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

Neuropeptides are present in nerve fibers of the upper and lower airways. Local release of these substances may be of importance for the pathophysiology of airway disorders and may play a role in responses to different stimuli. However, little is known about the distribution of neuropeptides in the larynx. The superior laryngeal nerve is one of the vagal branches supplying the larynx. The aim of the present study was to investigate the fiber composition of this nerve and to analyse the distribution of different neuropeptides and mast cells in the larynx. The internal and the external branches of the superior laryngeal nerve had a similar number and size of the nerve fibers. Numerous unmyelinated fibers were evenly distributed in the branches. A large majority of the fibers were sensory myelinated and unmyelinated fibers; only a few of the myelinated fibers of the external branch ( 2-10 %) were motor. About a quarter of the unmyelinated fibers of the internal and the external branches had their cell bodies in the brainstem, and single myelinated and unmyelinated fibers emanated from the superior cervical ganglion. In every superior laryngeal nerve examined one to three spherical paraganglia were observed. These paraganglia contained cells which were similar to the type I and type II cells found in the carotid body and the paraganglia of the recurrent laryngeal nerve. Thin-walled sinusoidal blood vessels which were sometimes fenestrated were also present The laryngeal mucosa was supplied with nerve fibers exhibiting substance P- and calcitonin gene-related peptide-like immunoreactivity with regional differences in the distribution. The laryngeal side of the epiglottis and the ventral recess were richly supplied, and the vocal cords showed no evidence of immunoreactive nerve fibers. The distribution of connective tissue mast cells and mucosal mast cells/globular leucocytes was similar to that of nerve fibers displaying substance P- and calcitonin gene-related peptide-like immunoreactivity. These cells were found in close approximation to nerve fibers. Acetylcholinesterase-positive ganglionic cells in the larynx showed vasoactive intestinal polypeptide-, neuropeptide Y-and enkephalin-like immunoreactivity. Neuropeptide Y-like immunoreactivity was co-localized with tyrosine-hydroxylase/dopamine beta-hydroxylase-like immunoreactivity in nerve fibers in some blood vessel walls. Enkephalin-like immunoreactivity was rarely found in this location and co-localization with tyrosine- hydroxylase-like immunoreactivity was not detected. In glands and some blood vessel walls neuropeptide Y- and enkephalin-like immunoreactivity were localized in nerve fibers showing a positive acetylcholinesterase reaction and vasoactive intestinal polypeptide-like immunoreactivity. Thus, this indicates that neuropeptide Y is present in both the sympathetic and parasympathetic nervous systems, while enkephalin and vasoactive intestinal polypeptide are confined to the parasympathetic nervous system in the rat larynx. The present study shows that the superior laryngeal nerve is mainly sensory, and the study also provides a morphological basis for neuropeptide effects in laryngeal physiology/pathophysiology. / <p>S. 1-27: sammanfattning, s. 29-97: 6 uppsatser</p> / digitalisering@umu

There is an unmet medical need for the efficient treatment of neuropathic pain, a condition that affects approximately 10% of the population worldwide. Current therapies need to be improved due to the associated side effects and lack of response in many patients. Moreover, neuropathic pain causes great suffering to patients and puts an economical burden on society. The work presented in this thesis addresses SP1-7, (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), a major metabolite of the pronociceptive neuropeptide Substance P (SP). SP is released in the spinal cord following a noxious stimulus and binds to the NK1 receptor. In contrast to SP, the degradation fragment SP1-7 is antinociceptive through binding to specific binding sites distinct from the NK1 receptor. The aim of this thesis was to investigate the impact of SP1-7 on neuropathic pain. To understand how SP1-7 exerts its effect, a series of N-truncated forms of the heptapeptide were biologically evaluated. A set of small high-affinity ligands was evaluated in animal models of neuropathic pain. To confirm a clinical relevance the levels of SP1-7 in human neuropathic pain were assessed incerebrospinal fluid (CSF) collected from neuropathic pain patients. The results showed that SP1-7 could alleviate thermal as well as mechanical hypersensitivity in three different animal models of neuropathic pain. C-terminal amidation was connected with increased efficacy. N-terminal truncation of SP1-7 indicated a necessity of five amino acids in order to retain biological effect. One small high-affinity ligand showed a significant anti-allodynic effect. CSF levels of SP1-7 in neuropathic pain patients were lower compared to controls. Taken together, these findings demonstrate that the formation of SP1-7 may be attenuated in neuropathic pain. C-terminal amidation and a majority of its amino acids are necessary for stability and permeability. Clearly, SP1-7 and SP1-7 mimetics with high affinity to the SP1-7 binding site ameliorate neuropathic pain-like behaviors in animal models of neuropathic pain. Overall, the findings presented in this thesis contribute to new knowledge regarding the role of SP1-7 and related analogues and fragments in neuropathic pain. In a future perspective, this could be essential for the development of efficient strategies for managing patients with neuropathic pain.

The abuse of anabolic androgenic steroids (AAS) is becoming increasingly common and may result in a range of physiological as well as psychological effects such as altered behavior in terms of increased aggression, cognitive dysfunction and addictive behavior. AAS comprise testosterone and its derivatives, of which nandrolone is one of the more common. Previous studies have shown nandrolone-induced effects in male rats on peptide levels within the Substance P (SP) system and the dynorphinergic system; these effects may be linked to some of the reported behavior alterations. The studies presented in this thesis aimed to investigate the mechanisms underlying these peptide alterations and also to further investigate neuropeptidergic effects attributed to nandrolone administration. The results display significant effects on the enzymatic conversion of SP and Dynorphin A into their bioactive metabolites SP(1-7) and Leu-enkephalin-Arg6, respectively, as a result of nandrolone treatment. More profound investigations on the dynorphinergic system displayed effects on the kappa opioid receptor density in various brain regions. There was also a significant increase in the expression of the gene transcript of prodynorphin in the hippocampus, a brain region associated with cognitive processes. In addition, impaired spatial learning and memory in the Morris water maze task following nandrolone administration was encountered. The results provide further understanding regarding neuropeptidergic mechanisms underlying AAS-induced behavioral effects.

Biologically active peptides are important for many physiological functions in the human body and therefore serve as interesting starting points in drug discovery processes. In this work the neuropeptide substance P 1–7 (SP1–7, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), which has been demonstrated to reduce neuropathic pain and attenuate opioid withdrawal symptoms in animal models, has been addressed in a medicinal chemistry program with the overall aim of transforming this bioactive peptide into more drug-like compounds. Specific binding sites for this neuropeptide have been detected in the brain and the spinal cord. Interestingly, the smaller neuropeptide endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) also interacts with these binding sites, although 10-fold less efficient. In this work the structure–activity relationship of SP1–7 and EM-2, regarding their affinity to the SP1–7 binding site was elucidated using alanine scans, truncation, and terminal modifications. The C-terminal part of both peptides, and especially the C-terminal phenylalanine, was crucial for binding affinity. Moreover, the C-terminal functional group should preferably be a primary amide. The truncation studies finally resulted in the remarkable discovery of H-Phe-Phe-NH2 as an equally good binder as the heptapeptide SP1–7. This dipeptide amide served as a lead compound for further studies. In order to improve the drug-like properties and to find a plausible bioactive conformation, a set of rigidified and methylated dipeptides of different stereochemistry, and analogs with reduced peptide character, were synthesized and evaluated regarding binding, metabolic stability and absorption. Small SP1–7 analogs with retained affinity and substantially improved permeability and metabolic stability were identified. Beside peptide chemistry the synthetic work included the development of a fast and convenient microwave-assisted protocol for direct arylation of imidazoles. Furthermore, microwave-assisted aminocarbonylation using Mo(CO)6 as a solid carbon monoxide source was investigated in the synthesis of MAP amides and for coupling of imidazoles with amino acids. In a future perspective the present findings, together with the fact that some of the SP1–7 analogs discovered herein have been shown to reproduce the biological effects of SP1-7 in animal studies related to neuropathic pain and opioid dependence, can ultimately have an impact on drug discovery in these two areas.

Submitted by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-23T13:14:13Z No. of bitstreams: 1 Publico-NOVO-05.pdf: 1218530 bytes, checksum: 4ed86e26ba531f290cfc9a29f57fba4d (MD5) / Approved for entry into archive by Maria Anália Conceição (marianaliaconceicao@gmail.com) on 2016-06-23T13:15:03Z (GMT) No. of bitstreams: 1 Publico-NOVO-05.pdf: 1218530 bytes, checksum: 4ed86e26ba531f290cfc9a29f57fba4d (MD5) / Made available in DSpace on 2016-06-23T13:15:03Z (GMT). No. of bitstreams: 1 Publico-NOVO-05.pdf: 1218530 bytes, checksum: 4ed86e26ba531f290cfc9a29f57fba4d (MD5) Previous issue date: 2014 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Introdução: A disfunção na liberação de neuropeptídeos acarreta alterações na pele, podendo gerar distúrbios no processo cicatricial e/ou afecções cutâneas. Sendo assim, a liberação de neuropeptídeos é estudada na literatura, relacionando a utilização de agentes eletrofísicos. Objetivo: Investigar o efeito da estimulação elétrica na liberação de neuropeptídeos SP e CGRP em pele de ratos. Métodos: Foram utilizados 28 animais distribuídos em 4 grupos, Grupo Controle (GC): as amostras foram coletadas após 60 minutos da tricotomia sem estímulo elétrico; Grupo Sham (GS): após 60min da tricotomia foram colocadas as placas de eletrodos com esponja umedecida com cloreto de sódio 0,9% e sobreposto na linha mediana dorsal e dispersivo na região ventral, com o aparelho desligado por 30minutos; Grupo Estimulação Elétrica com Polo Positivo (GPP): após 60 minutos da tricotomia, foi realizada a estimulação elétrica sobre a linha mediana dorsal com polaridade Positiva e o no Grupo Estimulação Elétrica com polaridade Negativa (GPN): os mesmos parâmetros utilizados acima, com a mudança, onde o polo negativo passou para linha mediana dorsal. Ao término da estimulação elétrica, foram coletadas amostras de pele, submetidas ao Western blotting para análise dos neuropeptídeos CGRP e SP. Para a análise estatística foi utilizado o teste de Análise Variância (ANOVA) para identificar a diferença entre os grupos. Resultado: Não causou diferença significante na liberação de CGRP e SP na pele de rato. Conclusão: A estimulação elétrica ultraexcitante não influenciou na liberação de neuropeptídeos, CGRP e SP, em pele de ratos. / Introduction: A dysfunction in the release of neuropeptides cause changes in the skin, and may cause disturbances in the healing process and/or skin disorders. Therefore the release of neuropeptides has been studied in the literature, connecting the use of electrophysical agents. Objective: To investigate the effect of electrical stimulation on the release of neuropeptides SP and CGRP in rat skin. Methods: 28 animals were randomly divided into 4 groups, Control Group (CG): the samples were collected 60 minutes after shaving without electrical stimulation; Sham Group (SG): after 60 minutes of trichotomy were placed electrodes plates with damp sponge with sodium chloride 0.9% and superimposed on the dorsal midline and dispersive on the ventral region, with the equipment off for 30 minutes; Electrical Stimulation Positive Pole Group (GPP): later 60 minutes of trichotomy, electrical stimulation was performed on the dorsal midline with positive polarity and the Electrical Stimulation Group with negative polarity (NPG): the same parameters used above, with the change where the negative pole was passed into the dorsal midline. At the end of electrical stimulation, skin samples, tested by Western Blotting for analysis of neuropeptides CGRP and SP, were collected. For statistical analysis, analysis of variance (ANOVA) was used to identify differences between the groups. Result: There was a minor release of neuropeptides from the negative and positive polarity, however, not significant. Conclusion: The ultra-exciting electrical stimulation did not influence the release of neuropeptides, SP and CGRP, in rat skin.

Objetivos: Avaliar a implicação da doença periodontal (DP) avançada, e da expressão da NOS e sP dos tecidos gengivais inflamados, na intensidade de dor e na qualidade de vida de pacientes com dor crônica crânio-facial. Casuística e Métodos: foram avaliados e tratados 20 pacientes com queixas de dores crônicas crânio-faciais e DP (Grupo de Estudo), comparativamente com 20 pacientes que tinham somente DP (Grupo Controle). Todos os pacientes receberam tratamento cirúrgico periodontal. A avaliação foi realizada pré e pós-tratamento periodontal (7, 30 e 180 dias). Instrumentos de avaliação: ficha clínica EDOF-HC, Escala Visual Analógica (EVA), questionário de dor McGill, Índices de Placa (IP), Sangramento (IS), Profundidade Clínica de Sondagem (PCS) e de Inserção (PCI); questionário de qualidade de vida WHOQOL-bref e análise imunohistoquímica para a expressão da óxido nítrico sintase (NOS) e da substância P do tecido gengival inflamado. A avaliação da dor foi realizada por investigador independente. Resultados: Os grupos apresentaram evolução clínica semelhante pelos parâmetros da DP: IP (p=0,0934), IS (p=0,8657), PCS (p=0,1728) e PCI (p=0,7406) nos três momentos avaliados. Houve redução da EVA no Grupo de Estudo aos 30 dias (p<0,05) e 180 dias (p<0,05), comparativamente ao pré-operatório. Aos 180 dias houve melhora significativa das queixas iniciais (p=0,005 para o Grupo de Estudo e p = 0,027 para o Grupo Controle), comparativamente ao pré-operatório, entretanto, houve diferença significativa entre os dois grupos nos três momentos da avaliação, seja para o percentual de melhora (p<0,001), seja para os descritores de melhora Edof-HC (p=0,004 aos 30 dias e p=0,001 aos 180 dias). No Grupo de Estudo, houve redução significativa dos descritores afetivos de dor (questionário de dor McGill) aos 180 dias (p = 0,014). Os escores de qualidade de vida do Grupo de Estudo foram inferiores aos do Grupo Controle nos domínios psicológico e físico (p<0,001 e p=0,007, respectivamente). As expressões da nNOS e da iNOS foram maiores nas gengivas inflamadas do Grupo de Estudo (p<0,001 e p= 0,003, respectivamente) e a expressão da sP foi semelhante nos dois grupos (p=0,363). Conclusões: Houve redução da intensidade da dor crônica crânio-facial dos pacientes que receberam tratamento para doença periodontal avançada. A expressão da substância P foi semelhante nos tecidos gengivais inflamados dos dois grupos, mas a expressão da nNOS e iNOS foi superior na gengiva inflamada dos pacientes com dor crônica crânio-facial. Pelos dados deste estudo a Doença Periodontal avançada pode ter implicação nas queixas de dor dos pacientes com dor crônica crânio-facial. / Aims: To assess the implications of advanced periodontal disease (PD), and the expression of NOS and sP of inflamed gingival tissue, in the intensity of pain and the quality of life in patients with chronic craniofacial pain. Casuistic and Methods: Were evaluated and treated 20 patients with complaints of chronic craniofacial pain and DP (Study Group), compared with 20 patients who had only DP (Control Group). All patients received surgical periodontal treatment. The evaluation was performed at pre-operative and post-operative periodontal treatment (7, 30 and 180 days). Tools for evaluation: clinical record EDOF-HC, Visual Analogic Scale (VAS), McGill Pain Questionnaire, Scores of Plaque (IP), Bleeding (IS), Clinical Probing Depth (CPD) and Insertion (CPI), the quality of life questionnaire WHOQOL-Bref and immunohistochemical analysis for the expression of nitric oxide synthase (NOS) and substance P from inflamed gingival tissue. The assessment of pain was conducted by independent researcher Results: The groups had similar clinical evolution by parameters of the PD: IP (p=0.0934), IS (p=0.8657), PCS (p=0.1728) and PCI (p=0.7406) in the three moments evaluated. There was reduction in the VAS of Study Group at 30 days (p<0.05) and 180 days (p<0.05), compared to preoperative. At 180 days there was significant improvement of the initial complaints (p=0.005 for the Study Group and p=0.027 for the Control Group), compared to preoperative, but there was significant difference between the two groups in the three moments of assessment, for the percentage of improvement (p<0.001), and either for the improvement descriptors Edof-HC (p=0.004 at 30 days and p=0.001 for 180 days). In Study Group, there was significant reduction of pain affective descriptors (McGill Pain Questionnaire) to 180 days (p=0.014). The scores of quality of life of the Study Group were lower than the Control Group in the psychological and physical (p<0.001 and p=0.007, respectively). The nNOS and iNOS expressions were higher in inflamed gingival Study Group (p<0.001 and p=0.003, respectively) and the expression of sP was similar in the two groups (p=0.363). Conclusions: There was reduction in the intensity of chronic craniofacial pain of patients receiving treatment for advanced periodontal disease. The expression of substance P was similar in inflamed gingival tissue of the two groups, but the expression of iNOS and nNOS was higher in inflamed gingival of patients with chronic craniofacial pain. The data of this study show that advanced periodontal disease can have involvement in complaints of pain in patients with chronic craniofacial pain.

O nervo depressor aórtico (NDA) é, primariamente, um conjunto de fibras aferentes que transmitem informações oriundas de alterações da pressão arterial (PA) a partir dos barorreceptores arteriais (mecanorreceptores localizados no arco da aorta ou seio carótico) aos centros de controle cardiovascular localizados no sistema nervoso central (SNC). Este mecanismo é responsável pela regulação reflexa da função cardíaca e vascular, promovendo ajustes nos centros vasoconstritor e vasodilatador, atuando simultaneamente sobre os sistemas simpático e parassimpático. Fato este que, contribui para o aumento da atividade vagal cardíaca e inibição de descargas simpáticas para vasos e coração, garantindo a manutenção dos níveis pressóricos dentro de uma faixa de normalidade. Diversos neurotransmissores foram descritos atuando nos centros de controle cardiocirculatório localizados no tronco encefálico, mais especificamente no bulbo, participando da regulação da PA. Nestas regiões centrais, os neurotransmissores glutamato, GABA (Àcido Gama Aminobutírico) e substância P (SP) foram amplamente investigados. Entretanto, em nenhum destes trabalhos foi realizado um estudo detalhado, investigando a presença da SP em nervos depressores aórticos de forma direta, sendo esta informação ainda desconhecida. Acredita-se que a SP seja um transmissor do reflexo barorreceptor, atuando na modulação deste circuito, na tentativa de atenuar elevações da pressão sanguínea. Existe portanto a necessidade de uma investigação morfológica e imunohistoquímica com o intuito de promover o esclarecimento sobre os neurotransmissores presentes no NDA. Os nervos frênicos foram utilizados como controle positivo, já que neste território a SP já se encontra caracterizada. Inúmeros são os estudos que descrevem a existência da SP em nervos frênicos, fato este que justifica a aplicação do referido nervo como controle do NDA, foco de estudo deste projeto. Baseados nestas necessidades, o objetivo do presente estudo foi primeiramente o de promover a padronização da técnica imunohistoquímica (IHQ), bem como a verificação da viabilidade de utilização do glutaraldeído à 2,5% como um fixativo primário, auxiliando na identificação de neurotransmissores dentro do sistema nervoso periférico. Em seguida, a identificação e quantificação da SP em NDA de ratos normotensos através do método imunohistoquímico indireto (3,3\'- Diaminobenzidina \"DAB\") foram realizados. O referido estudo foi desenvolvido em duas etapas. A primeira parte corresponde a padronização e otimização da técnica de imunohistoquímica em nervos frênicos de ratos Wistar através da localização e caracterização da SP e da enzima colina acetiltransferase (CAT). A segunda fase, trata-se da identificação e quantificação da SP no NDA, sendo este, um possível neurotransmissor ou neuromodulador do reflexo barorreceptor. Para este estudo foram utilizados no total 38 ratos da linhagem Wistar (Rattus Norvegicus), normotensos, com 20 semanas de idade, machos e fêmeas. Deste total, 16 animais machos foram destinados à padronização da técnica de IHQ em nervos frênicos. E para a caracterização e quantificação da SP no NDA foram utilizados 22 ratos Wistar, sendo 12 machos e 10 fêmeas. Nossos resultados demonstram de forma inédita a presença da SP em fibras amielínicas (tipo C) e fibras de pequeno diâmetro (A-delta) no NDA de forma bastante pontualizada em segmentos proximais e difusa distalmente, sugerindo a existência de subpopulações de fibras amielínicas do tipo C. Estes achados confirmam inúmeras suposições de que a SP atue como um dos neurotransmissores de aferências barorreceptoras, podendo participar na modulação do Sistema Nervoso Autônomo (SNA), uma vez que encontra-se localizada em centros responsáveis pela regulação reflexa da PA. Adicionalmente, a análise do percentual de marcação positiva à SP entre os gêneros apresentou um aparente predomínio da SP em machos mas sem diferença significativa entre os grupos. De forma semelhante, a padronização imunohistoquímica em cortes transversais e longitudinais de nervos frênicos apresentaram uma imunomarcação positiva e aleatória da SP em conjuntos de fibras amielínicas (tipo C) e em fibras de pequeno diâmetro localizadas próximo a periferia do espaço endoneural, corroborando com a localização relatada em estudos morfológicos e ultraestruturais, assegurando a especificidade e a reprodutibilidade do método. Distintamente, as fibras de grande e médio diâmetro (A-alfa, beta e gama), consideradas fibras mielinizadas de condução rápida, foram imunorreativas à CAT em nervos frênicos. Por fim, espera-se que a identificação deste neuropeptídeo sirva de gatilho para que futuras pesquisas envolvendo a liberação de neurotransmissores em aferências barorreceptoras sejam explorados. Fato este, que contribuirá para a agregação de informações pertinentes à modulação ou transmissão da informação neural, propiciando desta forma melhor entendimento da comunicação e atividades barorreflexas associadas a mecanismos cardiovasculares. / The aortic depressor nerve (ADN) is primarily a set of afferent fibers that transmit derived information of changes in arterial blood pressure (BP) from arterial baroreceptors (mechanoreceptors located in the aortic arch and carotid sinus) to sites of cardiovascular control located into central nervous system (CNS). This mechanism is responsible for the reflex regulation of cardiac and vascular function, promoting adjustments of vasoconstrictor and vasodilator centers, simultaneously acting on the sympathetic and parasympathetic systems. In addition, contributes to increased cardiac vagal activity and inhibition of sympathetic discharges to vessels and heart, ensuring the maintenance of blood pressure levels within the normal range. Many neurotransmitters have been described operating in cardio-circulatory control centers located in the brainstem, more specifically in the bulb, participating in the regulation of BP. In these central regions, the neurotransmitters glutamate, GABA (Gamma Aminobutyric Acid) and substance P (SP) have been widely investigated. However, none of these works was carried out a detailed study, investigating the presence of SP in aortic depressor nerves directly, and this information is still unknown. It is believed that SP can be a transmitter at the synapse of the baroreceptor reflex, operating in the modulation of this circuit in an attempt to attenuate elevation of blood pressure. Therefore, there is a need to investigate a morphological and immunohistochemical approach in order to promote the clarification on the present neurotransmitters into ADN. The phrenic nerves were used as a positive control, already as substance P (SP) is characterized in this territory. There have been numerous studies describing the existence of SP in phrenic nerves, a fact that justifies the application of the nerve as control of the ADN, study focus of this project. Based on these requirements, the aim of the present study is two-fold. Firstly, it attempts to promote the standardization of the immunohistochemical (IHC) technique as well as the verification of the feasibility of using glutaraldehyde fixative as a primary, assisting in the identification of neurotransmitters in the peripheral nervous system (PNS). Subsequently, the identification and quantification of SP immunoreactivity in the ADN of normotensive rats by indirect immunohistochemical method (3,3\'-Diaminobenzidine \"DAB\") were done. The study was developed in two stages. The first part corresponds to standardization and optimization of immunohistochemical technique in phrenic nerves of Wistar rats through location and characterization of the SP and enzyme choline acetyltransferase (ChAT). The second phase is about the identification and quantification of the SP into ADN, being a possible neurotransmitter or neuromodulator from the baroreceptor reflex. For this study we used a total of 38 Wistar rats (Rattus norvegicus), normotensive, 20 weeks old, male and female. From this total, 16 male animals were used for standardization of IHC technique in the phrenic nerves. Nonetheless, for the characterization and quantification of SP in ADN were used 22 Wistar rats, 12 males and 10 females. Our results showed an unprecedented manner the presence of SP in unmyelinated fibers (type C) and small diameter fibers (A-delta) into ADN, being quite focused on proximal segments and diffuse distally, suggesting the existence of subsets of unmyelinated fibers. These findings confirm numerous assumptions that the SP acts as a neurotransmitter from afferent baroreceptor and may participate in the modulation of the Autonomic Nervous System (ANS), since it is located in centers responsible for regulating reflex of BP. Further, an analysis of the percentage of positive SP staining between genders, presented an apparent predominance of SP in males but no significant difference between the groups were found. Similarly, IHC standardization in transverse and longitudinal sections of phrenic nerves showed a positive random and immunostaining of SP in sets of unmyelinated fibers (type C) and small diameter fibers located near the periphery of endoneural space, corroborating location reported on morphological and ultrastructural studies, ensuring the specificity and reproducibility of the method. Distinctly, the fibers of large and medium diameters (A-alpha, beta and gamma), considered myelinated fibers of fast conducting, were immunoreactive to ChAT in phrenic nerves. Finally, it is expected that the identification of neuropeptide serve as a trigger for that future studies involving the release of neurotransmitters into afferent baroreceptors be explored. These results could contribute to the aggregation of relevant information for the modulation and transmission of neural information, thus providing better understanding of communication and baroreflex activities associated with cardiovascular mechanisms.

The observations that substance P (SP) is localized in the anterior pituitary gland (AP) and is regulated by the hormonal status of the animal, as well as the demonstration of SP binding sites in the AP, have led to the idea that SP may participate in the regulation of AP function. Numerous and sometimes contradictory reports of SP effects on AP hormone secretion, particularly on luteinizing hormone (LH), left the question of whether SP acts directly at the level of the AP to regulate LH secretion still unanswered. To investigate a possible physiological function of SP in the AP, the effects of exogenous SP on LH secretion from AP cells from adult and prepubertal male and female rats in short term culture were studied. It was found that SP (100nM-1μM) significantly stimulates LH release in cultured AP cells and that this effect varies as a function of age and sex. SP has no significant effect on LH release from AP cells of male and female prepubertal rats. After day 30 a sharp increase in the response to SP occurs in both sexes. This level of responsiveness continues through adulthood in AP cells from the female rat. In contrast, AP cells from male rats failed to respond during adulthood (over 50 days of age) but were highly responsive during the peripubertal period (30-35 days). The possibility that the responsiveness to SP is influenced by the endocrine status of the animal was investigated by exposing AP cells from responding animals to androgens in vivo and in vitro. It was found that AP cells from female rats treated with androgen were less responsive to 100nM SP but did respond at higher doses of SP. SP effects on AP function were further analyzed in experiments using radioligand binding assays to assess possible changes in SP receptor number or affinity as related to age and sex. In AP membranes from female rats, maximum binding is 8-fold higher (Bmax=4.2 pmo1/mg membrane protein) than in AP membranes from male rats (Bmax=560fmo1/ mg membrane protein). These studies suggest a role for SP as a secondary regulator of LH secretion with possible physiological significance for reproductive function.

The distribution of neurokinin receptors in rat kidney, renal artery, renal vein, and proximal ureter was evaluated by autoradiography after in vitro labeling of NK1 sites with [125I]Bolton-Hunter substance P (BHSP) or NK3 sites with [125I][MePhe7]neurokinin B ([MePhe7NKB). Film autoradiography using [125I][MePhe7]NKB revealed specific binding sites associated with the renal vein and its large branches, the renal pelvis, the inner strip of outer renal medulla, and the proximal ureter. High-resolution autoradiograms demonstrated that these sites were localized to the smooth muscle layer in the veins, pelvis, and ureter. Neither the renal arterial system nor the renal cortex contained specific [125I][MePhe7]NKB binding sites although a high level of nonspecific binding was associated with the renal artery. Specific binding of [125I]BHSP was associated with the renal artery and renal pelvis but not the renal veins. Arterial NK1 receptors appeared to be localized to the adventitia. The results indicate that at least two types of tachykinin receptor are present in the rat kidney. The distinct localization observed for most of the NK1 and NK3 receptors suggests that they have different functions.