The L-type calcium channel (LTCC) provides trigger Ca2+ for sarcoplasmic reticulum Ca2+-release and LTCC function is influenced by interacting proteins including the LTCC Distal Carboxyl-terminus (DCT) and calmodulin. DCT is proteolytically cleaved, and re-associates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (IBa,L) in reconstituted channel complexes, yet the contribution of DCT to ICa,L in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte ICa,L. We measured LTCC current and Ca2+ transients with DCT co-expressed in murine cardiomyocytes. We also heterologously co-expressed DCT and CaV1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, CaV1.2Δ1801, and a shorter deletion corresponding to well-studied construct, CaV1.2Δ1733. DCT inhibited IBa,L in cardiomyocytes, and in HEK 293 cells expressing CaV1.2Δ1801 and CaV1.2Δ1733. Ca2+-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of IBa,L combined with Ca2+-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca2+. We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca2+, and show that DCT reduced diastolic Ca2+ at low stimulation frequencies but spared high frequency Ca2+-entry. DCT reduction of diastolic Ca2+ and relief of block at high pacing frequencies, and under conditions of supraphysiological bath Ca2+ suggests that a physiological function of DCT is to increase the dynamic range of Ca2+ transients in response to elevated pacing frequencies. Our data motivates the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.

Beta adrenergic antagonists and antianginal drugs are used with the aim to ultimately decrease mortality and enable patients to lead an improved quality of life by avoidance of anginal episodes. Each class of medications used for this purpose has a variety of actual or potential side effects associated with their use. Side effects and drug interactions involving these medications are discussed in the following chapter. Evidence presented should be used in the context of the patient populations described and may aid clinical decision making through avoidance or identification of actual or potential side effects. This review includes literature published from January 2019 to January 2020 written in English.

Kindling epileptogenesis has been associated with a number of different forms of neuroplasticity in the hippocampus, including mossy fibre sprouting and an increase in both intracellular calcium and zinc. The purpose of this thesis was to determine whether interfering with the influx of calcium via the voltage gated calcium channels would interfere with kindling- induced plasticity. Both kindled and control rats were injected with either 5 or 25mg/kg of the L-type voltage gated calcium channel blocker, nifedipine, or a control vehicle, DMSO (dimethylsulfoxide). The kindled groups received a kindling stimulation twice a day for 11 days. It was revealed that both doses of nifedipine significantly increased afterdischarge duration (p<0.001) and furthermore, both doses of nifedipine were capable of significantly interfering with the rate of kindling (p<0.001). Three weeks following the last kindling stimulation, rats were perfused and brain tissue was processed according to the Timm method. The density of Timm granules, an indication of the level of intracellular zinc in the mossy fibre pathway, was quantified. The results of this analysis revealed that 25mg/kg of nifedipine is capable of significantly reducing the amount of intracellular zinc in both the IML (p<0.04) and the CA3 (p<0.01) region of the mossy fibre pathway, regardless of whether the rats had received kindling stimulations or not. These results provide support for the notion that nifedipine (5 or 25mg/kg) is an effective anticonvulsant agent. These results also suggest that, at a sufficient dose (25mg/kg), nifedipine can reduce the amount of intracellular zinc in the mossy fibre pathway in both kindled and non-kindled animals, suggesting that nifedipine may be a useful therapeutic agent for pathologies that have been associated with zinc-induced neurotoxicity. / Thesis / Master of Science (MSc)

No description available.

No description available.

Tyrosine nitration results in altered function of smooth muscle voltage-gated L-type calcium channel. We explored the possibility that smooth muscle contains denitrase activity to allow functional recovery of the calcium channel without requiring synthesis of new channel proteins. Following peroxynitrite treatment of mouse colonic smooth muscle strips, CaCl2 (1 mM)-induced smooth muscle contraction was significantly reduced by 67% (P ≤ 0.05), which reversed by approximately 86% upon periodic washing within 2 hr period (P ≤ 0.001). The effect of the c-Src kinase inhibitor, PP2, on muscle contraction was also restored after 2 hr post-peroxynitrite treatment consistent with the thesis that recovery from tyrosine nitration allows for tyrosine phosphorylation of the calcium channel. In addition, sodium orthovanadate prevented nitration-induced inhibition of muscle contraction by approximately 90%. Moreover, denitration of nitrated proteins was observed by western blots in smooth muscle cells over 2 hr. Since nitrotyrosine formation interferes with tyrosine kinase pathways involved in cell signaling, the presence of denitrase activity in smooth muscle cells may have profound and important effects in restoring the function of nitrated proteins involved in cell signaling processes.

O processo de fertilização está intimamente relacionado ao íon cálcio. Há relatos que bloqueadores de canais de íons cálcio voltagem-dependentes, utilizados na terapia anti-hipertensiva, podem levar à infertilidade masculina, mas poucos relatos são os de seus efeitos sobre o oócito. O objetivo deste experimento foi verificar a influência dos bloqueadores de canais de cálcio voltagem-dependentes do tipo-L, verapamil, nifedipina e diltiazem, sobre os gametas masculino e feminino. Machos e fêmeas hamsters foram utilizados em testes in vivo e in vitro. Foram realizados tratamentos in vitro no Experimento I: espermatozóides foram capacitados em meios contendo três concentrações de bloqueadores de canais de cálcio e avaliados quanto à capacitação espermática, hiperatividade e fertilidade in vitro. Oócitos foram tratados previamente por 30 minutos com quatro concentrações de antagonistas de canais de cálcio e submetidos a teste de fertilização in vitro e à avaliação do comportamento dos grânulos corticais. No Experimento II, os bloqueadores de canais de cálcio foram administrados, duas vezes ao dia, por 60 dias nos machos e por 30 dias nas fêmeas, que foram analisados pela fertilidade in vivo, capacitação e hiperatividade espermáticas, fecundação in vitro e exocitose dos grânulos corticais, e microscopia eletrônica de transmissão nos oócitos tratados in vivo. No Experimento III, a concentração intracelular de cálcio foi mensurada em espermatozóides e oócitos na presença dos bloqueadores de canais de cálcio. Como resultados, no Experimento I, os fármacos atuaram de forma dose-dependente, os espermatozóides tratados apresentaram menor taxa de capacitação espermática, de hiperatividade e de fecundação in vitro. Os oócitos tratados mostraram menor taxa de fertilização in vitro em relação ao controle e, nos grupos verapamil e diltiazem, exocitose parcial dos grânulos corticais e ativação não completa dos oócitos. O Experimento II demonstrou que os machos dos grupos tratados tiveram menor taxa de capacitação espermática, de hiperatividade e de fertilidade in vitro em relação ao grupo controle, e nos grupos verapamil e nifedipina houve uma redução da fertilidade refletida no número de fêmeas paridas em relação ao grupo controle e diltiazem. A fecundação in vivo das fêmeas tratadas não variou entre os grupos; porém, a taxa de fertilização in vitro dos oócitos de fêmeas tratadas in vivo com os antagonistas foi menor em relação ao controle; tanto a exocitose dos grânulos corticais quanto a microscopia eletrônica não foram detectadas diferenças entre os grupos. As ninhadas de fêmeas que receberam a terapia no início da gestação apresentaram menor peso ao nascimento e maior taxa de mortalidade. No Experimento III, notou-se que os fármacos causaram um bloqueio do influxo de cálcio extracelular tanto em espermatozóides como em oócitos. Concluiu-se que, tanto espermatozóides como oócitos, possuem em sua membrana plasmática canais de íons cálcio sensíveis aos bloqueadores de canais de cálcio voltagem-dependentes do tipo-L. Nos tratamentos in vitro, estes fármacos atuaram negativamente na capacidade de fecundação dos espermatozóides e dos oócitos. A terapia com os bloqueadores de canais de cálcio reduziu a fertilidade nos machos, mas não nas fêmeas. A administração dos bloqueadores no início da gestação causou efeitos teratogênicos / Fertilization process is closely related with calcium ion. There are reports about voltage-dependent calcium ion channel blockers of anti-hypertension therapeutic use causing male infertility, but there are few reports about such effects on the oocyte. The main goal of this experiment was to check the influence of verapamil, nifedipine and diltiazem (L-type voltage-dependent calcium ion channel blockers) on spermatozoa and oocytes. Male and female hamsters were used for in vivo and in vitro experiment. In vitro treatments were preformed in the Experiment I, in which: spermatozoa were capacitated in three concentrations of the three calcium ion channel blockers and they were evaluated for sperm capacitation, hyperactivity and in vitro fertility. Oocytes were incubated for 30 minutes in four concentrations of the same calcium channel antagonists and submitted to in vitro fertilization and evaluation of cortical granules exocytosis. In the Experiment II, calcium channel blockers were supplied twice per day to males during 60 days and to females during 30 days. After this period, drug effects on the animals were evaluated through in vivo fertility, sperm capacitation and hyperactivity, in vitro fertilization, cortical granules exocytosis and transmission electronic microscopy of in vivo treated oocytes. In the Experiment III, the intracellular calcium concentrations were measured in spermatozoa and oocytes in the presence of calcium ion channel blockers. As results, in the Experiment I, the antagonists presented dose-dependent effects, affecting sperm capacitation rate, hyperactivity and in vitro fertilization rate on treated spermatozoa. Treated oocytes presented lower in vitro fertilization rate when compared to the control group and, in the verapamil and diltiazem groups, cortical granules exocytosis was partial and oocyte activation was not concluded. Experiment II showed that male hamsters of treatment groups presented lower sperm capacitation rate, hyperactivity and in vitro fertilization rate and there were fewer pregnant females, demonstrating a reducing male fertility in verapamil and nifedipine groups when compared to the control and diltiazem groups. In vivo fertility of treated females did not change among groups; however, in vitro fertilization rates in oocytes of calcium antagonists in vivo treated females were lower. Nor cortical granules exocytosis neither electronic microscopy detected differences among groups. Progeny of females that received the therapy in the beginning of pregnancy presented lower birth weight and higher mortality rate. In the Experiment III, the antagonists blocked extracellular calcium influx in spermatozoa and in oocytes. It is concluded that, there were calcium ion channels with sensibility to L-type voltage-dependent calcium ion channel blockers in the plasmatic membrane of spermatozoa and oocytes. In in vitro treatments, these antagonists influenced negatively spermatozoa and oocytes capacity of fertilization. Calcium channel blocker therapy reduced the fertility in males, but not in females. Calcium channel blockers supplied in the beginning pregnancy caused teratogenic effects

O processo de fertilização está intimamente relacionado ao íon cálcio. Há relatos que bloqueadores de canais de íons cálcio voltagem-dependentes, utilizados na terapia anti-hipertensiva, podem levar à infertilidade masculina, mas poucos relatos são os de seus efeitos sobre o oócito. O objetivo deste experimento foi verificar a influência dos bloqueadores de canais de cálcio voltagem-dependentes do tipo-L, verapamil, nifedipina e diltiazem, sobre os gametas masculino e feminino. Machos e fêmeas hamsters foram utilizados em testes in vivo e in vitro. Foram realizados tratamentos in vitro no Experimento I: espermatozóides foram capacitados em meios contendo três concentrações de bloqueadores de canais de cálcio e avaliados quanto à capacitação espermática, hiperatividade e fertilidade in vitro. Oócitos foram tratados previamente por 30 minutos com quatro concentrações de antagonistas de canais de cálcio e submetidos a teste de fertilização in vitro e à avaliação do comportamento dos grânulos corticais. No Experimento II, os bloqueadores de canais de cálcio foram administrados, duas vezes ao dia, por 60 dias nos machos e por 30 dias nas fêmeas, que foram analisados pela fertilidade in vivo, capacitação e hiperatividade espermáticas, fecundação in vitro e exocitose dos grânulos corticais, e microscopia eletrônica de transmissão nos oócitos tratados in vivo. No Experimento III, a concentração intracelular de cálcio foi mensurada em espermatozóides e oócitos na presença dos bloqueadores de canais de cálcio. Como resultados, no Experimento I, os fármacos atuaram de forma dose-dependente, os espermatozóides tratados apresentaram menor taxa de capacitação espermática, de hiperatividade e de fecundação in vitro. Os oócitos tratados mostraram menor taxa de fertilização in vitro em relação ao controle e, nos grupos verapamil e diltiazem, exocitose parcial dos grânulos corticais e ativação não completa dos oócitos. O Experimento II demonstrou que os machos dos grupos tratados tiveram menor taxa de capacitação espermática, de hiperatividade e de fertilidade in vitro em relação ao grupo controle, e nos grupos verapamil e nifedipina houve uma redução da fertilidade refletida no número de fêmeas paridas em relação ao grupo controle e diltiazem. A fecundação in vivo das fêmeas tratadas não variou entre os grupos; porém, a taxa de fertilização in vitro dos oócitos de fêmeas tratadas in vivo com os antagonistas foi menor em relação ao controle; tanto a exocitose dos grânulos corticais quanto a microscopia eletrônica não foram detectadas diferenças entre os grupos. As ninhadas de fêmeas que receberam a terapia no início da gestação apresentaram menor peso ao nascimento e maior taxa de mortalidade. No Experimento III, notou-se que os fármacos causaram um bloqueio do influxo de cálcio extracelular tanto em espermatozóides como em oócitos. Concluiu-se que, tanto espermatozóides como oócitos, possuem em sua membrana plasmática canais de íons cálcio sensíveis aos bloqueadores de canais de cálcio voltagem-dependentes do tipo-L. Nos tratamentos in vitro, estes fármacos atuaram negativamente na capacidade de fecundação dos espermatozóides e dos oócitos. A terapia com os bloqueadores de canais de cálcio reduziu a fertilidade nos machos, mas não nas fêmeas. A administração dos bloqueadores no início da gestação causou efeitos teratogênicos / Fertilization process is closely related with calcium ion. There are reports about voltage-dependent calcium ion channel blockers of anti-hypertension therapeutic use causing male infertility, but there are few reports about such effects on the oocyte. The main goal of this experiment was to check the influence of verapamil, nifedipine and diltiazem (L-type voltage-dependent calcium ion channel blockers) on spermatozoa and oocytes. Male and female hamsters were used for in vivo and in vitro experiment. In vitro treatments were preformed in the Experiment I, in which: spermatozoa were capacitated in three concentrations of the three calcium ion channel blockers and they were evaluated for sperm capacitation, hyperactivity and in vitro fertility. Oocytes were incubated for 30 minutes in four concentrations of the same calcium channel antagonists and submitted to in vitro fertilization and evaluation of cortical granules exocytosis. In the Experiment II, calcium channel blockers were supplied twice per day to males during 60 days and to females during 30 days. After this period, drug effects on the animals were evaluated through in vivo fertility, sperm capacitation and hyperactivity, in vitro fertilization, cortical granules exocytosis and transmission electronic microscopy of in vivo treated oocytes. In the Experiment III, the intracellular calcium concentrations were measured in spermatozoa and oocytes in the presence of calcium ion channel blockers. As results, in the Experiment I, the antagonists presented dose-dependent effects, affecting sperm capacitation rate, hyperactivity and in vitro fertilization rate on treated spermatozoa. Treated oocytes presented lower in vitro fertilization rate when compared to the control group and, in the verapamil and diltiazem groups, cortical granules exocytosis was partial and oocyte activation was not concluded. Experiment II showed that male hamsters of treatment groups presented lower sperm capacitation rate, hyperactivity and in vitro fertilization rate and there were fewer pregnant females, demonstrating a reducing male fertility in verapamil and nifedipine groups when compared to the control and diltiazem groups. In vivo fertility of treated females did not change among groups; however, in vitro fertilization rates in oocytes of calcium antagonists in vivo treated females were lower. Nor cortical granules exocytosis neither electronic microscopy detected differences among groups. Progeny of females that received the therapy in the beginning of pregnancy presented lower birth weight and higher mortality rate. In the Experiment III, the antagonists blocked extracellular calcium influx in spermatozoa and in oocytes. It is concluded that, there were calcium ion channels with sensibility to L-type voltage-dependent calcium ion channel blockers in the plasmatic membrane of spermatozoa and oocytes. In in vitro treatments, these antagonists influenced negatively spermatozoa and oocytes capacity of fertilization. Calcium channel blocker therapy reduced the fertility in males, but not in females. Calcium channel blockers supplied in the beginning pregnancy caused teratogenic effects

To identify novel therapeutic targets for basal-like breast cancer (BLBC) subtype, we investigated several DNA repair mechanisms associated with maintenance of high genomic instability for cell survival in cancer cells. We identified that the mismatch repair proteins, MSH2 and MSH6 (referred to as MSH2/6 hereafter), are highly elevated across BLBC samples. High expression level of MSH2/6 in BLBC is associated with worse prognosis and survivability for patients. Therefore, we knocked out MSH2 in BLBC cell lines and performed in vivo xenograft and syngeneic mice model studies to find significant attenuation of tumor growth in MSH2 KO group. Also, MSH2-deficient BLBC cells have increased rate of new mutations. Additionally, we tested the efficacy of conventional chemotherapeutics and radiation treatment that would further tip the genomic instability in MSH2-deficient BLBC cells towards cell death, but found them to be ineffective. Next, we performed high-throughput screening of 1280 FDA-approved compounds to discover that calcium channel blockers preferentially kill MSH2-deficient BLBC cells. This was likely due to association of significantly mutated pathways that involved calcium ion binding and calmodulin binding sites. Here we provide evidence of an alternative therapeutic strategy targeting DNA repair genes in BLBC patients utilizing bioinformatics analysis, high-throughput drug screening, in vitro,and vivoexperimentalmodels.

Research Objective Patients with chronic kidney disease (CKD) are at increased risk for atherosclerosis and peripheral artery disease (PAD). PAD has received far less attention than coronary artery disease (CAD) in CKD patients. Few studies have examined risk factors for PAD in CKD. We studied the possible related risk factors and benefit of hypertension treatment in CKD patients with PAD. Data Sources We included 129 patients of both sexes with stages 3 to 5 of CKD, as described by the Kidney Outcome Quality Initiatives (K/DOQI ) classification, without receiving dialysis therapy, not previously diagnosed with PAD. Study Design The following information were collected within six month period, including demographic characteristics, history of hypertension, anti-hypertension drug, diabetes, smoking, and pre-existing cardiovascular disease, body mass index (BMI), fasting blood glucose, HbA1c, total cholesterol, triglyceride(TG), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol(LDL), calcium (Ca), phosphate(P), Albumin, uric acid, urine protein. Ankle-brachial index (ABI) is a noninvasive diagnostic test that is efficient in detecting asymptomatic PAD with ABI <0.9. Findings There were 22 (17.7 %) participants with PAD. Higher systolic blood pressure (SBP), higher diastolic blood pressure (DBP), higher pulse pressure showed strong association with PAD in CKD patients. On further analysis, significant fewer patients treated with calcium channel blocker (CCB) in hypertensive CKD patients with PAD (£q2 =7.055, p =0.008). The multivariate logistic regression analysis in hypertensive patients demonstrated the risk factors for PAD was pulse pressure, and Calcium channel blocker treatment may correlate with decreasing PAD formation (odds ratio= 0.232, 95% CI=0.07-0.73, p =0.013) in CKD patients. Conclusion There is a high prevalence rate of PAD in population of CKD, especially those with hypertension. ABI should be routinely examined in these patients who can benefit earlier from therapeutic measures.