Evidence of calpain/cdk5 pathway inhibition by lithium in 3-nitropropionic acid toxicity in vivo and in vitro

Lithium reduced striatal neurodegeneration induced in rats by 3-nitropropionic acid inhibiting calpain activation. Lithium prevented an increase in cdk5 activity, as shown by the levels of the co-activator p35. Myocite enhancer factor 2 (MEF2), a downstream substrate for cdk5 with pro-survival activity, showed increased phosphorylation. In primary cultures of neurons treated with 3-NP, lithium also reduced protease activity mediated by calpain, cdk5 activation and cellular death. These observations indicate that lithium has a neuroprotective effect. Lithium treatment also reduced the intracellular increase in calcium induced by 3-NP. The finding that lithium mediates the modulation of the calpain/cdk5 pathway further supports its use in the treatment of neurodegenerative diseases.     

Glucose inhibits glucagon secretion by a direct effect on mouse pancreatic alpha cells

Aims/hypothesis The mechanisms by which glucose regulates glucagon release are poorly understood. The present study aimed to clarify the direct effects of glucose on the glucagon-releasing alpha cells and those effects mediated by paracrine islet factors. Materials and methods Glucagon, insulin and somatostatin release were measured from incubated mouse pancreatic islets and the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) recorded in isolated mouse alpha cells. Results Glucose inhibited glucagon release with maximal effect at 7 mmol/l. Since this concentration corresponded to threshold stimulation of insulin secretion, it is unlikely that inhibition of glucagon secretion is mediated by beta cell factors. Although somatostatin secretion data seemed consistent with a role of this hormone in glucose-inhibited glucagon release, a somatostatin receptor type 2 antagonist stimulated glucagon release without diminishing the inhibitory effect of glucose. In islets exposed to tolbutamide plus 8 mmol/l K⁺, glucose inhibited glucagon secretion without stimulating the release of insulin and somatostatin, indicating a direct inhibitory effect on the alpha cells that was independent of ATP-sensitive K⁺ channels. Glucose lowered [Ca²⁺]_i of individual alpha cells independently of somatostatin and beta cell factors (insulin, Zn²⁺ and γ-aminobutyric acid). Glucose suppression of glucagon release was prevented by inhibitors of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, which abolished the [Ca²⁺]_i-lowering effect of glucose on isolated alpha cells. Conclusions/interpretation Beta cell factors or somatostatin do not seem to mediate glucose inhibition of glucagon secretion. We instead propose that glucose has a direct inhibitory effect on mouse alpha cells by suppressing a depolarising Ca²⁺ store-operated current.     

Effects of rapid and slow potassium repolarization currents and calcium dynamics on hysteresis in restitution of action potential duration

We used a mathematical model to investigate effects of repolarizing currents I_kr and I_ks, calcium (Ca) current I_CaL, and Ca dynamics in network sarcoplasmic reticulum and junctional sarcoplasmic reticulum (JSR) on hysteresis in restitution of action potential duration. Enhanced I_kr increased slope of restitution, hysteresis loop thickness, and delay between peaks of diastolic intervals and action potential duration. Increase in I_ks decreased loop thickness and peak delay. Decrease in I_CaL had effects similar to increasing I_kr, except slope of restitution decreased markedly. Uptake of Ca into the network sarcoplasmic reticulum had less effect on hysteresis than transfer of Ca into JSR. Faster transfer of Ca into JSR markedly decreased loop thickness and peak delay. Our results provide insight into mechanisms responsible for this newly identified property of restitution. Such information will be valuable in studies where modification of hysteresis is used to investigate its role in arrhythmogenesis.     

Prediction of all-cause mortality in a patient population with hypertension and type 2 DM by using traditional risk factors and serum-phosphate,-calcium and-magnesium

The aim of this study is to investigate whether the prediction of all-cause mortality from traditional risk factors is improved by adding electrolytes (serum-phosphate (S-P), serum-calcium (S-Ca) and serum-magnesium (S-Mg)) in a Cox regression. The study uses an 18-year follow-up of patients (n=2504) referred by physicians in primary health care and hospitals to the Vindeln Patient Education (VPE) Center, mainly with a diagnosis of hypertension (HT), type 2 diabetes mellitus (DM) and/or obesity. Cox regression, with the latest registered value and baseline values for risk factors, was used to study all-cause mortality in men and women. 221 out of 1096 men and 157 out of 1408 women died during the 18-year follow-up (20% and 11% respectively). The Cox regression analysis reveals that high blood glucose (B-Glu) and low S-Mg were significantly associated with increased all-cause mortality in the whole patient population as well as in men and women separately. Among women, type 2 DM and systolic blood pressure (SBP) and among men, high S-Ca, S-P, S-urate and body mass index (BMI) were the main predictors of all-cause mortality. There is significantly improved prediction of all-cause mortality with electrolytes added to the traditional risk factors. High B-Glu and low S-Mg in both men and women, and high S-Ca and S-P in men, are significantly associated with all-cause mortality. The metabolic disturbance in this high-risk group of patients can be more fully understood if ionic imbalance is included in the prediction of mortatlity.     

美托洛尔对家兔心力衰竭心肌细胞钙调控蛋白表达的影响

目的观察美托洛尔对家兔心力衰竭（心衰）心肌细胞钙调控蛋白表达的影响,探讨其改善心衰的可能分子机制。方法30只家兔随机分为3组,即假手术组（n=11）、心衰组（n=11）、美托洛尔干预组（n=8）。心衰组和美托洛尔干预组家兔先建立实验性主动脉瓣关闭不全,2周后行腹主动脉缩窄,利用心脏多普勒观察术前后家兔心脏功能的变化,共观察6周。采用常规酶解法分离心室肌细胞,经Fluo-3／AM负载后,利用激光共聚焦显微技术,观察咖啡因诱导的钙瞬变过程中细胞内钙浓度的动态变化。从左心室心肌组织提取膜蛋白后,采用Western blot法测定钙调控蛋白表达水平,并应用UVIDoc成像仪进行蛋白表达半定量分析。结果假手术组与心衰组射血分数分别为（72．6±5．0）％、（45．7±3．0）％（P〈0．01）,咖啡因诱导的钙瞬变幅度（FI）分别为43．5±6．2、16．0±3．5（P〈0．01）,峰值到达时间分别为（52．2±7．4）s、（129．8±14．5）s（P〈0．01）,兰尼碱受体表达量分别为0．203±0．021、0．106±0．007（P〈0．01）及肌浆网钙泵与钠钙交换体（SERCA2a／NCX）表达量比值分别为1．96±0．12、1．22±0．23（P〈0．01）。与心衰组相比,美托洛尔干预组射血分数值[（60．2±5．1）％,P〈0．05]明显增加,钙瞬变幅度增加（32．8±5．4,P〈0．05）,峰值到达时间缩短（91．4±10．9）s,P〈0．05],兰尼碱受体表达量（0．164±0．016,P〈0．05）和SERCA2a／NCX表达量比值（1．68±0．17,P〈0．05）增加。结论美托洛尔可延缓心衰心肌细胞钙调控蛋白表达的改变,进而改善钙瞬变,这可能是长期应用β受体阻滞剂改善心衰患者心功能的分子机制之一。     

Independent FHC-related cardiac troponin T mutations exhibit specific alterations in myocellular contractility and calcium kinetics

Mutations in cardiac troponin T (cTnT) are linked to a severe form of Familial Hypertrophic Cardiomyopathy. Patients carrying mutations flanking the tropomyosin-binding domain of cTnT (R92L and Delta160E) develop distinct clinical syndromes. In order to better understand the cellular pathophysiology underlying these clinically relevant differences, we studied isolated adult left ventricular myocytes from independent transgenic cTnT mouse lines carrying either a 35% (Delta160E) or 50% (R92L) replacement of the endogenous cTnT with the mutant forms. Measurement of baseline myocellular contraction revealed that the Delta160E cells had significant decreases in the peak rate of contraction and percent shortening as compared to either R92L or Non-TG myocytes. In addition, while both Delta160E and R92L myocytes demonstrated a decrease in the peak rate of relaxation as compared to Non-TG, the magnitude of the difference was significantly greater in Delta160E cells. Concurrent myocyte [Ca2+](i) transient measurements revealed that while the alterations in the peak rates and times of the rise and decline of the [Ca2+](i) transient were similar to the changes in the respective measures of sarcomeric mechanics, R92L cells also exhibited reduced rates of the rise and decline of the [Ca2+](i) transient but did not exhibit these reductions in terms of sarcomeric mechanics. Of note, only Delta160E, and not R92L myocytes, demonstrated significant reductions in SR Ca2+ load and uptake, corresponding to the impairments seen in the [Ca2+](i) and mechanical transients. Finally, Western analysis revealed a significant Delta160E-specific reduction in the SERCA2a/PLB ratio, which may well underlie the observed alterations in Ca2+ homeostasis. Therefore, independent cTnT mutations result in significant mutation-specific effects in Ca2+ handling that may, in part, contribute to the observed clinical variability in cTnT-related FHC.     

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart

The Na(+)/Ca(2+) exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate at least in part for the rudimentary sarcoplasmic reticulum in the developing mouse heart. However, to date little is known about the functional expression of NCX during development. This prompted us to investigate the NCX current (I(NCX)) in very early (embryonic day E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. For standard I(NCX) measurements, [Ca(2+)](i) was buffered to 150 nmol/l and voltage ramps were applied from +60 mV to -120 mV. At very early stages of development, we observed a prominent role of the I(NCX) Ca(2+) inward mode in elevating the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Accordingly, a high I(NCX) density was observed (+60 mV: 4.6+/-0.7 pA/pF, n=14). Likewise, we found a strong Ca(2+) outward mode of I(NCX) (-120 mV: -3.9+/-0.7 pA/pF, n=14). At later stages, however, I(NCX) Ca(2+) inward mode was reduced by 54+/-6% (n=15, p<0.0001) in ventricular and 68+/-10% (n=9, p<0.0006) in atrial cells. For the outward mode, a reduction by 43+/-10% (n=15, p<0.01) in ventricular and 62+/-11% (n=9, p<0.004) in atrial cardiomyocytes was observed. By contrast, NCX isoform expression and the reversal potential did not significantly change during development. Thus, NCX displays a prominent Ca(2+) inward and outward mode during early embryonic heart development pointing to its important contribution to maintain [Ca(2+)](i) homeostasis. The functional and protein expression of NCX declines during further development.     

Immunoreactivity of calcium binding protein secretagogin in the human hippocampus is restricted to pyramidal neurons

Disturbed calcium homeostasis plays a crucial role in the aetiology of Alzheimer's disease (AD) and the aging process. We evaluated immunoreactivity of secretagogin, a recently cloned calcium binding protein, in hippocampus and adjacent entorhinal cortex of 30 neuropathologically examined post mortem brains (m:f=12:18; mean age, 79.8+/-15.1 years). The study group consisted of 15 cases fulfilling the criteria for high probability of AD according to the NIA-Reagan Institute Criteria and 15 cases with no to medium probability. Sections were incubated with secretagogin-specific antibodies and the number of immunoreactive neurons as well as staining intensities in both neurons and neuropil were assessed. Both cellular and neuropil immunoreactivity were restricted to subiculum and Ammons horn. Cellular immunoreactivity was further restricted to pyramidal neurons and showed a hierarchical distribution: the mean percentage of immunoreactive neurons was highest in sector CA3 (64.41%), followed by CA2 (44.09%), CA4 (34.38%), CA1 (10.9%), and the subiculum (2.92%; P<0.001, except CA2-CA4, P>0.05), while it did not differ significantly between groups with different degrees of AD pathology. The pattern of secretagogin immunoreactivity resembles that of calcium sensor proteins as it is restricted to a subset of neurons and therefore secretagogin could serve highly specialized tasks in neuronal calcium signalling.     

Influence of cilnidipine or nisoldipine on sympathetic activity in healthy male subjects

The aim of this study was to investigate whether cilnidipine, an N- and L-type calcium channel blocker, and nisoldipine, an L-type calcium channel blocker, have different effects on sympathetic activity, using an identical group of healthy male subjects. Eight healthy men (22–28 years) were given 10 mg of cilnidipine or 10 mg of nisoldipine in a randomized crossover design. In each trial, in subjects without medication on day 1 (control) and with medication on day 2, we measured heart rate (HR), low frequency (LF)/high frequency (HF) of HR variability, and plasma noradrenaline (NA) in a resting supine position and during head-up tilt, and palmar sweating during a mental arithmetic test, before and at 1, 2, 4, 6, and 8 h after administration. Time-plasma concentration profiles of the two drugs were similar. Measurements in controls on the two days showed no significant difference in any of these parameters. Nisoldipine, but not cilnidipine, slightly increased HR and LF/HF at rest. Head-up tilt increased HR, LF/HF, and plasma NA. As evaluated with repeated-measures analysis of variance, head-up tilt induced a significant increase in LF/HF, but not HR or plasma NA, and the effect of cilnidipine was significantly less than that of nisoldipine (P = 0.017). Postural hypotension was not observed. There was no difference in mental arithmetic-induced sweating between the two drugs. Cilnidipine, but not nisoldipine, might have a weak inhibitory effect on reflex sympathetic activity.