Efecto de la dibucaína sobre la SERCA del músculo pterigoideo interno / Drug action of dibucaine on the SERCA from medial pterygoid muscle

Objetivo: Determinar el efecto del anestésico local di-bucaína sobre las principales isoformas de la SERCA (calcio ATPasa de retículo sarco-endoplásmico) pre-sentes en músculo pterigoideo interno. Métodos: Se aislaron por centrifugación diferencial membranas de retículo sarcoplásmico de pterigoideo interno de conejo neozelandés macho (n=5). Se separaron las isoformas SERCA1a, 2a y 2b por cromatografía de afinidad. Se determinó in vitro la actividad enzimá-tica en presencia de diferentes concentraciones de dibucaína (0-90 mM) por el método de Fiske y Subba-row, realizando 5 experimentos por duplicado y en paralelo para cada isoforma. Se calculó la media y ES de la CI50 (mM) del anestésico para cada isofor-ma y éstas se compararon por ANOVA de una vía (p<0,05), y prueba Student-Newman-Keuls de com-paraciones múltiples. Resultados: Dibucaína inhibió la actividad enzimática en función de su concentra-ción en las tres isoformas en estudio. Las CI50 fueron: SERCA1a 20,02 ± 0,64 mM, SERCA2a 15,03 ± 0,52 mM y SERCA2b 16,00 ± 0,51 mM y resultaron signi-ficativamente diferentes (F2,27 = 11,08, p<0,001). La prueba post hoc identificó diferencias significativas entre SERCA1a y 2a, 1a y 2b. El efecto inhibitorio re-sultó significativamente mayor sobre las isoformas 2a y 2b, cuya presencia es sustancialmente mayor en músculos masticadores. Conclusión: La dibucaína inhibe a la SERCA de pterigoideo interno a concen-traciones menores que las usadas en clínica médica (29 mM). Es un anestésico local con potencial efecto miotóxico derivado de la inhibición de la SERCA (AU)

Aim: To test the effect of the local anesthetic dibu-caine on the main isoforms of the SERCA (sarco-endosplasmic reticulum calcium-ATPase) in medial pterygoid muscle. Methods: Sarcoplasmic reticulum membranes from male New Zealand rabbits (n=5) were isolated from medial pterygoid muscle by ul-tracentrifugation. The isoforms SERCA1a, 2a and 2b were separated using high affinity chromatography. In vitro enzymatic activity determinations were per-formed in the presence of different dibucaine con-centrations (0-90 mM) using the colorimetric method described by Fiske & Subbarow. Five assays in dupli-cate and run in parallel were performed for each of the isoforms. Mean and SEM of the IC50 (mM) for the effect of the anesthetic on each isoform were calcu-lated and compared by one-way ANOVA (p<0.05), and Student-Newman-Keuls multiple comparisons test. Results: Dibucaine inhibited the enzymatic activity in a concentration-dependent manner for the three studied isoforms. The IC50 values were: SERCA1a 20.02 ± 0.64 mM, SERCA2a 15.03 ± 0.52 mM and SER-CA2b 16.00 ± 0.51 mM. The values were significantly different (F2.27 = 11.08, p<0.001). The post hoc test revealed significant differences between SERCA1a and 2a, 1a and 2b. The inhibitory effect was signifi-cantly higher on 2a and 2b isoforms, whose presence is substantially higher in masticatory muscles. Con-clusion: Dibucaine inhibits SERCA in medial pterygoid muscle at concentrations lower than those used in clinical medicine (29 mM). It is a potentially myotoxic local anesthetic whose toxic effect may derive from SERCA inhibition (AU)

Effect of tetrahydroquinoline derivatives on the intracellular Ca2+ homeostasis in breast cancer cells (MCF-7) and its relationship with apoptosis / Efecto de derivados de tetrahidroquinolinas sobre la homeostasis del Ca2+ intracelular en células de cáncer de mama (MCF-7) y su relación con la apoptosis

Abstract Tetrahydroquinoline derivatives are interesting structures exhibiting a wide range of biological activities, including antitumor effects. In this investigation, the effect of the synthesized tetrahydroquinolines JS-56 and JS-92 on apoptosis, intracellular Ca2+ concentration ([Ca2+]i), and the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity was determined on MCF-7 breast cancer cells. Colorimetric assays were used to assess MCF-7 cells viability and SERCA activity. Fura-2 and rhodamine 123 were used to measure the intracellular Ca2+ concentration and the mitochondrial electrochemical potential, respec tively. TUNEL assay was used to analyze DNA fragmentation, while caspase activity and NF-κB-dependent gene expression were assessed by luminescence. In silico models were used for molecular docking analysis. These compounds increase intracellular Ca2+ concentration; the main contribution is the Ca2+ entry from the extracellular milieu. Both JS-56 and JS-92 inhibit the activity of SERCA and dissipate the mitochondrial electrochemical potential through processes dependent and independent of the Ca2+ uptake by this organelle. Furthermore, JS-56 and JS-92 generate cytotoxicity in MCF-7 cells. The effect of JS-92 is higher than JS-56. Both compounds activate caspases 7 and 9, cause DNA fragmentation, and potentiate the effect of phorbol 12-myristate-13-acetate on NF-κB-dependent gene expression. Molecular docking analysis suggests that both compounds have a high interaction for SERCA, similar to thapsigargin. Both tetrahydroquinoline derivatives induced cell death through a combination of apoptotic events, increase [Ca2+]i, and inhibit SERCA activity by direct interaction.

Resumen Los derivados de tetrahidroquinolina son estructuras interesantes que exhiben una amplia gama de actividades biológicas, incluyendo efectos antitumorales. Se determinó el efecto de las tetrahidroquinolinas sintetizadas JS-56 y JS-92 sobre la apoptosis, concentración intracelular de Ca2+ ([Ca2+]i) y la actividad Ca2+-ATPasa del retículo sarco(endo)plásmico (SERCA) en células de cáncer de mama MCF-7. Se usaron ensayos colorimétricos para evaluar la viabilidad de las células MCF-7 y la actividad SERCA. Se emplearon Fura-2 y rodamina 123 para medir la concentración de Ca2+ intracelular y el potencial electroquímico mitocondrial, respectivamente. El ensayo TUNEL se utilizó para analizar la fragmentación del ADN, mientras que la actividad de caspasas y la expresión génica dependiente de NF-κB se evaluaron mediante luminiscencia. Modelos in silico permitieron el análisis del acoplamiento molecular. Estos compuestos aumentan la concentración de Ca2+ intracelular; la principal contribución es la entrada de Ca2+ desde el medio extracelular. Tanto JS-56 como JS-92 inhiben la actividad de SERCA y disipan el potencial electroquímico mitocondrial a través de procesos dependientes e independientes de la captación de Ca2+ por este orgánulo. Además, JS-56 y JS-92 generan citotoxicidad en células MCF-7. El efecto de JS-92 es mayor que JS-56. Ambos compuestos activan las caspasas 7 y 9, provocan la fragmentación del ADN y potencian el efecto del 12-miristato-13-acetato de forbol en la expresión génica dependiente de NF-κB. El análisis de acoplamiento molecular sugiere que ambos compuestos tienen una alta interacción con SERCA, similar a la tapsigargina. Ambos derivados de tetrahidroquinolina indujeron la muerte celular a través de una combinación de eventos apoptóticos, aumento de [Ca2+]i e inhibición de la actividad SERCA por interacción directa.

Theoretical Study of the Function of the IP3 Receptor/BK Channel Complex in a Single Neuron / Estudio Teórico de la Función del Complejo Receptor-IP3/Canales BK en Neurona Única

ABSTRACT Large conductance calcium-activated potassium (BK) channels carry out many functions in the central nervous system. These channels open in response to increased cytosolic calcium ([Ca2+]cyt) concentration. The influx of calcium ions to the cytosol can occur through voltage-gated calcium channels (VGCCs) on the plasma membrane and/ or through IP3 receptors (IP3-Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum membrane. The BK channel/IP3-R/RyR interaction has been widely reported in smooth muscle but scarcely investigated in relation to neurons. The aim of this study was to theoretically explore the function of the BK/IP3-R complex by means of a computational model of a neuron that replicates the interaction between the release of Ca2+ from the endoplasmic reticulum (through IP3-Rs) and the opening of the BK channels. The mathematical models are based on the Hodgkin-Huxley formalism and the Goldbeter model. These models were implemented on Visual Basic® and differential equations were solved numerically. Distinct conditions were contemplated for BK conductance and the efflux of endoplasmic Ca2+ to the cytosol. An abrupt rise in [Ca2+]cyt (≥ 5 μM) and short duration (spark) was found to activate BK channels and either pause or stop the action potential train.

RESUMEN Los canales de potasio activados por calcio de gran conductancia (canales BK) cumplen múltiples funciones en el sistema nervioso central. Estos canales se abren en respuesta al incremento de la concentración de calcio citosólico ([Ca2+]cyt). La entrada de Ca2+ puede ocurrir a través de canales de calcio dependientes de voltaje (VGCCs) localizados en la membrana plasmática y por eflujo de Ca2+ del retículo endoplásmico (ER) causado por 1,4,5-Trifosfato (IP3) o rianodina (RyR). La interacción BK/IP3/RyR ha sido ampliamente estudiada en músculo liso, pero escasamente en neuronas. El objetivo de este estudio fue explorar teóricamente la función del complejo BK/IP3-R mediante un modelo computacional de una neurona que replica la interacción entre la liberación de Ca2+ del retículo endoplásmico (a través de IP3-Rs) y la apertura de los canales BK. Los modelos matemáticos se basan en el formalismo de Hodgkin-Huxley y el modelo de Goldbeter. Estos modelos fueron implementados en Visual Basic® y las ecuaciones diferenciales fueron resueltas por métodos numéricos. Se contemplaron distintas condiciones para la conductancia del canal BK y la salida de Ca2+ endoplásmico al citosol. Los resultados muestran que un incremento abrupto de [Ca2+] cyt (≥ 5 μM) y de corta duración (spark) activa los canales BK y producen una pausa o detiene el tren de potenciales de acción.

Research progress of serca2a in heart failure / 中国药理学通报

Heart failure from various cardiovascular diseases is a serious threat to human health. Systolic and diastolic dysfunction are the basic characteristics of heart failure. SERCA2a, a key enzyme for calcium transport, regulates intracellular free calcium ion concentration, affecting the myocardial diastolic process. This article mainly summarized the structure and function of SERCA2a gene, the expression and regulation of SERCA2a in heart failure, and the current situation of drug therapy, gene therapy and clinical research targeting SERCA2a gene.

Effect of Stem Cells and Gene Transfected Stem Cells Therapy on the Pancreas of Experimentally Induced Type 1 Diabetes

BACKGROUND AND OBJECTIVES: Insulin secretion entirely depends on Ca²⁺ influx and sequestration into endoplasmic reticulum (ER) of β-cells, performed by Sarco-ER Ca²⁺-ATPase 2b (SERCA2b). In diabetes, SERCA2b is decreased in the β-cells leading to impaired intracellular Ca²⁺ homeostasis and insulin secretion. Adipose mesenchymal stem cells (AMSCs) play a potential role in transplantation in animal models. The present study aimed at investigating and comparing the therapeutic effect of non-transfected AMSCs and SERCA2b gene transfected AMSCs on the pancreas of induced diabetes type 1 in rat. METHODS AND RESULTS: 58 adult male albino rats were divided into: Donor group: 22 rats, 2 for isolation, propagation and characterization of AMSCs and SERCA2b transfected AMSCs, in addition 20 for isolated islet calcium level assessment. Group I (Control Group): 6 rats, Group II (Diabetic Group): 10 rats, 50 mg streptozotocin (STZ) were injected intraperitoneal (IP), Group III (AMSCs Group): 10 rats, 1×10⁶ AMSCs were injected intravenous and Group IV (SERCA2b transfected AMSCs Group): 10 rats, 1×10⁶SERCA2b transfected AMSCs were injected as in group III. Groups I, II, III and IV were sacrified 3 weeks following confirmation of diabetes. Serological, histological, morphometric studies and quantitative polymerase chain reaction (qPCR) were performed. Nuclear, cytoplasmic degenerative and extensive fibrotic changes were detected in the islets of group II that regressed in groups III and IV. Isolated islet calcium, blood glucose, plasma insulin and qPCR were confirmative. CONCLUSIONS: AMSCs and SERCA2b gene transfected AMSCs therapy proved definite therapeutic effect, more obvious in response to SERCA2b gene transfected AMSCs.

Crotonaldehyde inhibits myocardial contractile function in mice / 基础医学与临床

Objective To study the effect of an essential α,β-unsaturated aldehyde from cigarette smoke crotonal-dehyde on myocardial contractile function and intracellular Ca2+function in mice. Methods Hearts of from male C57BL/6 mice were digested by Langendorff to islate the cardiomyocytes. The cardiomyocytes of mice were then in-cubated with crotonaldehyde(1,10,25 and 50 μmol/L) for 6 h,and the control group was treated without croton-aldehyde,then they were evaluated including peak shortening(PS),maximal velocity of shortening/relengthening (±dL/dt),time-to-PS(TPS),time-to-90% relengthening(TR90),fura-2 fluorescence intensity(FFI),intracel-lular Ca2+decay and SERCA 45Ca2+uptake and the expression of Na+-Ca2+exchange were evaluated by Western blot analysis. Results Compared with the control group, the higher concentrations of the crotonaldehyde(25 and 50 μmol/L) groups significantly diminished the PS, ±dL/dt,ΔFFI,SERCA activity and Ca2+decay (P<0.05), as well as prolonged the TR90(P<0.05);however the crotonaldehyde with different concentrations had no effect on the expression of Na+-Ca2+exchange in cardiomyocytes. Conclusions Crotonaldehyde may inhibit cardiomyocyte contraction by suppressing SERCA activity and compromising intracellular Ca2+handling.

Construction of SERCA Interference Lentivirus Expression Vector and Establishment of Stable PC 12 Cell Line / 中国药学杂志

OBJECTIVE: To construct the SERCA gene interference lentivirus expression vector and establish stable transfected PC 12 cell line. METHODS: The interference sequence targeting at rat SERCA gene was designed and synthesized. pGag/Pol, pRev, and pVSV-G were co-transfected into 293T cells. The lentivirus particles were packaged and generated. The virus titer was detected. PC 12 cells were transfected for establishing the stable cell line; RT-PCR and Western blot were used to detect SERCA gene and protein expression in stable PC 12 cells,and the RESULTS were compared with those in the control group. RESULTS: The lentivirus expression vector targeted at SERCA was successfully constructed and the virus titer was 3×108 U•mL-1. Stable transfected PC 12 cells line was established. The effective interference verification revealed that shSERCA could significantly reduce the mRNA and protein levels of SERCA (P<0.01). CONCLUSION: The shRNA lentiviral expression vector of SERCA gene is successfully constructed and the PC 12 cell line stably interfering with SERCA expresion is established.

rAAV1 mediated SER CA2a gene transfer improves heart failure canine cardiac function by down-regulat-ing MMP-9 expression / 实用医学杂志

Objective To investigate the effect and mechanism of recombinant adenovirus associated vi-rus type 1 (rAAV1) mediated SERCA2a gene transfer on cardiac function in canine with heart failure (HF). Methods 20 healthy male beagle dogs were randomly divided into control group,HF group,HF+EGFP group and HF+SERCA2a group,5 dogs in each group. The canine cardiac function,apoptosis and MMP-9 expression in cardiac myocytes were detected by TUNEL and immunohistochemistry methods. Results The left ventricular dia-stolic diameter (LVDD),left ventricular systolic diameter (LVSD) and left ventricular posterior wall dimension (LVPWD)in the HF and HF+EGFP groups,were significantly higher than those of the control and HF+SERCA2a groups,and the ejection fraction(EF)in the former two groups was significantly lower than that of the control group and HF+SERCA2a group(P<0.05). The left ventricular systolic pressure(LVSP)and left ventricular maximal rate of pressure rise(+dp/dtmax)in the HF and HF+EGFP groups were both significantly lower than those of the control group and HF+SERCA2a group,while left ventricular end diastolic pressure(LVEDP)and left ventricular maximal rate of pressure decline(-dp/dtmax)significantly higher than those of the control and HF+SERCA2a groups(P<0.05). The apoptosis index of HF+SERCA2a group was significantly lower than that of group HF and group HF+EGFP(P<0.05). The MMP-9 light density of HF+SERCA2a group was significantly higher than that of the control group(P < 0.05). Conclusion RAAV1 mediated CA2a SER gene transfer can effectively improve the cardiac function of HF dog,probably by inhibiting the apoptosis of cardiac muscle cells and down-regulating the expression of MMP-9.

rAAV1 mediated SER CA2a gene transfer improves heart failure canine cardiac function by down-regulat-ing MMP-9 expression / 实用医学杂志

Objective To investigate the effect and mechanism of recombinant adenovirus associated vi-rus type 1 (rAAV1) mediated SERCA2a gene transfer on cardiac function in canine with heart failure (HF). Methods 20 healthy male beagle dogs were randomly divided into control group,HF group,HF+EGFP group and HF+SERCA2a group,5 dogs in each group. The canine cardiac function,apoptosis and MMP-9 expression in cardiac myocytes were detected by TUNEL and immunohistochemistry methods. Results The left ventricular dia-stolic diameter (LVDD),left ventricular systolic diameter (LVSD) and left ventricular posterior wall dimension (LVPWD)in the HF and HF+EGFP groups,were significantly higher than those of the control and HF+SERCA2a groups,and the ejection fraction(EF)in the former two groups was significantly lower than that of the control group and HF+SERCA2a group(P<0.05). The left ventricular systolic pressure(LVSP)and left ventricular maximal rate of pressure rise(+dp/dtmax)in the HF and HF+EGFP groups were both significantly lower than those of the control group and HF+SERCA2a group,while left ventricular end diastolic pressure(LVEDP)and left ventricular maximal rate of pressure decline(-dp/dtmax)significantly higher than those of the control and HF+SERCA2a groups(P<0.05). The apoptosis index of HF+SERCA2a group was significantly lower than that of group HF and group HF+EGFP(P<0.05). The MMP-9 light density of HF+SERCA2a group was significantly higher than that of the control group(P < 0.05). Conclusion RAAV1 mediated CA2a SER gene transfer can effectively improve the cardiac function of HF dog,probably by inhibiting the apoptosis of cardiac muscle cells and down-regulating the expression of MMP-9.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.

El Ca2+ y los esfingolípidos como moduladores de la apoptosis y el cáncer / Ca2+ and sphingolipids as modulators for apoptosis and cancer

El Ca2+ es un segundo mensajero que regula funciones directamente relacionadas con el cáncer como la proliferación, diferenciación y la apoptosis. La concentración intracelular de Ca2+ ([Ca2+]i) está altamente regulada por diversos mecanismos entre los que destacan canales iónicos, la Ca2+-ATPasa del retículo endoplasmático (SERCA) y de la membrana plasmática (PMCA), y el transporte de Ca2+ mitocondrial. En el cáncer, la célula tumoral prolifera sin control tras su incapacidad de reconocer señales apoptóticas. La apoptosis es mediada a través de cambios en la actividad de ciertas proteínas como las caspasas y miembros de la familia Bcl-2. Adicionalmente, el “estrés del retículo”, promovido por la acumulación y agregación de proteínas mal plegadas en el interior del retículo endoplasmático (RE), puede desencadenar la apoptosis. El “estrés del retículo” es inducido por una variedad de agentes, entre los que destaca la tapsigargina, inhibidor específico de la SERCA, la cual promueve un notable aumento en la [Ca2+]i, induciendo además apoptosis. En consecuencia, actualmente se están ensayando exitosamente derivados de la tapsigargina como agentes antineoplásicos. El Ca2+ es transferido a la mitocondria desencadenando señales apoptóticas. Por otra parte, los esfingolípidos, como la ceramida y la esfingosina, y sus derivados fosforilados, la ceramida-1-fosfato y la esfingosina-1-fosfato, los cuales regulan la [Ca2+]i, también están estrechamente vinculados con la señalización intracelular en procesos relacionados con el cáncer. Esta revisión discute nuevas evidencias sobre el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y su conexión con la apoptosis y el cáncer.

Ca2+ is a second messenger which regulates many functions directly related with cancer such as proliferation, differentiation and apoptosis. The intracellular Ca2+ concentration ([Ca2+]i) is finely regulated by several mechanisms, among them ionic channels, the endoplasmic reticulum Ca2+-ATPase (SERCA), the plasma membrane calcium pump (PMCA) and the mitochondrial Ca2+ transport. In cancer, the tumour cell proliferates without control since the capacity to recognize apoptotic signals has been lost. The apoptosis is regulated by changes in several proteins, as caspases and the Bcl-2 family members, among others. Additionally, the “reticulum stress”, promoted by the accumulation and aggregation of unfolded proteins in the interior of the endoplasmic reticulum (ER), ussually leads to apoptosis. The “reticulum stress” can be induced by several agents, remarkably with thapsigargin, a selective inhibitor of the SERCA, which in turn induces a large increment in [Ca2+]I, leading to apoptosis. As a consequence, currently, derivatives of thapsigargin are successfully been assayed as anti-neoplastic agents. Ca2+ is then transferred to the mitochondria, where it is known to constitute a main apoptotic signal. On the other hand, several sphingolipids, such as ceramide and sphingosine, and their phosphorylated derivatives ceramide-1-phosphate and sphingosine-1-phosphate, directly involved in the [Ca2+]I regulation, are also recognized as signal messengers related with cancer processes. In this review we discuss new evidences on the effect of several sphingolipids in the intracellular Ca2+ homeostasis and its relationship with apoptosis and cancer.

Manipulating thyroid status alters endoplasmic reticulum calcium homeostasis in rat cerebellum.

Thyroid-related hormones regulate the efficiency and expression of sarco-endoplasmic reticulum calcium ATPases in cardiac and skeletal muscle. However, little is known about the relationship between thyroid hormones and calcium (Ca2+) homeostasis in the brain. It is hypothesized that manipulating rat thyroid hormone levels would induce significant brain Ca2+ adaptations consistent with clinical findings. Adult male Sprague-Dawley rats were assigned to one of three treatment groups for 28 days: control, hypothyroid (6-n-propyl-2-thiouracil (PTU), an inhibitor of thyroxine (T4) synthesis), and hyperthyroid (T4). Throughout, rats were given weekly behavioral tests. Ca2+ accumulation decreased in the cerebellum in both hyper- and hypothyroid animals. This was specific to different ER pools of calcium with regional heterogeneity in the response to thyroid hormone manipulation. Behavioral tasks demonstrated sensitivity to thyroid manipulation, and corresponded to alterations in calcium homeostasis. Ca2+ accumulation heterogeneity in chronic hyper- and hypothyroid animals potentially explains clinical manifestations of altered thyroid status.

Inhibitory effect of Suaeda asparagoides (Miq.) extract on the motility of rat gastric antrum is mediated by beta-adrenoceptor / 한국실험동물학회지

Suaeda asparagoides (Miq.) has long been used as a Korean folk herbal medicine for the treatment of functional gastrointestinal disorders. However, reports on its pharmacological activity on gastrointestinal motility are scarce. The present study investigated the effects of Suaeda asparagoides water fraction of the extract (SAWF) on antral motility in vitro. Muscle strips from rat gastric antrum were set up in an organ bath in a circular orientation. SAWF (100 microg/mL) inhibited the spontaneous contraction of antral circular muscle strips. These inhibitory effects were not significantly affected by tetrodotoxin (1 microM), N omega-Nitro-L-arginine methyl ester hydrochloride (100 microM), 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 microM), ryanodine (10 microM) and phentolamine (10 microM). SAWF-induced inhibition was mostly restored by cyclopiazonic acid (10 microM). Furthermore, the beta-adrenergic receptor antagonist, propranolol (10 microM), abolished SAWF-induced inhibition. These results suggest that SAWF may exert its activity on gastrointestinal smooth muscle via a-adrenergic receptors and sarcoplasmic reticulum Ca2+ ATPase.

Immediate response of myocardium to pressure overload includes transient regulation of genes associated with mitochondrial bioenergetics and calcium availability

Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca+2 ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50 percent diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50 percent food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

NCX and SERCA2a expression in myocardial tissues in rats with pressure overload-induced left ventricular hypertrophy / 基础医学与临床

Objective To observe the abnormity of heart function in rats with pressure overload-induced left ventricular hypertrophy and the changes of NCX,SERCA2a expression in myocardial tissues. Methods Cardiac hypertrophy was induced by clipping the abdominal aorta in rats. The cardiac hypertrophy was evaluated by Left ventricular weight index(LVWI,left ventricular weight/body weight). NCX, SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and protein,respectively. Results LVSP and LVEDP were obviously enhanced(P

Effect of vitamin E on detrusor protection in BOO rabbits / 中华泌尿外科杂志

Objective To explore the protective effect of vitamin E on the rabbit bladder after partial outlet obstruction artificially setup. Methods A total of 28 New Zealand white male rabbits were divided into 4 groups (group A in 6,group B in 6,group C in 8 and group D in 8).Group A,B and C were fed a regular diet,and group D were placed on a diet enriched with 600 mg vitamin E.After 4 weeks partial outlet obstruction was created in groups C and D,while group B underwent sham operation. After 4 weeks of obstruction each rabbit was sedated and cystometry was repeated.After cystometry the bladder was weighed.The gene expression of sarcoplasmic endoplasmic reticulum,calcium,magnesium,adenosine triphosphatase (SERCA2) in bladder was detected by using RT-PCR assay,while the protein level of SERCA2 was measured by Western blot analysis. Results All parameters measured were approximately identical in nomal rabbits(Group A) and shum operation rabbits(group B).Thus,these 2 groups were combined as the control group(Group A and B).Partial outlet obstruction resulted in bladder weight increased significantly in obstructed groups given vehicle group C(13.07?1.71)g and those vitamin E group D(11.80?2.01)g,4-fold higher than in the control group (2.81?0.30)g(P

The Effect of Mesenchymal Stem Cells Transplantation on Heart Function of Rat Myocardial Infarction / 中国医师杂志

Objective To investigate the effect of mesenchymal stem cells transplantation on heart function and the expression of Ca 2+ -ATPase (ERCA) mRNA after rat myocardial infarction. Methods Wistar inbred rat mesenchymal stem cells were cultivated, proliferated and labeled in vitro. Myocardial infarct models were set up by liquid nitrogen cryoinjuring the free wall of left ventricle. 4 weeks later, 2?10 6 mesenchymal stem cells or cold D-Hanks solution were injected into several different points of infarcted myocardial area of experiment group and control group rats. In addition, 5 Wistar inbred rats served as normal group only got thoracotomy twice. 4 weeks after transplantation, all rats received cardiac function test and specimens from infarcted area were got for the expression assay of SERCA2 mRNA by RT-PCR. Results Mesenchymal stem cells were alive at 1, 2 and 4 weeks after transplantation and had a trend toward differentiation and maturation in vivo. Traumatic dynamic measurement was applied to assay heart function by Buxco system, which showed that SLVP and +dp/dt were the highest in normal group, next in experiment group, and the lowest in control group, which LVEDP was the lowest in normal group, next in experiment group, and the highest in control group. The expression level of SERCA2 mRNA in control group was much lower than that in normal group(0.988?0.004 VS 1.824?0.009,P