRNA, a marker of neuronal activation, in brain regions involved in footshock-induced reinstatement. Pretreatment with a CRF antagonist blocked NA-induced reinstatement. In contrast, pretreatment with the α2-adrenoceptor agonist, clonidine, failed to block CRF-induced reinstatement. Taken together, these findings suggest a functional interaction between NA and CRF systems in mediating stress-induced reinstatement of cocaine seeking, whereby activation of CRF receptors occurs subsequent to, and downstream of, the sites of action of NA. A second series of experiments examined the role of D1- and D2-like receptors in CRF-induced reinstatement. Pretreatment with the D1- or D2-like receptor antagonists, SCH23390 and raclopride, respectively, dose-dependently blocked CRF-induced reinstatement of cocaine seeking. Taken together with previous findings, these results suggest that CRF-induced reinstatement of cocaine seeking likely involves DAergic signaling via D1- and D2-like receptors, subsequent to activation of CRF receptors. The final series of experiments investigated the neuropharmacology of yohimbine-induced reinstatement, focusing on the roles of α2-adrenoceptors, D1- and D2-like receptors. These experiments were prompted by an unexpected finding in the first series of experiments, in which a CRF antagonist failed to interfere in yohimbine-induced reinstatement of cocaine seeking. Results showed that pretreatment with the α2-adrenoceptor agonist, clonidine, or raclopride, prior to tests for yohimbine-induced reinstatement failed to influence responding. In contrast, pretreatment with SCH23390 blocked yohimbine-induced reinstatement. Taken together, these findings suggest that yohimbine may act through system(s) other than NA to have its effects.

People who endure horrific, life-threatening experiences are at risk for developing post-traumatic stress disorder (PTSD). However, only about 25% of all individuals who experience trauma develop PTSD. Recent research indicates that the presence of certain physiological conditions, such as reduced cortisol and parasympathetic inhibition, during trauma may increase one's susceptibility to developing PTSD. Thus, I attempted to develop a novel animal model of PTSD and test the hypothesis that reduced adrenal and parasympathetic activity during stress would exacerbate its long-term effects on behavior.In Experiment One, adult male rats were exposed to two stress sessions, each involving one hour of immobilization plus cat exposure. Before each session, rats were injected with vehicle, metyrapone, AF-DX 116, or both drugs. The second session occurred 10 days after the first and served to model a traumatic flashback. Stressed rats endured unstable housing conditions throughout t he experiment to add an element of daily anxiety. Three weeks after the second session, all rats underwent a battery of tests to examine the lasting effects of stress on physiology and behavior. The results indicated that stressed rats exhibited heightened anxiety on the elevated plus maze, an exaggerated startle response, and greater blood pressure, relative to controls. Moreover, metyrapone, when combined with stress, led to significant short- and long-term spatial memory impairments. Experiment Two assessed the effects of the same stress paradigm on rats' sensitivity to yohimbine, an alpha-2 adrenergic receptor antagonist. Yohimbine induces flashbacks and panic attacks in patients with PTSD; thus, I hypothesized that stressed rats would react abnormally to this agent. Stressed and unstressed rats were administered vehicle or yohimbine (1 mg/kg) 30 min prior to behavioral testing. The results indicated that stressed rats were hyperresponsive to yohimbine, as evidenced by a greater su ppression of rearing, greater avoidance of the center of the open field, and a greater suppression of activity on the elevated plus maze, relative to controls. Collectively, the findings of these studies indicate that uncontrollable and unpredictable psychological stress produces lasting changes in the physiology and behavior of rats that resemble symptoms commonly observed in people with PTSD.

Summary: The effects of an alpha2 adrenoceptor blocker, yohimbine, and an alpha1 adrenoceptor blocker, phenoxybenzamine, and the central alpha2 adrenoceptor agonist, clonidine, on changes in arterial blood pressure and heart rate were studied during fatiguing muscular contractions to determine whether an adrenergic-opioidergic system might be involved in the mediation of cardiovascular function. Fatiguing contractions of the gastrocnemius and plantaris muscles of cats caused an increase in mean arterial blood pressure to 150-170 mmHg from resting values of 110-120 mmHg. Injection of clonidine into the cerebral aqueduct eliminated the increase in blood pressure; this effect was dose dependent. Naloxone antagonised the effects of the highest dose of clonidine (5 μg). Injections of yohimbine (1 μg) into the cerebral aqueduct had no significant effect on this pressor response. Yohimbine (1 μg) effectively counteracted the antipressor effects of clonidine when the two drugs were injected together until higher doses of clonidine (2-5 μg) were used. Phenoxybenzamine had no effect on the pressor response itself but unlike yohimbine was able to attenuate the effects of clonidine only when injected together. These data suggest that activation of muscle ergoreceptor afferent nerve fibres (group III and IV fibres) during muscular contractions may cause an increase in arterial blood pressure by interfering with an inhibitory adrenergic-endorphinergic pathway in the medullary region of the brainstem.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.