In the heart, the primary role of the L-type calcium channel (LTCC) CaV1.2 is to conduct calcium into cardiomyocytes and initiate contraction. However, part of the CaV1.2 channel itself, the cleaved carboxyl-terminus (CCt) can also localize to the nucleus and regulate gene transcription. Therefore, the goal of this dissertation project was to determine the role and regulation of CCt in the embryonic and adult heart. The global hypothesis of my dissertation project is that CCt localizes to the nucleus in embryonic and adult cardiomyocytes via a calcium-mediated mechanism and regulates transcription. A model of pharmacological LTCC block-induced perturbation of murine embryonic heart development was first utilized to study the role of CCt. Pharmacological block at embryonic day 10 perturbed cardiogenesis and increased CaV1.2 expression. This result was not mimicked by removal of extracellular calcium or inhibition of calcium release from the sarcoplasmic reticulum. Co-currently, pharmacological block decreased CCt nuclear localization in embryonic cardiomyocytes. At the transcriptional level, CCt suppressed the CaV1.2 promoter. This indicated that the observed upregulation of CaV1.2 induced by pharmacological block may be caused by nuclear localization of the transcriptional repressor, CCt. Therefore, the conclusion was made that pharmacological LTCC block perturbed embryonic cardiogenesis by decreasing nuclear localization of the transcription factor CCt; implying a role for CCt in embryonic heart development. Next, CCt regulation was studied in the adult heart. Similar to the embryonic heart, pharmacological LTCC block decreased nuclear localization of CCt. Inhibition of the calcium activated phosphatase calcineurin also decreased CCt nuclear localization. To determine a role for CCt in the adult heart, CCt nuclear localization was measured in response to hypertrophic stimuli. Serum-induced cardiomyocyte hypertrophy significantly increased nuclear localization of CCt. In conclusion, this dissertation supports the hypothesis that CCt localizes to the nucleus in embryonic and adult cardiomyocytes, and that this regulation is mediated by calcium entry into the cardiomyocyte. Furthermore, data from this dissertation suggests that CCt nuclear localization may play an important role in embryonic heart development and adult cardiac hypertrophy.

The role of Ca++ in olfactory responses was investigated with inorganic and organic calcium channel antagonists. Electrophysiological responses to odorants were recorded from frog olfactory mucosa before and after aerosol application of different agents. Electroolfactogram responses were blocked by certain inorganic ions with the order of effectiveness Zn++ >Ln+++>Cd++>Ca++>Co++>Sr++>Mg++. Ba++ potentiated olfactory responses, and is known to potentiate calcium channel-mediated responses in other tissues. Certain local anesthetics which are thought to act through calcium channel blockade were inhibitory to olfactory responses, with the order of effectiveness being dibucaine>tetracaine>procaine. These data support the idea that Ca++ is involved in olfaction, perhaps acting as a current carrier and/or a second messenger. Preliminary experiments on channel localization were performed using a silicon-labeled amine. Attempts to localize the silicon label were inconclusive, although silicon was detected in the olfactory tissue.

Orientador: André Sampaio Pupo / Banca: Wilma de Grava Kempinas / Banca: Edson Antunes / Resumo: A ejaculação precoce é a disfunção sexual masculina com maiores taxas de incidência e prevalência. De etiologia complexa, as principais abordagens farmacológicas desta condição têm sido a utilização de anestésicos locais, inibidores seletivos da recaptura de serotonina e mais recentemente, inibidores da fosfodiesterase tipo 5. O reflexo ejaculatório é um processo altamente organizado com a interação de áreas centrais encefálicas e espinhais, centros autonômicos simpáticos e parasimpáticos além de reflexos somáticos. O sistema nervoso autônomo simpático, através da ativação de adrenoceptores 1 ( 1-ARs), particularmente o subtipo 1A, desempenha papel fundamental na fase de emissão do reflexo ejaculatório através da modulação da contratilidade de órgãos como o ducto deferente, vesícula seminal e próstata. Além disso, a participação do influxo de cálcio extracelular via canais de cálcio dependentes de voltagem tipo L (canais de cálcio tipo L) na atividade contrátil destes órgãos é bem caracterizada. Devido à importância dos 1-ARs e canais tipo L no reflexo ejaculatório, este trabalho avaliou a eficácia do antagonista de 1-ARs Tamsulosin e das diidropiridinas bloqueadoras de canais de cálcio Nifedipina e (S)-(+)-Niguldipina no retardo da ejaculação em ratos. Os resultados obtidos demonstram que a atividade contrátil do ducto deferente e vesícula seminal ao estímulo adrenérgico via ativação de 1-ARs in vitro é fortemente inibida pelo antagonista de 1-ARs Tamsulosin e pelas bloqueadoras de canais de cálcio Nifedipina e (S)-(+)-Niguldipina. Entretanto, embora a contração destes órgãos e, portanto, a fase de emissão do reflexo ejaculatório tenha sido afetada, a latência ejaculatória dos animais não foi alterada. Estes resultados indicam que o bloqueio de canais de cálcio tipo L ou o antagonismo de 1-ARs no ducto deferente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Premature ejaculation is one of the most common male sexual dysfunctions. With complex aetiology, the main pharmacological approaches involve local anesthetics, selective serotonin reuptake inhibitors and, more recently, phosphodiesterase 5 inhibitors. The sympathetic nervous system plays a crucial role in the ejaculation, controlling vasa deferentia, seminal vesicles and prostate contraction through 1- adrenoceptor ( 1-AR) activation. In addition, the contractions of these organs are dependent on influx of extracellular calcium through L-type voltage-dependent calcium channels (L-type calcium channels). Due to the involvement of 1-ARs and L-type calcium channels in the ejaculatory reflex, this dissertation evaluated the efficacy of the 1-AR antagonist Tamsulosin and of dihydropyridine calcium channel blockers Nifedipine and (S)-(+)-Niguldipine in the delay of ejaculation in rats. The results showed that the vas deferens and seminal vesicle contraction in response to 1-AR activation is potently inhibited by Tamsulosin, Nifedipine and (S)-(+)- Niguldipine. However, although vas deferens and seminal vesicles contractions have been inhibited, the ejaculatory latency in vivo was not affected. These results show that although L-type calcium channel blockade and 1-ARs antagonism in the vas deferens and seminal vesicle are effective in the inhibition of events related to the seminal emission phase of ejaculation, they are not able to delay the ejaculation. The present results indicate that modulation of contractions of organs involved in seminal emission is not a suitable strategy to retard of ejaculation / Mestre

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Alpinia zerumbet à uma planta da famÃlia Zingiberaceae, denominada popularmente âcolÃniaâ no Nordeste do Brasil. à utilizada com fins medicinais no tratamento de hipertensÃo e tem sido estudada em relaÃÃo as suas propriedades farmacolÃgicas. Esta pesquisa teve por objetivo analisar o efeito farmacolÃgico das fraÃÃes hexÃnica (FHOEAz), clorofÃrmica (FCOEAz) e metanÃlica (FMOEAz) do Ãleo essencial da Alpinia zerumbet (OEAz) na reatividade vascular in vitro e nos parÃmetros cardiovasculares in vivo. O projeto de pesquisa foi aprovado pela ComissÃo de Ãtica em Pesquisa Animal, da Universidade Federal do CearÃ, sob os protocolos n 55/10 e n 18/2011, de acordo com as normas de boas prÃticas que envolvem o uso de animais experimentais. Para todos os experimentos foram utilizados ratos Wistar machos. Realizaram-se experimentos de reatividade vascular no banho de ÃrgÃos, com preparaÃÃes de aorta isolada de rato, com endotÃlio Ãntegro e desnudo, mantidos em carbogÃnio e soluÃÃo de Krebs-Henseleit com concentraÃÃo em mmol/L: NaCl: 118,0; KCl: 4,7; KH2PO4: 1,2; MgSO4.7H2O; 1,2; NaHCO3: 15,0; CaCl2: 2,5 e Glicose: 5,5. Foi observada a variaÃÃo da tensÃo isomÃtrica e utilizados diferentes fÃrmacos inibidores especÃficos para anÃlise do mecanismo de aÃÃo do efeito vasodilatador. Analisou-se a pressÃo arterial indireta por pletismografia de cauda em ratos submetidos ao modelo de hipertensÃo por inibiÃÃo crÃnica do Ãxido nÃtrico, atravÃs da administraÃÃo do L-NAME, obtendo-se a pressÃo arterial sistÃlica, diastÃlica e mÃdia, frequÃncia cardÃaca, alÃm da variaÃÃo do peso corporal. Constatou-se que o OEAz, FHOEAz, FCOEAz e FMOEAz induzem relaxamento de anÃis aÃrticos prÃ-contraÃdos com Fenilefrina (1 mol/L), de forma dependente da dose, sendo a menor CE50 a da FMOEAz (150,45 g/mL). A administraÃÃo por gavagem de 100 mg/kg de OEAz, FHOEAz, FCOEAz e FMOEAz reduziu a pressÃo arterial em ratos hipertensos pelo modelo de inibiÃÃo crÃnica do Ãxido nÃtrico, um efeito que foi superior ao controle negativo com Ãgua destilada e inferior aos controles positivos com Captopril e Nifedipina. A FMOEAz (0,1 - 3000 g/mL) induz relaxamento dependente da dose em anÃis aÃrticos prÃ-contraÃdos com Fenilefrina (1 mol/L) ou KCl (80 mmol/L), com endotÃlio intacto ou desnudo. Os estudos de mobilizaÃÃo de cÃlcio mostraram que a FMOEAz inibe o influxo de Ca2+ do meio extracelular, bem como interfere na contraÃÃo induzida pela liberaÃÃo de Ca2+ dos estoques intracelulares pela Fenilefrina (1 mol/L) ou CafeÃna (30 mmol/L). A 4-aminopiridina (1 mmol/L) e a Iberiotoxina (30 nmol/L) aumentam a CE50 da FMOEAz, mas nÃo interferem no seu efeito vasodilatador final. A prÃ-incubaÃÃo com L-NAME (100 mol/L), ODQ (10 mol/L); Indometacina (10 mol/L), Atropina (1 mol/L), Catalase (500 U/ml), SOD (300 U/mL); Wortmannina (0,5 mol/L), TetraetilamÃnio (10 mmol/L), Apamina (1 mol/L); Caribdotoxina (15 nmol/L) e Glibenclamida (10 mol/L) nÃo interferiram no relaxamento induzido pela FMOEAz. Concluiu-se que o OEAz, FHOEAz, FCOEAz e FMOEAz possuem efeito hipotensor in vivo e vasodilatador in vitro, e que o mecanismo de aÃÃo da FMOEAz, provavelmente, envolve o antagonismo aos canais de cÃlcio dependentes de voltagem, aos canais de cÃlcio operados por receptor, interferindo tambÃm na liberaÃÃo de cÃlcio dos estoques intracelulares. / The Alpinia zerumbet is a plant of the family Zingiberaceae, popularly called "colÃnia" in Northeastern Brazil. It is used for medicinal purposes to treat hypertension and has been studied in relation to its pharmacological properties. This study aimed to analyze the pharmacological effect of hexanic (HFEOAz), chloroformic (CFEOAz), and methanolic (MFEOAz) fractions of the essential oil of Alpinia zerumbet (EOAz) in the vascular reactivity in vitro and cardiovascular parameters in vivo. The research project was approved by the Ethics Committee on Animal Research, of the Federal University of CearÃ, under protocol numbers 55/10 and 18/2011, according to the standards of good laboratory practice involving the use of experimental animals. For all experiments male Wistar rats were used. Experiments of vascular reactivity were conducted in organ bath, with preparations of isolated rat aorta with intact endothelium and desnuded, kept in carbogen and Krebs-Henseleit solution with a concentration in mmol/L: NaCl: 118,0; KCl: 4,7; KH2PO4: 1,2; MgSO4.7H2O; 1,2; NaHCO3: 15,0; CaCl2: 2,5 e Glicose: 5,5. Was observed variation in isometric tension and used different specific inhibitors to analyze the mechanism of action of the vasodilator effect. We analyzed blood pressure indirectly by tail plethysmography in rats submitted to chronic hypertension by inhibition of nitric oxide by the administration of L-NAME, obtaining the systolic, diastolic and mean blood pressure, heart rate, beyond variation in body weight. It was found that the EOAz, HFEOAz, CFEOAz MFEOAz induced relaxation of aortic rings pre-contracted with Phenylephrine (1 mol/L), of dose-dependent manner, with the smallest of the EC50 MFEOAz (150.45 mg/mL). The administration by gavage of 100 mg/kg EOAz, HFEOAz, CFEOAz MFEOAz reduced blood pressure in hypertensive rats by the model of chronic inhibition of nitric oxide, an effect that was greater than the negative control with distilled water and less than the positive controls with Captopril and Nifedipine. The MFEOAz (0.1 - 3000 Âg/mL) concentration dependently relaxed Phenylephrine (1 mol/L) and KCl (80 mmol/L) contracted rings with intact or denuded endothelium. Studies of calcium mobilization showed that FMOEAz inhibits the influx of Ca2+ from the extracellular environment and interferes with the contraction-induced Ca2+ release from intracellular stocks by Phenylephrine (1 mol/L) or Caffeine (30 mmol/L). The 4-aminopyridine (1 mmol/L) and Iberiotoxina (30 nmol/L) increased the EC50 of MFEOAz but do not interfere in its final vasodilator effect. The pre-incubation with L-NAME (100 mol/L), ODQ (10 mol/L), Indomethacin (10 mol/L), Atropine (1 mol/L), Catalase (500 U/mL), SOD (300 U/mL), Wortmannin (0.5 mol/L), Tetraethylammonium (10 mmol/L), Apamin (1 mol/L); Caribdotoxin (15 nmol/L) and Glibenclamide (10 mol/L) did not interfere with the relaxation induced by MFEOAz. It was concluded that the EOAz, HFEOAz, CFEOAz and MFEOAz have hypotensive effect in vivo and vasodilator effect in vitro, and that the mechanism of action of MFEOAz probably involves the antagonism of calcium channels voltage-dependent, the calcium channel operated by receptor, also interfering in the release of calcium from intracellular stores.

Sigma receptors have been implicated in the regulation of the cardiovascular system. Some of the cardiovascular effects of sigma receptors may be through the modulation of autonomic neurons. Studies on the expression and cellular function of sigma receptors in autonomic neurons were conducted in neonatal rat intracardiac (ICG) and superior cervical ganglia (SCG). Individual neurons from SCG and ICG were shown to express transcripts encoding the sigma-1 receptor. The effects of sigma receptors activation on high-voltage-activated Ca2+ channels was studied in isolated neurons of these ganglia. Bath application of sigma receptor agonists depressed peak calcium channel currents in a dose-dependent manner and the rank order potency of haloperidol> ibogaine > (+)-pentazocine > DTG is consistent with the effects being mediated by a sigma-2 receptor. Sigma receptor antagonist, metaphit, blocked DTG-mediated inhibition of Ca2+ current. Sigma ligands also altered the biophysical properties of these channels. Activation of sigma receptors reversibly blocked delayed outwardly rectifying potassium channels, large conductance Ca2+-sensitive K+ channels, and the M-current with maximal inhibition >80%. The rank order potency of different sigma ligands suggests that the effect is mediated by sigma-1 receptor. While bath application of sigma ligands depolarized ICG neurons, the number of action potentials (AP) fired by the cells in response to depolarizing current pulses was decreased. Experiments on the signal transduction cascade mediated the inhibition of K+ and Ca2+ channels by sigma ligands showed that the signal transduction pathway does not involve a diffusible cytosolic second messenger or a G-protein. Sigma ligands also modulate voltage-gated Na+ channels (VGSC) in ICG neurons. Bath application of sigma ligands inhibited VGSC current with maximal inhibition >90% and altered the biophysical properties of VGSC. The latency of AP generation during depolarizing current ramp was increased by sigma ligands and this effect is through the inhibition of VGSC. These data suggest that activation of sigma receptors on autonomic neurons modulates voltage-gated Ca2+, K+ and Na+ channels and as a result, the generation of AP is inhibited in these neurons. Sigma receptors are likely altering the cell-to-cell signaling in autonomic ganglia and thus regulating cardiac function by the peripheral nervous system.

Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions.

No description available.

The leaner mouse carries an autosomal recessive mutation in the α1A subunit of neuronal P/Q-type voltage gated calcium ion channels. Due to this mutation, the leaner mouse exhibits severe ataxia, absence seizures and paroxysmal dyskinesia. Mutations in this same gene in humans cause: episodic ataxia type 2, familial hemiplegic migraine, spinocerebellar ataxia type 6 and probably the newly recognized form of human inherited epilepsy. Decreased amplitude of calcium current in cerebellar Purkinje cells and decreased calcium buffering capacity suggest that failure of calcium homeostasis may lead to the neurodegeneration observed in these mutant mice. Both sexes are affected. Despite their neurological dysfunction, homozygous leaner mice are able to breed and produce viable offspring. The survival rate for these pups is highly correlated with early fostering to normal lactating dams. This thesis studies the reproductive dysfunction observed in female homozygous leaner mice and is divided into four parts: onset of puberty, estrous cycle, pregnancy and litter assessment, and hormone levels. We have discovered that the onset of puberty is precocious in leaner females compared to age-matched wild type females, and leaner mice spend more time in estrous than age-matched wild type females. Also, we have observed that leaner mice became pregnant less readily than wild type mice, but once pregnant, female leaner mice produced more pups per litter compared with wild type mice. The number of corpora lutea observed in leaner mice is greater than in wild type mice. In leaner mice, the number of corpora lutea in the ovary corresponding to the uterine horn with the highest number of offspring is larger than the number of corpora lutea found in the ovary corresponding to the other uterine horn. Radioimmunoassays of estradiol hormone levels at postnatal day 28 shows higher levels in leaner compared to age-matched wild type mice. However, at postnatal day 28, the luteinizing hormone levels are similar in both categories of mice. This study of reproductive dysfunction in leaner mice was performed to gain further understanding about the role of intracellular calcium ion signaling in neuronal regulation of reproductive processes in females.

Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions.

Invertebrate T-type calcium channels cloned from the great pond snail, Lymnaea Stagnalis (LCav3) possess highly sodium permeant ion channel currents by means of alternative splicing of exon 12. Exon 12 is located on the extracellular turret and the descending helix between segments 5 and segments 6, upstream of the ion selectivity filter in Domain II. Highly-sodium permeant T-type channels are generated without altering the selectivity filter locus, the primary regulatory domain known to govern ion selectivity for calcium and sodium channels. Comparisons of exon 12 sequences between invertebrates and vertebrate T-type channels reveals a conserved pattern of cysteine residues. Calcium-selective mammalian T-type channels possess a single cysteine in exon 12 in comparison to invertebrate T-type channels with either a tri- or penta- cysteine framework. Cysteine residues in exon 12 were substituted with a neutral amino acid, alanine in LCav3 channels harbouring exon 12a and 12b to mimic the turret structure of vertebrate T-type channels. The results generated T-type channels that were even more sodium-permeable than the native T-type channels in snails. Furthermore, permeant divalent ions similar in structure to calcium (eg. barium) were unable to sufficiently block the monovalent ion current of channels lacking cysteines in Domain II, suggesting that the pore is highly sodium permeant, and has weak affinity and block by permeant divalent ions other than calcium. Besides ion selectivity, the cysteine mutated T-type channels were 10 to 100 fold more sensitive to inhibition by nickel and zinc, respectively. The cysteine mutation data highly suggests that the cysteines form an extracellular structure that regulates ion selectivity and shields T-type channels from block by nickel and zinc. In addition, we replaced exon 12 from the sodium permeant snail T-type channel with exon 12 from human Cav3.2 channels. The snail T-type channel with exon 12 from human T-type channels produced a T-type channel that was modestly sodium permeable, but did not confer the high calcium permeability of Cav3.2 channels. These findings suggest that the cysteine containing extracellular domains in exon 12 are not sufficient to generate calcium selective channels similar to human Cav3.2 and likely work in concert with other extracellular domains to regulate the calcium or sodium selectivity of T-type channels.