Inhibition Of Voltage-dependent Calcium Channels Research Articles

Co-localization of influenza A virus and voltage-dependent calcium channels provides new perspectives on the internalization process in pigs

Influenza A virus (IAV) is an RNA virus that causes respiratory disease in a wide range of mammals including humans and pigs. Cav1.2 is a specific voltage-dependent calcium channel (VDCC) important for the internalization of IAV and VDCC inhibitors can decrease IAV disease severity in mice. In this paper, the distribution pattern of a range of VDCCs by immunohistochemistry and Cav1.2 by in situ hybridization in the porcine respiratory tract is documented for the first time. Furthermore, we showed co-localization of VDCC-positive and IAV-positive cells in experimentally infected pigs. These findings provide new perspectives on the IAV internalization process and pave the way for further research investigating the effect of VDDC inhibitors on the IAV infection dynamics in pigs, which could have relevance to humans too.

7-Hydroxy Frullanolide Ameliorates Isoproterenol-Induced Myocardial Injury through Modification of iNOS and Nrf2 Genes.

Myocardial infarction (MI) is the principal cause of premature death. Protecting myocardium from ischemia is the main focus of intense research. 7-hydroxy frullanolide (7-HF) is a potent anti-inflammatory agent, showing its efficacy in different acute and chronic inflammatory disorders such as atherosclerosis, suggesting it can be a potential cardioprotective agent. For the induction of MI, Sprague-Dawley rats (n = 5) were administered isoproterenol (ISO) 85 mg/kg s.c at 24 h intervals for two days. The potential cardioprotective effect of 7-HF and its mechanisms were explored by in vivo and in vitro methods. 7-HF significantly prevented the extent of myocardial injury by decreasing the infarct size, preserving the histology of myocardial tissue, and reducing the release of cardiac biomarkers. Further, 7-HF increased the mRNA expression of cardioprotective gene Nrf2 and reduced the mRNA expression of iNOS. 7-HF also improved cardiac function by decreasing the cardiac workload through its negative chronotropic and negative ionotropic effect, as well as by reducing peripheral vascular resistance due to the inhibition of voltage-dependent calcium channels and the release of calcium from intracellular calcium stores. In conclusion, 7-HF showed cardioprotective effects in the MI model, which might be due to modulating the expression of iNOS and Nrf2 genes as well as improving cardiac functions.

PFI-3 induces vasorelaxation with potency to reduce extracellular calcium influx in rat mesenteric artery.

PFI-3 is a small-molecule inhibitor that targets the bromodomains (BRDs) of Brahma-related gene 1 (BRG1). This monomeric compound, which has high selectivity and potent cellular effects, has recently been developed. Although PFI-3 has been reported as a potential therapeutic agent targeting thrombomodulin, its role in the regulation of vascular function remains unknown. Therefore, we aimed to investigate the impact of PFI-3 on arterial vessel tone. A microvascular tension measurement device (DMT) was utilized to identify alterations in vascular tension within the mesenteric artery. To detect variations in cytosolic [Ca2+]i, a Fluo-3/AM fluorescent probe and fluorescence microscope were employed. Additionally, whole-cell patch clamp techniques were utilized to evaluate the activity of L-type voltage-dependent calcium channels (VDCCs) in cultured arterial smooth muscle cells (A10 cells). PFI-3 exerted a dose-dependent relaxation effect on rat mesenteric arteries with both intact and denuded endothelium after phenylephrine (PE)- and high-K+-induced constriction. PFI-3-induced vasorelaxation was not affected by the presence of L-NAME/ODQ or K+ channel blockers (Gli/TEA). PFI-3 abolished Ca2+-induced contraction on endothelium-denuded mesenteric arteries preincubated by PE in Ca2+-free solution. Incubation with TG had no impact on PFI-3-induced vasorelaxation pre-contracted by PE. PFI-3 reduced Ca2+-induced contraction on endothelium-denuded mesenteric arteries pre-incubated by KCl (60mM) in Ca2+-free solution. PFI-3 declined extracellular calcium influx in A10 cells detected by Fluo-3/AM fluorescent probe and fluorescence microscope. Furthermore, we observed that PFI-3 decreased the current densities of L-type VDCC by whole-cell patch clamp techniques. PFI-3 blunted PE and high K+-induced vasoconstriction independent of endothelium on rat mesenteric artery. The vasodilatory effect of PFI-3 may be attributed to its inhibition of VDCCs and receptor-operated calcium channels (ROCCs) on vascular smooth muscle cells (VSMCs).

Design and Applications of Genetically-Encoded Voltage-Dependent Calcium Channel Inhibitors.

Ca2+ influx through high-voltage-gated Ca2+ channels (HVGCCs; CaV1/CaV2) is an exceptionally powerful and versatile signal that controls numerous cell and physiological functions including neurotransmission, muscle contraction, and regulation of gene expression. The impressive ability of a singular signal, Ca2+ influx, to have such a plethora of functional outcomes is enabled by: molecular diversity of HVGCC pore-forming α1 and auxiliary subunits; organization of HVGCCs with extrinsic modulatory and effector protein to form discrete macromolecular complexes with unique properties; distinctive distribution of HVGCCs into separate subcellular compartments; and varying expression profiles of HVGCC isoforms among different tissues and organs. The capacity to block HVGCCs with selectivity and specificity with respect to the different levels oftheir organization is critical for fully understanding the scope of functional consequences of Ca2+ influx through them, and is also important for realizing their full potential as therapeutic targets. In this review, we discuss the gaps in the current landscape of small-molecule HVGCC blockers and how these may be addressed with designer genetically-encoded Ca2+ channel inhibitors (GECCIs) that draw inspiration from physiological protein inhibitors of HVGCCs.

Downregulation of CYP17A1 by 20-hydroxyecdysone: plasma progesterone and its vasodilatory properties.

Aim:To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions.Methods:Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC–MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography.Results:CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition.Conclusion:In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Designer genetically encoded voltage-dependent calcium channel inhibitors inspired by RGK GTPases.

High‐voltage‐activated calcium (CaV1/CaV2) channels translate action potentials into Ca2+ influx in excitable cells to control essential biological processes that include; muscle contraction, synaptic transmission, hormone secretion and activity‐dependent regulation of gene expression. Modulation of CaV1/CaV2 channel activity is a powerful mechanism to regulate physiology, and there are a host of intracellular signalling molecules that tune different aspects of CaV channel trafficking and gating for this purpose. Beyond normal physiological regulation, the diverse CaV channel modulatory mechanisms may potentially be co‐opted or interfered with for therapeutic benefits. CaV1/CaV2 channels are potently inhibited by a four‐member sub‐family of Ras‐like GTPases known as RGK (Rad, Rem, Rem2, Gem/Kir) proteins. Understanding the mechanisms by which RGK proteins inhibit CaV1/CaV2 channels has led to the development of novel genetically encoded CaV channel blockers with unique properties; including, chemo‐ and optogenetic control of channel activity, and blocking channels either on the basis of their subcellular localization or by targeting an auxiliary subunit. These genetically encoded CaV channel inhibitors have outstanding utility as enabling research tools and potential therapeutics.

Gonadotrope androgen receptor mediates pituitary responsiveness to hormones and androgen-induced subfertility

Many women with hyperandrogenemia suffer from irregular menses and infertility. However, it is unknown whether androgens directly affect reproduction. Since animal models of hyperandrogenemia-induced infertility are associated with obesity, which may impact reproductive function, we have created a lean mouse model of elevated androgen using implantation of low dose dihydrotestosterone (DHT) pellets to separate the effects of elevated androgen from obesity. The hypothalamic-pituitary-gonadal axis controls reproduction. While we have demonstrated that androgen impairs ovarian function, androgen could also disrupt neuroendocrine function at the level of brain and/or pituitary to cause infertility. To understand how elevated androgens might act on pituitary gonadotropes to influence reproductive function, female mice with disruption of the androgen receptor (Ar) gene specifically in pituitary gonadotropes (PitARKO) were produced. DHT treated control mice with intact pituitary Ar (Con-DHT) exhibit disrupted estrous cyclicity and fertility with reduced pituitary responsiveness to GnRH at the level of both calcium signaling and LH secretion. These effects were ameliorated in DHT treated PitARKO mice. Calcium signaling controls GnRH regulation of LH vesicle exotocysis. Our data implicated upregulation of GEM (a voltage-dependent calcium channel inhibitor) in the pituitary as a potential mechanism for androgen's pathological effects. These results demonstrate that gonadotrope AR, as an extra-ovarian regulator, plays an important role in reproductive pathophysiology.

Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2a or a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding-independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

Verapamil augments carmustine- and irradiation-induced senescence in glioma cells by reducing intracellular reactive oxygen species and calcium ion levels.

Resistance to conventional therapies and frequent recurrence are the major obstacles to the treatment of high-grade gliomas, including glioblastoma. Thus, the development of new therapeutic strategies to overcome these obstacles is necessary to improve the treatment outcomes. In this study, we found that verapamil, a pan-adenosine triphosphate-binding cassette transporter and L-type voltage-dependent calcium channel inhibitor, sensitized U87MG glioma cells to carmustine- and irradiation-induced senescence. Furthermore, our results indicated that verapamil treatment, in combination with carmustine and irradiation, rendered U87MG glioma cells and several patient-derived glioma stem cells more sensitive to therapy-induced senescence than individual or dual-combination treatments. When investigating the underlying mechanism, we found that verapamil treatment markedly decreased intracellular reactive oxygen species and calcium ion levels. Reactive oxygen species reduction with N-acetylcysteine, a reactive oxygen species scavenger, rendered U87MG glioma cells more sensitive to carmustine and irradiation whereas the protein kinase C agonist, phorbol 12-myristate 13-acetate, mitigated the effects of carmustine and irradiation. Taken together, our results indicate that verapamil may be a potent therapeutic sensitizer for increasing the effectiveness of glioblastoma treatment.

Broad-range TRP channel inhibitors (2-APB, flufenamic acid, SKF-96365) affect differently contraction of resistance and conduit femoral arteries of rat

Transient receptor potential (TRP) channels are proposed to contribute to membrane depolarization and Ca2+ influx into vascular smooth muscle (VSM) cells. Our aim was to study the effects of widely used broad-range TRP channel inhibitors – 2-aminoethoxydiphenyl borate (2-APB), flufenamic acid (FFA) and SKF-96365 – on the contraction of freshly isolated small and large arteries.Endothelium-denuded resistance (≈250µm) and conduit (≈1000µm) femoral arteries were isolated from adult Wistar rats and mounted in wire myograph. The effects of the above mentioned TRP channel inhibitors and voltage-dependent calcium channel inhibitor nifedipine were studied on arterial contractions induced by phenylephrine, U-46619 or K+. Phenylephrine-induced contractions were also studied in the absence of extracellular Na+. mRNA expression of particular canonical and melastatin TRP channel subunits in femoral vascular bed was determined.TRP channel inhibitors attenuated K+-induced contraction less than nifedipine. Phenylephrine-induced contraction was more influenced by 2-APB in resistance arteries, while FFA completely prevented U-46619-induced contraction in both sizes of arteries. The absence of extracellular Na+ prevented the inhibitory effects of 2-APB, but not those of FFA.The observed effects of broad-range TRP channel inhibitors, which were dependent on the size of the artery, confirmed the involvement of TRP channels in agonist-induced contractions. The inhibitory effects of 2-APB (but not those of FFA or SKF-96365) were dependent on the presence of extracellular Na+.

Abstract P196: Chemerin Activates Procontractile Pathways in Isolated Arteries

In the vasculature, the adipokine chemerin is present in perivascular adipose tissue and causes arterial contraction through activation of the heptahelical receptor ChemR23. We presently test the hypothesis that ChemR23 is linked to procontractile signaling pathways in isolated arteries. The isolated, endothelium-denuded thoracic aorta of both male and female rats was used for measurement of isometric contraction and biochemical analyses. The peptide analog chemerin-9 (1 nm -3 μM) caused concentration-dependent contraction in both isolated aorta from male and female, with a maximum of ~80% of a phenylephrine (10 μM) contraction. The L type voltage dependent calcium channel inhibitors verapamil (1 μM) and nifedipine (1 μM), as well as the protein kinase C (PKC) inhibitor chelerythrine chloride (10 μM), abolished chemerin-9 induced contraction relative to vehicle-incubated tissues (male and female). Next most effective in inhibiting chemerin-9-induced contraction were the Rho Kinase inhibitors Y27632 (1 μM) and fasudil (1 μM), both effective in male and female aorta. The p38 MAPK inhibitor SB203850 (10 μM) and phosphoinositide-3-kinase inhibitor LY294002 (10 μM) were significantly less effective in reducing chemerin-9-induced contraction in both sexes, reducing maximum contraction in male and female by less than 50%. Finally, the phospholipase C inhibitor U73122 (5 μM) and Erk MAPK inhibitor PD098059 (1 μM) did not shift or reduce chemerin-9-induced contraction compared to control. Western analyses of chemerin-9-contracted tissues validated activation of phosphoinositide-3-kinase and lack of activation of the Erk MAPKs. These data are the first to associate ChemR23 with L type channel and Rho Kinase activation, and support the hypothesis that ChemR23 taps into some but not all of the well-recognized contractile pathways. This is important knowledge for understanding the mechanisms by which chemerin could support obesity-associated hypertension

Voltage-Dependent Calcium Channels in Chondrocytes: Roles in Health and Disease

Chondrocytes, the single cell type in adult articular cartilage, have conventionally been considered to be non-excitable cells. However, recent evidence suggests that their resting membrane potential (RMP) is less negative than that of excitable cells, and they are fully equipped with channels that control ion, water and osmolyte movement across the chondrocyte membrane. Amongst calcium-specific ion channels, members of the voltage-dependent calcium channel (VDCC) family are expressed in chondrocytes where they are functionally active. L-type VDCC inhibitors such as nifedipine and verapamil have contributed to our understanding of the roles of these ion channels in chondrogenesis, chondrocyte signalling and mechanotransduction. In this narrative review, we discuss published data indicating that VDCC function is vital for chondrocyte health, especially in regulating proliferation and maturation. We also highlight the fact that activation of VDCC function appears to accompany various inflammatory aspects of osteoarthritis (OA) and, based on in vitro data, the application of nifedipine and/or verapamil may be a promising approach for ameliorating OA severity. However, very few studies on clinical outcomes are available regarding the influence of calcium antagonists, which are used primarily for treating cardiovascular conditions in OA patients. This review is intended to stimulate further research on the chondrocyte 'channelome', contribute to the development of novel therapeutic strategies and facilitate the retargeting and repositioning of existing pharmacological agents currently used for other comorbidities for the treatment of OA.

Fluoride affects calcium homeostasis and osteogenic transcription factor expressions through L-type calcium channels in osteoblast cell line.

Osteoblast L-type voltage-dependent calcium channels (VDCC) play important roles in maintaining intracellular homeostasis and influencing multiple cellular processes. In particular, they contribute to the activities and functions of osteoblasts (OBs). In order to study how L-type VDCC modulate calcium ion (Ca(2+)) homeostasis and the expression of osteogenic transcription factors in OBs exposed to fluoride, MC3T3-E1 cells were exposed to a gradient of concentrations of fluoride (0, 2.0, 5.0, 10.0 mg/L) in combination with 10 μM nifedipine, a specific inhibitor of VDCC, for 48 h. We examined messenger RNA (mRNA) and protein levels of Cav1.2, the main subunit of VDCC, and c-fos, c-jun, runt-related transcription factor 2 (Runx2), osterix (OSX), and intracellular free Ca(2+) ([Ca(2+)]i) concentrations in MC3T3-E1 cells. Our results showed that [Ca(2+)]i levels increased in a dose-dependent manner with increase in concentration of fluoride. Meantime, results indicated that lower concentrations of fluoride (less than 5 mg/L, especially 2 mg/L) can lead to high expression of Cav1.2 and enhance osteogenic function, while high concentration of fluoride (10 mg/L) can induce decreased Cav1.2 and osteogenic transcriptional factors in MC3T3E1 cells exposed to fluoride. However, the levels of [Ca(2+)]i, Cav1.2, c-fos, c-jun, Runx2, and OSX induced by fluoride were significantly altered and even reversed in the presence of nifedipine. These results demonstrate that L-type calcium channels play a crucial role in Ca(2+) homeostasis and they affect the expression of osteogenic transcription factors in fluoride-treated osteoblasts.

Chemical Analysis and Calcium Channel Blocking Activity of the Essential Oil of Perovskia abrotanoides

The aim of this study was to investigate the chemical composition and provide a pharmacological base for the medicinal use of the essential oil of Perovskia abrotanoides (Pa.Oil) in gastrointestinal disorders, such as colic. The chemical investigation resulted in the identification of 26 compounds, of which tricyclene, beta-trans-ocimene, terpinene-4-acetate, terpinen-4-ol, caran-3beta-ol, linalyl acetate, beta-caryophyllene oxide and alpha-elemene had not previously been reported from P. abrotanoides. Major constituents were 1,8-cineol and delta-3-carene, which constituting 50% of the oil. In the isolated rabbit jejunum preparation Pa.Oil caused inhibition of spontaneous and high K+ (80 mM)-induced contractions, with respective EC50 values of 0.13 (0.08-0.20; n = 4) and 0.90 mg/mL (0.50-1.60; n = 5), thus showing that spasmolytic activity is mediated possibly through calcium channel blockade (CCB). The CCB activity was confirmed when pre-treatment of the tissue with Pa.Oil (0.03-0.1 mg/mL) caused a rightward shift in the Ca++ concentration-response curves, similar to that caused by verapamil, a standard calcium channel blocker. These data indicate that the essential oil of P. abrotanoides possesses spasmolytic activity mediated possibly through inhibition of voltage-dependent calcium channels, which may explain its medicinal use in colic and possibly diarrhea.

Inhibition of dendritic Ca2+ spikes by GABAB receptors in cortical pyramidal neurons is mediated by a direct Gi/o‐βγ‐subunit interaction with Cav1 channels

Voltage-dependent calcium channels (VDCCs) serve a wide range of physiological functions and their activity is modulated by different neurotransmitter systems. GABAergic inhibition of VDCCs in neurons has an important impact in controlling transmitter release, neuronal plasticity, gene expression and neuronal excitability. We investigated the molecular signalling mechanisms by which GABA(B) receptors inhibit calcium-mediated electrogenesis (Ca(2+) spikes) in the distal apical dendrite of cortical layer 5 pyramidal neurons. Ca(2+) spikes are the basis of coincidence detection and signal amplification of distal tuft synaptic inputs characteristic for the computational function of cortical pyramidal neurons. By combining dendritic whole-cell recordings with two-photon fluorescence Ca(2+) imaging we found that all subtypes of VDCCs were present in the Ca(2+) spike initiation zone, but that they contribute differently to the initiation and sustaining of dendritic Ca(2+) spikes. Particularly, Ca(v)1 VDCCs are the most abundant VDCC present in this dendritic compartment and they generated the sustained plateau potential characteristic for the Ca(2+) spike. Activation of GABA(B) receptors specifically inhibited Ca(v)1 channels. This inhibition of L-type Ca(2+) currents was transiently relieved by strong depolarization but did not depend on protein kinase activity. Therefore, our findings suggest a novel membrane-delimited interaction of the G(i/o)-βγ-subunit with Ca(v)1 channels identifying this mechanism as the general pathway of GABA(B) receptor-mediated inhibition of VDCCs. Furthermore, the characterization of the contribution of the different VDCCs to the generation of the Ca(2+) spike provides new insights into the molecular mechanism of dendritic computation.

Somatic and dendritic GABAB receptors regulate neuronal excitability via different mechanisms

GABA(B) receptors play a key role in regulating neuronal excitability in the brain. Whereas the impact of somatic GABA(B) receptors on neuronal excitability has been studied in some detail, much less is known about the role of dendritic GABA(B) receptors. Here, we investigate the impact of GABA(B) receptor activation on the somato-dendritic excitability of layer 5 pyramidal neurons in the rat barrel cortex. Activation of GABA(B) receptors led to hyperpolarization and a decrease in membrane resistance that was greatest at somatic and proximal dendritic locations. These effects were occluded by low concentrations of barium (100 μM), suggesting that they are mediated by potassium channels. In contrast, activation of dendritic GABA(B) receptors decreased the width of backpropagating action potential (APs) and abolished dendritic calcium electrogenesis, indicating that dendritic GABA(B) receptors regulate excitability, primarily via inhibition of voltage-dependent calcium channels. These distinct actions of somatic and dendritic GABA(B) receptors regulated neuronal output in different ways. Activation of somatic GABA(B) receptors led to a reduction in neuronal output, primarily by increasing the AP rheobase, whereas activation of dendritic GABA(B) receptors blocked burst firing, decreasing AP output in the absence of a significant change in somatic membrane properties. Taken together, our results show that GABA(B) receptors regulate somatic and dendritic excitability of cortical pyramidal neurons via different cellular mechanisms. Somatic GABA(B) receptors activate potassium channels, leading primarily to a subtractive or shunting form of inhibition, whereas dendritic GABA(B) receptors inhibit dendritic calcium electrogenesis, leading to a reduction in bursting firing.

Calcium channel blocking drugs ameliorates the experimental septic cardiomyopathy induced by cecal ligation and puncture in mice

ObjectiveThis study tested the hypothesis that verapamil (VP), an L‐type voltage‐dependent calcium channel inhibitor, and dantrolene (DT), a sarcoplasmic reticulum calcium inhibitor, could attenuate calpain overexpression, dystrophin loss/reduction and cardiac dysfunction in experimental sepsis by CLP.Methods and ResultsMale C57Bl/6 mice were subjected to sham and severe septic injury (SSI) induced by CLP; half of animals from each group were treated with either verapamil (5mg/kg, SSI+VP) or dantrolene (10mg/kg, SSI+DT). Reduced myocardial levels of NF‐kB and calpain‐1 were detected in SSI+DT and SSI+VP mice associated with increased actin and myosin expression as compared to SSI mice. Consonantly, DT and VP significantly prevented the loss of dystrophin in comparison with SSI mice. Concurrently, SSI+DT and SSI+VP mice presented ejection fraction and fractional shortening improvement associated with increased survival rate.ConclusionsThese observations give support to the opinion that increased intracellular calcium concentration induces calpain activation that causes dystrophin loss/reduction and consequent myocardial dysfunction in CLP experimental sepsis. Studies are needed to further elucidate these new possible interventional pathways to prevent septic cardiomyopathy/heart failure.

New developments in the treatment of partial-onset epilepsy

Although most people presenting with partial-onset seizures will achieve control with antiepileptic medication, a considerable minority will have difficult-to-treat epilepsy that is resistant to existing medication. Over the last few years, a large number of new antiepileptic drugs have been developed. Some of these have a novel mode of action. Many of the older antiepileptic drugs act through sodium channels or by enhancement of gamma amino butyric acid (GABA). Lamotrigine has sodium-channel blocking properties but also has other important modes of action, indicated by efficacy in treating not only partial-onset but also generalized seizures. Vigabatrin and tiagabine both increase GABA activity, by inhibiting GABA transaminase and limiting GABA reuptake, respectively. The main mode of action of gabapentin and pregabalin is not via GABA but through a selective inhibitory effect on voltage-gated calcium channels containing the α2δ-1 subunit. Levetiracetam inhibits the recycling of SV2A (synaptic vesicle protein 2A) neurotransmitter vesicles but also has other effects, including inhibition of voltage-dependent calcium channels. Some drugs, eg, felbamate, zonisamide, and topiramate, have multiple modes of action. In many cases, although the main mode of action may have been identified, other modes of action also play a role. Two recently developed antiepileptic drugs appear to have completely novel primary modes of action; retigabine (ezogabine) and perampanel act on the potassium channel and on AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, respectively. The hope is that antiepileptic drugs with a novel mode of action will be effective where previous drugs have failed and will not have unacceptable adverse effects. However, experience with these medications is too limited to allow any conclusions to be drawn at present.

Calcium/calmodulin-dependent kinase IV controls glucose-induced Irs2 expression in mouse beta cells via activation of cAMP response element-binding protein

Irs2, which is upregulated by glucose, is important for beta cell plasticity. Cyclic AMP response element-binding protein (CREB) stimulates beta cell Irs2 expression and is a major calcium/calmodulin-dependent kinase (CaMK)(IV) target in neurons. We therefore hypothesised that CaMK(IV) mediates glucose-induced Irs2 expression in beta cells via CREB activation. The functions of CaMK(IV) and CREB were investigated in MIN6 beta cells and mouse islets using the CaMK inhibitor KN62, the calcium chelator bapta-(AM) and the voltage-dependent calcium channel inhibitor nifedipine. Small interfering RNAs were used to silence endogenous CaMK(IV) production and expression vectors to overproduce constitutively active and dominant negative forms of CaMK(IV) and CREB. Irs1 and Irs2 expression were determined by quantitative PCR and Western blotting, and the role of CREB was also investigated by assessing its phosphorylation on serine 133. Increasing the glucose concentration from 2.5 to 25mmol/l stimulated CREB phosphorylation on serine 133 and specifically stimulated Irs2 but not Irs1 expression. Similarly, overproduction of a constitutively active form of CaMK(IV) promoted sustained CREB phosphorylation and a significant increase in Irs2 but not Irs1 expression. In contrast, these stimulatory effects of glucose were all suppressed by overproducing an inactive CaMK(IV) mutant. Inhibition of glucose-induced calcium influx with nifedipine or chelation of intracellular calcium with bapta-(AM), as well as silencing of CaMK(IV) or inhibition of its activity with KN62 resulted in similar observations. Finally, overproduction of a dominant negative form of CREB completely suppressed glucose and CaMK(IV) stimulation of Irs2 expression. Our results suggest that the Ca(2+)/CaMK(IV)/CREB cascade plays a critical role in the regulation of Irs2 expression in beta cells.