RESUMO
The antimalarial agent, mefloquine, inhibits the function of connexin Cx36 gap junctions and hemichannels and has thus become a tool to investigate their physiological relevance in pancreatic islets. In view of earlier reports on a KATP channel-block by mefloquine, the specificity of mefloquine as a pharmacological tool was investigated. Mouse pancreatic islets and single beta cells were used to measure membrane potential, whole cell currents, Ca2+ channel activity, cytosolic Ca2+ concentration ([Ca2+]i) and insulin secretion. Mefloquine was tested in the concentration range of 5-50 µM 25 µM mefloquine was as effective as 500 µM tolbutamide to depolarize the plasma membrane of beta cells, but did not induce action potentials. Rather, it abolished tolbutamide-induced action potentials and the associated increase of [Ca2+]i. In the range of 5-50 µM mefloquine inhibited voltage-dependent Ca2+ currents in primary beta cells as effectively as 1 µM nisoldipine, a specific blocker of L-type Ca2+ channels. The Ca2+ channel opening effect of Bay K8644 was completely antagonized by mefloquine. Likewise, the increase of [Ca2+]i and of insulin secretion stimulated by 40 mM KCl, but not that by 30 mM glucose was antagonized by 50 µM mefloquine. Neither at 5 µM nor at 50 µM did mefloquin stimulate insulin secretion at basal glucose. In conclusion, mefloquine blocks KATP channels and L-type Ca2+ channels in pancreatic beta cells in the range from 5 to 50 µM. Thus it inhibits depolarization-induced insulin secretion, but in the presence of a stimulatory glucose concentration additional effects of mefloquine, possibly on intracellular Ca2+ mobilization, and the metabolic amplification by glucose permit a sustained rate of secretion.
Assuntos
Canais de Cálcio/metabolismo , Conexinas/antagonistas & inibidores , Secreção de Insulina/efeitos dos fármacos , Mefloquina/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Conexinas/metabolismo , Di-Hidropiridinas/farmacologia , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Nisoldipino/farmacologia , Cloreto de Potássio/farmacologia , Tolbutamida/farmacologia , Proteína delta-2 de Junções ComunicantesRESUMO
The voltage-gated Ca(2+) (CaV) channel acts as a key player in ß cell physiology and pathophysiology. ß cell CaV channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca(2+) concentration and thereby Ca(2+)-triggered ß cell apoptosis. The present study was aimed at revealing the subtypes of CaV1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of ß cell CaV1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic ß cells from CaV1 channel knockout and corresponding control mice. We now show that functional CaV1.3 channels are expressed in a subgroup of islet ß cells from CaV1.2 knockout mice (CaV1.2(-/-)). T1D serum enhanced whole-cell CaV currents in islet ß cells from CaV1.3 knockout mice (CaV1.3(-/-)). T1D serum increased the open probability and number of functional unitary CaV1 channels in CaV1.2(-/-) and CaV1.3(-/-) ß cells. These data demonstrate that T1D serum hyperactivates both CaV1.2 and CaV1.3 channels by increasing their conductivity and number. These findings suggest CaV1.2 and CaV1.3 channels as potential targets for anti-diabetes therapy.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Diabetes Mellitus Tipo 1/sangue , Células Secretoras de Insulina/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Masculino , Camundongos , Camundongos KnockoutRESUMO
Insulin production is the central feature of functionally mature and differentiated pancreatic ß-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote ß-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 ß-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of ß-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect ß-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and ß-cell differentiation status. In particular, our unbiased screen identified a novel role for a ß-cell sodium channel gene in insulin production.
RESUMO
Mouse pancreatic ß- and α-cells are equipped with voltage-gated Na(+) currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at -100 mV and -50 mV in ß- and α-cells, respectively). Single-cell PCR analyses show that both α- and ß-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: ß-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na(+) current by 80%. In ß-cells, knockout of Scn9a lowers the Na(+) current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a(-/-) islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na(+) channel α subunit in both α- and ß-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in ß-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.
Assuntos
Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.3/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sódio/fisiologia , Animais , Regulação da Expressão Gênica , Células Secretoras de Glucagon/efeitos dos fármacos , Glucose , Células HEK293 , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Neurotoxinas/farmacologia , Isoformas de Proteínas , Subunidades ProteicasRESUMO
Neuropathic pain and pain arising from local inflammation are characterized by increased release of inflammatory mediators like interleukin-6 (IL-6) by immune cells. The levels of IL-6 is increased in various painfull conditions and correlates with the severity of thermal and mechanical hypersensitivity. Deletion of the IL-6 signal transducer glycoprotein 130 (gp130) reduces inflammation associated with hypersensitivity to thermal and mechanical stimuli. In this study, we show that nociceptor-specific deletion of gp130 alters excitability parameters that are linked to changes in the potassium conductance. In SNS-gp130(-/-) sensory neurons, the resting membrane potential was reduced. Moreover the repolarization speed of the action potential and afterhypolarization was augmented, however, voltage-gated Na(+) and Ca(2+) current were not obviously altered. The main difference between gp130-deficient and control neurons was a significant increase in the conductance of both delayed rectifier as well as A-type potassium currents. Taqman RT-PCR analysis revealed significantly higher levels of Kcna4 mRNA, encoding A-type Kv1.4 potassium channel, in neuron cultures from SNS-gp130(-/-) versus control mice, which may account for the electrophysiological data. No difference in other voltage-gated ion channel mRNAs was observed. The present data show for the first time increased A-type K(+) currents and expression of voltage-gated potassium channel Kcna4 (Kv1.4) in SNS-gp130(-/-) nociceptors. This suggests that gp130 acts as a break for the expression of potassium channels and important regulator hub for nociceptor excitability.
Assuntos
Glicoproteínas/deficiência , Canal de Potássio Kv1.4/metabolismo , Nociceptores/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Cálcio/metabolismo , Glicoproteínas/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Canal de Potássio Kv1.4/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Mensageiro/genética , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Sódio/metabolismo , Regulação para CimaRESUMO
Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Mutação , Células-Tronco Pluripotentes , Potenciais de Ação/genética , Adulto , Células Cultivadas , Canal de Potássio ERG1 , Células-Tronco Embrionárias/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Células-Tronco Pluripotentes/fisiologia , Transporte Proteico/genética , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: The physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis. RESEARCH DESIGN AND METHODS: Gene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKIα and Iß rescue mice [α/ßRM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²(+) ([Ca²(+)](i)) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo. RESULTS: Immunohistochemistry showed that cGKI is present in α- but not in ß-cells in islets of Langerhans. Mice that lack α-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²(+)](i) levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²(+)](i) or hormone release in cGKI-deficient islets. CONCLUSIONS: We propose that cGKI modulates glucagon release by suppression of [Ca²(+)](i) in α-cells.
Assuntos
Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Guanilato Quinases/metabolismo , Animais , Glicemia/metabolismo , Cálcio/fisiologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/deficiência , Proteínas Quinases Dependentes de GMP Cíclico/genética , Primers do DNA , Amplificação de Genes , Genes Reporter , Glucagon/sangue , Células Secretoras de Glucagon/enzimologia , Teste de Tolerância a Glucose , Homeostase , Hipoxantina Fosforribosiltransferase/genética , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/citologia , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/fisiologia , Músculo Liso/enzimologia , RNA/genética , RNA/isolamento & purificação , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Long-QT syndromes are heritable diseases associated with prolongation of the QT interval on an electrocardiogram and a high risk of sudden cardiac death due to ventricular tachyarrhythmia. In long-QT syndrome type 1, mutations occur in the KCNQ1 gene, which encodes the repolarizing potassium channel mediating the delayed rectifier I(Ks) current. METHODS: We screened a family affected by long-QT syndrome type 1 and identified an autosomal dominant missense mutation (R190Q) in the KCNQ1 gene. We obtained dermal fibroblasts from two family members and two healthy controls and infected them with retroviral vectors encoding the human transcription factors OCT3/4, SOX2, KLF4, and c-MYC to generate pluripotent stem cells. With the use of a specific protocol, these cells were then directed to differentiate into cardiac myocytes. RESULTS: Induced pluripotent stem cells maintained the disease genotype of long-QT syndrome type 1 and generated functional myocytes. Individual cells showed a "ventricular," "atrial," or "nodal" phenotype, as evidenced by the expression of cell-typespecific markers and as seen in recordings of the action potentials in single cells. The duration of the action potential was markedly prolonged in "ventricular" and "atrial" cells derived from patients with long-QT syndrome type 1, as compared with cells from control subjects. Further characterization of the role of the R190QKCNQ1 mutation in the pathogenesis of long-QT syndrome type 1 revealed a dominant negative trafficking defect associated with a 70 to 80% reduction in I(Ks) current and altered channel activation and deactivation properties. Moreover, we showed that myocytes derived from patients with long-QT syndrome type 1 had an increased susceptibility to catecholamine-induced tachyarrhythmia and that beta-blockade attenuated this phenotype. CONCLUSIONS: We generated patient-specific pluripotent stem cells from members of a family affected by long-QT syndrome type 1 and induced them to differentiate into functional cardiac myocytes. The patient-derived cells recapitulated the electrophysiological features of the disorder. (Funded by the European Research Council and others.)
Assuntos
Potenciais de Ação , Células-Tronco Pluripotentes Induzidas/fisiologia , Canal de Potássio KCNQ1/genética , Miócitos Cardíacos/citologia , Síndrome de Romano-Ward/fisiopatologia , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Cardiotônicos/farmacologia , Criança , Feminino , Fibroblastos/citologia , Expressão Gênica , Humanos , Isoproterenol/farmacologia , Fator 4 Semelhante a Kruppel , Masculino , Mutação de Sentido Incorreto , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Linhagem , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Romano-Ward/tratamento farmacológico , Síndrome de Romano-Ward/genéticaRESUMO
Activity-dependent means of altering calcium (Ca(2)(+)) influx are assumed to be of great physiological consequence, although definitive tests of this assumption have only begun to emerge. Facilitation and inactivation offer two opposing, activity-dependent means of altering Ca(2+) influx via cardiac Ca(v)1.2 calcium channels. Voltage- and frequency-dependent facilitation of Ca(v)1.2 has been reported to depend on Calmodulin (CaM) and/or the activity of Calmodulin kinase II (CaMKII). Several sites within the cardiac L-type calcium channel complex have been proposed as the targets of CaMKII. Here, we generated mice with knockin mutations of alpha(1)1.2 S1512 and S1570 phosphorylation sites [sine facilitation (SF) mice]. Homocygote SF mice were viable and reproduced in a Mendelian ratio. Voltage-dependent facilitation in ventricular cardiomyocytes carrying the SF mutation was decreased from 1.58- to 1.18-fold. The CaMKII inhibitor KN-93 reduced facilitation to 1.28 in control cardiomyocytes. SF mutation negatively shifted the voltage-dependent inactivation and slowed recovery from inactivation, thereby making fewer channels available for activation. Telemetric ECG recordings at different heart rates showed that QT time decreased significantly more in SF than in control mice at higher rates. Our results strongly support the notion that CaMKII-dependent phosphorylation of Cav1.2 at S1512 and S1570 mediates Ca(2+) current facilitation in the murine heart.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Substituição de Aminoácidos , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Técnicas de Introdução de Genes , Ativação do Canal Iônico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação , Subunidades ProteicasRESUMO
UNLABELLED: There is a need for in vivo monitoring of cell engraftment and survival after cardiac cell transplantation therapy. This study assessed the feasibility and usefulness of combined PET and MRI for monitoring cell engraftment and survival after cell transplantation. METHODS: Human endothelial progenitor cells (HEPCs), derived from CD34+ mononuclear cells of umbilical cord blood, were retrovirally transduced with the sodium iodide symporter (NIS) gene for reporter gene imaging by (124)I-PET and labeled with iron oxides for visualization by MRI. Imaging and histologic analysis were performed on 3 groups of nude rats on days 1, 3, and 7 after intramyocardial injection of 4 million HEPCs. RESULTS: In vitro studies demonstrated stable expression of functional NIS protein and normal viability of HEPCs after transduction. On day 1, after intramyocardial transplantation, iron- and NIS-labeled HEPCs were visualized successfully on MRI as a regional signal void in the healthy myocardium and on PET as (124)I accumulation. The (124)I uptake decreased on day 3 and was undetectable on day 7, and the MRI signal remained unchanged throughout the follow-up period. Histologic analysis with CD31 and CD68 antibodies confirmed the presence of either labeled or nonlabeled control transplanted HEPCs at the site of injection on day 1 but not on day 7, when only iron-loaded macrophages were seen. Furthermore, deoxyuride-5'-triphosphate biotin nick end labeling showed extensive apoptosis at the site of transplantation. CONCLUSION: The combination of MRI and PET allows imaging of localization and survival of transplanted HEPCs together with morphologic information about the heart. Although iron labeling rapidly loses specificity for cell viability because of phagocytosis of iron particles released from dead cells, reporter gene expression provided specific information on the number of surviving cells. This multimodality approach allows complementary analysis of cell localization and viability.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/diagnóstico por imagem , Compostos Férricos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Sobrevivência Celular , Meios de Contraste , Células Endoteliais/fisiologia , Genes Reporter , Células-Tronco Hematopoéticas/fisiologia , Humanos , Masculino , Ratos , Ratos Nus , Cirurgia Assistida por Computador/métodos , Simportadores/genéticaRESUMO
Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Receptores Adrenérgicos beta/metabolismo , Alanina/química , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ecocardiografia , Eletrofisiologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação , Miócitos Cardíacos/metabolismo , Fosforilação , Serina/químicaRESUMO
In arterial vascular smooth muscle cells (VSMCs), Ca(2+) sparks stimulate nearby Ca(2+)-activated K(+) (BK) channels that hyperpolarize the membrane and close L-type Ca(2+) channels. We tested the contribution of L-type Ca(v)1.2 channels to Ca(2+) spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca(v)1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca(v)1.2 channel gene inactivation reduced Ca(2+) spark frequency and amplitude by approximately 50% and approximately 80%, respectively. These effects were associated with lower global cytosolic Ca(2+) levels and reduced sarcoplasmic reticulum (SR) Ca(2+) load. Elevating cytosolic Ca(2+) levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca(v)1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca(2+) sparks. Instead, Ca(v)1.2 channels contribute to global cytosolic [Ca(2+)], which in turn influences luminal SR calcium and thus Ca(2+) sparks.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Miócitos de Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Artéria Basilar/metabolismo , Células Cultivadas , Di-Hidropiridinas , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Artérias da Tíbia/metabolismoRESUMO
Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Animais , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Linhagem Celular , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Miocárdio/metabolismo , Peptídeos/química , Fosforilação , Estrutura Terciária de ProteínaRESUMO
Insulin and glucagon are the major hormones of the islets of Langerhans that are stored and released from the B- and A-cells, respectively. Both hormones are secreted when the intracellular cytosolic Ca2+ concentration ([Ca2+]i) increases. The [Ca2+]i is modulated by mutual inhibition and activation of different voltage-gated ion channels. The precise interplay of these ion channels in either glucagon or insulin release is unknown, owing in part to the difficulties in distinguishing A- from B-cells in electrophysiological experiments. We have established a single-cell RT-PCR method to identify A- and B-cells from the mouse. A combination of PCR, RT-PCR, electrophysiology and pharmacology enabled us to characterize the different sodium and calcium channels in mouse islet cells. In both A- and B-cells, 60% of the inward calcium current (I(Ca)) is carried by L-type calcium channels. In B-cells, the predominant calcium channel is Ca(v)1.2, whereas Ca(v)1.2 and Ca(v)1.3 were identified in A-cells. These results were confirmed by using mice carrying A- or B-cell-specific inactivation of the Ca(v)1.2 gene. In B-cells, the remaining I(Ca) flows in equal amounts through Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3. In A-cells, 30 and 15% of I(Ca) is due to Ca(v)2.3 and Ca(v)2.1 activity, respectively, whereas Ca(v)2.2 current was not found in these cells. Low-voltage-activated T-type calcium channels could not be identified in A- and B-cells. Instead, two TTX-sensitive sodium currents were found: an early inactivating and a residual current. The residual current was only recovered in a subpopulation of B-cells. A putative genetic background for these currents is Na(v)1.7.
Assuntos
Canais de Cálcio/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Canais de Sódio/metabolismo , Animais , Células Cultivadas , Camundongos , Distribuição TecidualRESUMO
The role of T-type Ca2+ channels for cardiovascular physiology, in particular blood pressure regulation, is controversial. Selective blockade of T-type Ca2+ channels in resistance arteries has been proposed to explain the effect of the antihypertensive drug mibefradil. In the present study, we used a third generation, time- and tissue-specific conditional knockout model of the L-type Ca2+ channel Cav1.2 (Cav1.2SMAKO mice) to genetically dissect the effects of mibefradil on T- and L-type Ca2+ channels. Myogenic tone and phenylephrine-induced contraction in hindlimb perfusion experiments were sensitive to mibefradil in control mice, whereas the drug showed no effect in Cav1.2-deficient animals. Mean arterial blood pressure in awake, freely moving control mice was reduced by 38+/-2.5 mm Hg at a dose of 1.25 mg/kg bodyweight mibefradil, but not changed in Cav1.2SMAKO mice. These results demonstrate that the effect of the putative T-type Ca2+ channel-selective blocker mibefradil on blood pressure and small vessel myogenic tone is mediated by the Cav1.2 L-type Ca2+ channel.
Assuntos
Anti-Hipertensivos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo T/fisiologia , Mibefradil/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/fisiologiaRESUMO
Mice deficient in the smooth muscle Cav1.2 calcium channel (SMACKO, smooth muscle alpha1c-subunit calcium channel knockout) have a severely reduced micturition and an increased bladder mass. L-type calcium current, protein, and spontaneous contractile activity were absent in the bladder of SMACKO mice. K+ and carbachol (CCh)-induced contractions were reduced to 10-fold in detrusor muscles from SMACKO mice. The dihydropyridine isradipine inhibited K+- and CCh-induced contractions of muscles from CTR but had no effect in muscles from SMACKO mice. CCh-induced contraction was blocked by removing extracellular Ca2+ but was unaffected by the PLC inhibitor U73122 or depletion of intracellular Ca2+ stores by thapsigargin. In muscles from CTR and SMACKO mice, CCh-induced contraction was partially inhibited by the Rho-kinase inhibitor Y27632. These results show that the Cav1.2 Ca2+ channel is essential for normal bladder function. The Rho-kinase and Ca2+-release pathways cannot compensate the lack of the L-type Ca2+ channel.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Músculo Liso/efeitos dos fármacos , Bexiga Urinária/fisiologia , Amidas/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo L/genética , Carbacol/farmacologia , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Ativação do Canal Iônico , Transporte de Íons/efeitos dos fármacos , Isradipino/farmacologia , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiopatologia , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Piridinas/farmacologia , Pirrolidinonas/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tapsigargina/farmacologia , Transtornos Urinários/genética , Transtornos Urinários/fisiopatologia , Quinases Associadas a rhoRESUMO
Blood pressure is regulated by a number of key molecules involving G-protein-coupled receptors, ion channels and monomeric small G-proteins. The relative contribution of these different signaling pathways to blood pressure regulation remains to be determined. Tamoxifen-induced, smooth muscle-specific inactivation of the L-type Cav1.2 Ca2+ channel gene in mice (SMAKO) reduced mean arterial blood pressure (MAP) in awake, freely moving animals from 120 +/- 4.5 to 87 +/- 8 mmHg. Phenylephrine (PE)- and angiotensin 2 (AT2)-induced MAP increases were blunted in SMAKO mice, whereas the Rho-kinase inhibitor Y-27632 reduced MAP to the same extent in control and SMAKO mice. Depolarization-induced contraction was abolished in tibialis arteries of SMAKO mice, and development of myogenic tone in response to intravascular pressure (Bayliss effect) was absent. Hind limb perfusion experiments suggested that 50% of the PE-induced resistance is due to calcium influx through the Cav1.2 channel. These results show that Cav1.2 calcium channels are key players in the hormonal regulation of blood pressure and development of myogenic tone.
Assuntos
Pressão Sanguínea/fisiologia , Canais de Cálcio Tipo L/metabolismo , Músculo Liso/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Camundongos , Camundongos Knockout , Músculo Liso/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
Voltage-gated calcium (Ca2+) channels play a key role in the control of heart contraction and are essential for normal heart development. The Cav1.2 L-type calcium channel is the predominant isoform in cardiomyocytes and is essential for excitation-contraction coupling. Although the inactivation of the Cav1.2 gene caused embryonic lethality before embryonic day E14.5, hearts were contracting before E14 depending on a dihydropyridine-sensitive calcium influx. We analyzed the consequences of the deletion of the Cav1.2 channel on the expression level of other voltage-gated calcium channels in the embryonic mouse heart and isolated cardiomyocytes. A strong compensatory up-regulation of the Cav1.3 calcium channel was observed on the mRNA as well as on the protein level. Reverse transcriptase PCR indicated that the recently identified new Cav1.3(1b) isoform was strongly up-regulated, whereas a more moderate increase was found for the Cav1.3(1a) variant. Heterologous expression of Cav1.3(1b) in HEK293 cells induced Ba2+ currents with properties similar to those found in Cav1.2 (-/-) cardiomyocytes, suggesting that this isoform constitutes a major component of the residual L-type calcium current in Cav1.2 (-/-) cardiomyocytes. In summary, our results imply that calcium channel expression is dynamically regulated during heart development and that the Cav1.3 channel may substitute for Cav1.2 during early embryogenesis.
Assuntos
Canais de Cálcio Tipo L/genética , Canais de Cálcio/biossíntese , Processamento Alternativo , Animais , Western Blotting , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/fisiologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Concentração Inibidora 50 , Camundongos , Modelos Genéticos , Miocárdio/citologia , Miocárdio/metabolismo , Isoformas de Proteínas , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para CimaRESUMO
In the heart, where Ca(2+) influx across the sarcolemma is essential for contraction, L-type Ca(2+) channels represent the major entry pathway of Ca(2+). Mice with a homozygous deletion of the L-type Ca(v)1.2 Ca(2+) channel gene die before day 14.5 p.c. Electrophysiological and pharmacological investigations on Ca(v)1.2-/- cardiomyocytes demonstrated that contractions depended on the influx of Ca(2+) through an L-type-like Ca(2+) channel. We analyzed now the expression pattern of various L-type Ca(2+) channels. Amplification of the alternative exons 1a and 1b revealed that embryonic cardiac cells express both Ca(v)1.2a and Ca(v)1.2b subunits. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifications indicated the expression of Ca(v)1.1 and Ca(v)1.3 in about a 1:10 ratio in Ca(v)1.2-/- embryos. Two different amino termini of the Ca(v)1.3 cDNA were found in the embryonic heart, which both gave rise to functional channels. Ca(v)1.3(1a) and Ca(v)1.3(1b) channels have similar current kinetics and voltage-dependencies as described for Ca(v)1.3(8A) channels [J. Biol. Chem. 276 (2001) 22100], but the properties of Ca(v)1.3(1a) or Ca(v)1.3(1b) channels are different from that of the L-type-like current in Ca(v)1.2-/- cardiomyocytes. The I(Ba) of Ca(v)1.3(1a) was blocked by the dihydropyridine nisoldipine with an IC(50) value of 0.13 microM at a holding potential of -80 mV. In embryonic Ca(v)1.2+/+ cardiomyocytes, I(Ba) was blocked by nisoldipine with an IC(50) value of 0.1 microM. Although the expressed Ca(v)1.3 channel has a similar affinity for nisoldipine as Ca(v)1.2+/+ cardiomyocytes, the L-type-like Ca(2+) channel found in Ca(v)1.2+/+ and -/- cardiomyocytes is not identical with the new Ca(v)1.3 splice variants.