Molecular Basis for Ligand Modulation of a Mammalian Voltage-Gated Ca2+ Channel.

The L-type voltage-gated Ca2+ (Cav) channels are modulated by various compounds exemplified by 1,4-dihydropyridines (DHP), benzothiazepines (BTZ), and phenylalkylamines (PAA), many of which have been used for characterizing channel properties and for treatment of hypertension and other disorders. Here, we report the cryoelectron microscopy (cryo-EM) structures of Cav1.1 in complex with archetypal antagonistic drugs, nifedipine, diltiazem, and verapamil, at resolutions of 2.9 Å, 3.0 Å, and 2.7 Å, respectively, and with a DHP agonist Bay K 8644 at 2.8 Å. Diltiazem and verapamil traverse the central cavity of the pore domain, directly blocking ion permeation. Although nifedipine and Bay K 8644 occupy the same fenestration site at the interface of repeats III and IV, the coordination details support previous functional observations that Bay K 8644 is less favored in the inactivated state. These structures elucidate the modes of action of different Cav ligands and establish a framework for structure-guided drug discovery.

Electrographic seizures and brain hyperoxia may be key etiological factors for postconcussive deficits.

Repetitive mild traumatic brain injuries (RmTBIs) are increasingly recognized to have long-term neurological sequelae in a significant proportion of patients. Individuals that have had RmTBIs exhibit a variety of sensory, cognitive, or behavioral consequences that can negatively impact quality of life. Brain tissue oxygen levels ([Formula: see text]) are normally maintained through exquisite regulation of blood supply to stay within the normoxic zone (18-30 mmHg in the rat hippocampus). However, during neurological events in which brain tissue oxygen levels leave the normoxic zone, neuronal dysfunction and behavioral deficits have been observed, and are frequently related to poorer prognoses. The oxygenation response in the brain after RmTBIs/repeated concussions has been poorly characterized, with most preliminary research limited to the neocortex. Furthermore, the mechanisms by which RmTBIs impact changes to brain oxygenation and vice versa remain to be determined. In the current study, we demonstrate that upon receiving RmTBIs, rats exhibit posttraumatic, electrographic seizures in the hippocampus, without behavioral (clinical) seizures, that are accompanied by a long-lasting period of hyperoxygenation. These electrographic seizures and the ensuing hyperoxic episodes are associated with deficits in working memory and motor coordination that were reversible through attenuation of the posttraumatic and postictal (postseizure) hyperoxia, via administration of a vasoconstricting agent, the calcium channel agonist Bay K8644. We propose that the posttraumatic period characterized by brain oxygenation levels well above the normoxic zone, may be the basis for some of the common symptoms associated with RmTBIs.NEW & NOTEWORTHY We monitor oxygenation and electrographic activity in the hippocampus, immediately before and after mild traumatic brain injury. We demonstrate that as the number of injuries increases from 1 to 3, the proportion of rats that exhibit electrographic seizures and hyperoxia increases. Moreover, the presence of electrographic seizures and hyperoxia are associated with postinjury behavioral impairments, and if the hyperoxia is blocked with Bay K8644, the behavioral deficits are eliminated.

Assays for L-type voltage gated calcium channels.

Voltage gated calcium channels (VGCCs) are pursued as drug targets for neurodegenerative and cardiovascular diseases. High throughput drug screening targeting VGCCs depends on patch-clamp electrophysiology or fluorophore-based calcium imaging that requires powerful equipment and specialized expertise thus leading to cost escalation. Moreover, VGCC needs to be transfected into cell lines such as HEK-293. We report the presence of L-type VGCC (L-VGCC) subunit proteins, Cav1.2, α2δ and ß in HEK-293 cells and the application of simple methods for its assay. Endogenous expression of the channel in HEK-293 cells overcomes the need for transfection. L-VGCC in HEK-293 cells was activated either by the agonist, BayK8644 or by KCl-mediated depolarization. Activity was detected using the calcium sensing probe, GCaMP6m by live imaging. L-VGCC activity induced enhancement in GCaMP6m fluorescence returned to baseline corresponding to channel-closure. Activity was also shown using a methodology involving end-point detection of the calcium dependent interaction of α-CaMKII with NMDA receptor subunit GluN2B sequence. This methodology further simplifies the assay as it eliminates the need for real time imaging. Activation was blocked by the specific L-type VGCC antagonist, nifedipine. Finding the protein and activity of L-VGCC in HEK-293 cells offers commercially viable assays for drug screening.

Cav1.4 dysfunction and congenital stationary night blindness type 2.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

CaMKII inhibition has dual effects on spontaneous Ca2+ release and Ca2+ alternans in ventricular cardiomyocytes from mice with a gain-of-function RyR2 mutation.

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. We studied spontaneous Ca2+ release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+ release events in RyR2-R2474S cardiomyocytes exposed to the ß-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+ release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca2+ release but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

Neuroprotective Effects of Early Brain Injury after Subarachnoid Hemorrhage in Rats by Calcium Channel Mediating Hydrogen Sulfide.

The present study explored the modulating apoptosis effect of hydrogen sulfide (H2S) in subarachnoid hemorrhage (SAH) rats and its exact mechanism. A rat SAH model established by intravascular puncturing was used for the present study. After giving NaHS (donor of H2S), an L-type calcium channel opener (Bay K8644), or a calcium channel agonist (nifedipine), the neurological function of the rats, associated pathological changes, and expression of apoptosis-related proteins (Bcl-2, Bax, and caspase-3) and microtubule-associated protein (MAP-2) were examined. The concentration of H2S and expression of cystathionine beta synthase in the hippocampus changed upon early brain injury (EBI) after SAH. Compared with the SAH group, the neurological function of the rats and microstructure observed by electron microscopy were better in the SAH + NaHS group and SAH + Bay K8644 group. It was observed that apoptosis was more obvious in the SAH group than in the control group and was alleviated in the SAH + NaHS group. Furthermore, the alleviating effect of NaHS was partially weakened by nifedipine, indicating that the effect of anti-apoptosis in H2S might be correlated with the calcium channel. The expression of Bax and caspase-3 was elevated, while the expression of Bcl-2 decreased in the SAH group but improved in the SAH + NaHS and SAH + Bay K8644 group. Compared with the SAH + NaHS group, the expression of pro-apoptotic proteins was higher in the SAH + NaHS + nifedipine group. Therefore, upon EBI following SAH, the H2S system plays an important neurological protective effect by modulating the function of the L-type calcium channel and inhibiting apoptosis.

The phytochemical p-hydroxycinnamic acid suppresses the growth and stimulates the death in human liver cancer HepG2 cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases and causes a third of cancer-related death. The prognosis and effective treatment of advanced HCC remains poor in spite of the development of novel therapeutic strategies. In the present study, we investigate anticancer effects of the botanical molecule p-hydroxycinnamic acid (HCA) in the HepG2 liver cancer model in vitro. Culturing with HCA (10-1000 nM) suppressed colony formation and growth of HepG2 cells. Mechanistically, culturing with HCA decreased levels of Ras, PI3K, Akt, MAPK, NF-κB p65 and ß-catenin, which are linked to processes of cell signaling and transcription, and increased levels of retinoblastoma and regucalcin, which are suppressors for carcinogenesis. These alterations may lead to the suppression of cell growth. Furthermore, culturing with HCA (10-1000 nM) stimulated cell death due to increased caspase-3 levels. Interestingly, the effects of HCA on the growth and death of HepG2 cells were inhibited by culturing with CH223191, an antagonist of aryl hydrocarbon receptor (AHR), suggesting that the flavonoid effects are, at least partly, mediated by activation of AHR signaling. Notably, HCA blocked stimulatory effects of Bay K 8644, an agonist of L-type calcium channel, on the growth of HepG2 cells. Thus, our study demonstrates that HCA suppresses the growth and stimulates the death of human liver cancer HepG2 cells in vitro. The botanical molecule HCA may therefore be a useful tool in the treatment of HCC, providing a novel strategy for the therapy of human liver cancers.

Structural Basis of the Modulation of the Voltage-Gated Calcium Ion Channel Cav 1.1 by Dihydropyridine Compounds*.

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav ) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. The structure of rabbit Cav 1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remained elusive. Herein, we report five cryo-EM structures of nanodisc-embedded Cav 1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 Å. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that (S)-(-)-Bay K8644, when acting as an agonist, is insufficient to lock the activated state of the channel for a prolonged duration.

Mechano-Pharmacological Testing of L-Type Ca2+ Channel Modulators via Human Vascular Celldrum Model.

BACKGROUND/AIMS: This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology. METHODS: The so-called CellDrum technology was applied to analyse the biomechanical properties of confluent human aorta muscle cells (haSMC) in monolayer. The cell generated tensions deviations in the range of a few N/m² are evaluated by the CellDrum technology. This study focuses on the dilative and contractive effects of L-type Ca2+ channel agonists and antagonists, respectively. We analyzed the effects of Bay K8644, nifedipine and verapamil. Three different measurement modes were developed and applied to determine the most appropriate analysis technique for the study purpose. These three operation modes are called, particular time mode" (PTM), "long term mode" (LTM) and "real-time mode" (RTM). RESULTS: It was possible to quantify the biomechanical response of haSMCs to the addition of vasoactive agents using CellDrum technology. Due to the supplementation of 100nM Bay K8644, the tension increased approximately 10.6% from initial tension maximum, whereas, the treatment with nifedipine and verapamil caused a significant decrease in cellular tension: 10nM nifedipine decreased the biomechanical stress around 6,5% and 50nM verapamil by 2,8%, compared to the initial tension maximum. Additionally, all tested measurement modes provide similar results while focusing on different analysis parameters. CONCLUSION: The CellDrum technology allows highly sensitive biomechanical stress measurements of cultured haSMC monolayers. The mechanical stress responses evoked by the application of vasoactive calcium channel modulators were quantified functionally (N/m²). All tested operation modes resulted in equal findings, whereas each mode features operation-related data analysis.

The calcium channel agonist Bay K 8644 promotes the growth of human liver cancer HepG2 cells in vitro: suppression with overexpressed regucalcin.

Hepatocellular carcinoma is one of the most prevalent malignant diseases and causes a third of cancer-related death. The consequences of altered calcium homeostasis in cancer cells may contribute to tumor progression. Regucalcin plays an inhibitory role in calcium signaling linked to transcription regulation. Regucalcin gene expression is downregulated in the tumor tissues of liver cancer patients, suggesting an involvement as a suppressor in hepatocarcinogenesis. We investigated whether Bay K 8644, an agonist of the L-type Ca2+ channel, promotes the growth of human liver cancer and if the effect of Bay K 8644 is suppressed by overexpressed regucalcin using the HepG2 cell model. The colony formation and growth of HepG2 cells were promoted by culturing with Bay K 8644 (0.1-10 nM). This effect was suppressed by inhibitors of signaling processes linked to cell proliferation, including PD98059 and wortmannin. Death of HepG2 cells was stimulated by Bay K 8644 with higher concentrations (25 and 100 nM). The effects of Bay K 8644 on cell growth and death were abolished by verapamil, an antagonist of calcium channel. Mechanistically, culturing with Bay K 8644 increased levels of mitogen-activated protein kinase (MAPK) and phospho-MAPK. Notably, overexpressed regucalcin suppressed Bay K 8644-promoted growth and death of HepG2 cells. Furthermore, overexpressed regucalcin prevented growth and increased death induced by thapsigargin, which induces the release of intracellular stored calcium. Thus, higher regucalcin expression suppresses calcium signaling linked to the growth of liver cancer cells, providing a novel strategy in treatment of hepatocellular carcinoma with delivery of the regucalcin gene.

Ritanserin blocks CaV1.2 channels in rat artery smooth muscles: electrophysiological, functional, and computational studies.

CaV1.2 channel blockers or 5-HT2 receptor antagonists constitute effective therapy for Raynaud's syndrome. A functional link between the inhibition of 5-HT2 receptors and CaV1.2 channel blockade in arterial smooth muscles has been hypothesized. Therefore, the effects of ritanserin, a nonselective 5-HT2 receptor antagonist, on vascular CaV1.2 channels were investigated through electrophysiological, functional, and computational studies. Ritanserin blocked CaV1.2 channel currents (ICa1.2) in a concentration-dependent manner (Kr = 3.61 µM); ICa1.2 inhibition was antagonized by Bay K 8644 and partially reverted upon washout. Conversely, the ritanserin analog ketanserin (100 µM) inhibited ICa1.2 by ~50%. Ritanserin concentration-dependently shifted the voltage dependence of the steady-state inactivation curve to more negative potentials (Ki = 1.58 µM) without affecting the slope of inactivation and the activation curve, and decreased ICa1.2 progressively during repetitive (1 Hz) step depolarizations (use-dependent block). The addition of ritanserin caused the contraction of single myocytes not yet dialyzed with the conventional method. Furthermore, in depolarized rings, ritanserin, and to a lesser extent, ketanserin, caused a concentration-dependent relaxation, which was antagonized by Bay K 8644. Ritanserin and ketanserin were docked at a region of the CaV1.2 α1C subunit nearby that of Bay K 8644; however, only ritanserin and Bay K 8644 formed a hydrogen bond with key residue Tyr-1489. In conclusion, ritanserin caused in vitro vasodilation, accomplished through the blockade of CaV1.2 channels, which was achieved preferentially in the inactivated and/or resting state of the channel. This novel activity encourages the development of ritanserin derivatives for their potential use in the treatment of Raynaud's syndrome.

Vasorelaxing Activity of Stilbenoid and Phenanthrene Derivatives from Brasiliorchis porphyrostele: Involvement of Smooth Muscle CaV1.2 Channels.

Five compounds, 3,4'-dihydroxy-3',5,5'-trimethoxydihydrostilbene, 1: ; 3,4'-ihydroxy-3',5'-dimethoxydihydrostilbene, 2: ; 3,4'-dihydroxy-5,5'-dimethoxydihydrostilbene, 3: ; 9,10-dihydro-2,7-dihydroxy-4,6-dimethoxyphenanthrene, 4: ; and the previously unreported 1,2,6,7-tetrahydroxy-4-methoxyphenanthrene, 5: were isolated from the South American orchid, Brasiliorchis porphyrostele. An in-depth analysis of their vascular effects was performed on in vitro rat aorta rings and tail main artery myocytes. Compounds 1:  - 4: were shown to possess vasorelaxant activity on rings pre-contracted by the α 1 receptor agonist phenylephrine, the CaV1.2 stimulator (S)-(-)-Bay K 8644, or depolarized with high K+ concentrations. However, compound 5: was active solely on rings stimulated by 25 mM but not 60 mM K+. The spasmolytic activity of compounds 1: and 4: was significantly affected by the presence of an intact endothelium. The KATP channel blocker glibenclamide and the KV channel blocker 4-aminopyridine significantly antagonized the vasorelaxant activity of compounds 4: and 1: , respectively. In patch-clamp experiments, compounds 1:  - 4: inhibited Ba2+ current through CaV1.2 channels in a concentration-dependent manner, whereas neither compound 4: nor compound 1: affected K+ currents through KATP and KV channels, respectively. The present in vitro, comprehensive study demonstrates that Brasiliorchis porphyrostele may represent a source of vasoactive agents potentially useful for the development of novel antihypertensive agents that has now to be validated in vivo in animal models of hypertension.

Vasorelaxing Activity of R-(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol from Dalbergia tonkinensis: Involvement of Smooth Muscle CaV1.2 Channels.

Dalbergia species heartwood, widely used in traditional medicine to treat various cardiovascular diseases, might represent a rich source of vasoactive agents. In Vietnam, Dalbergia tonkinensis is an endemic tree. Therefore, the aim of the present work was to investigate the vascular activity of R-(-)-3'-hydroxy-2,4,5-trimethoxydalbergiquinol isolated from the heartwood of D. tonkinensis and to provide circular dichroism features of its R absolute configuration. The vascular effects of R-(-)-3'-hydroxy-2,4,5-trimethoxydalbergiquinol were assessed on the in vitro mechanical activity of rat aorta rings, under isometric conditions, and on whole-cell Ba2+ currents through CaV1.2 channels (IBa1.2) recorded in single, rat tail main artery myocytes by means of the patch-clamp technique. R-(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol showed concentration-dependent, vasorelaxant activity on both endothelium-deprived and endothelium intact rings precontracted with the α 1 receptor agonist phenylephrine. Neither the NO (nitric oxide) synthase inhibitor Nω-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin affected its spasmolytic activity. R-(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol-induced vasorelaxation was antagonized by (S)-(-)-Bay K 8644 and unaffected by tetraethylammonium plus glibenclamide. In patch-clamp experiments, R-(-)-3'-hydroxy-2,4,5-trimethoxydalbergiquinol inhibited IBa1.2 in a concentration-dependent manner and significantly decreased the time constant of current inactivation. R-(-)-3'-Hydroxy-2,4,5-trimethoxydalbergiquinol likely stabilized the channel in its closed state, as suggested by molecular modelling and docking simulation to the CaV1.2 channel α 1c subunit. In conclusion, D. tonkinensis species may represent a source of agents potentially useful for the development of novel antihypertensive drugs.

Rho kinase inhibitors reduce voltage-dependent Ca2+ channel signaling in aortic and renal microvascular smooth muscle cells.

Voltage-dependent L-type Ca2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 (P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction (P < 0.05). Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca2+]i in A7r5 cells was suppressed by 1-10 µM Y-27632 (P < 0.05), but 10 µM Y-27632 was required to suppress Ca2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca2+]i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca2+]i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.

Neuromuscular junction abnormalities in a zebrafish loss-of-function model of TDP-43.

Almost 90% of amyotrophic lateral sclerosis (ALS) cases are characterized by the presence of aggregates of insoluble, misfolded cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43). Distal axonopathy with impaired neuromuscular junctions (NMJs) before motor neuron degeneration or clinical onset of symptoms has been hypothesized as an early pathology in ALS. However, synaptic defects at the NMJ caused by TDP-43 mutations have not been characterized. In this study, we examined a previously reported zebrafish line expressing the tardbpY220X/Y220X variant, which results in an unstable and degraded protein. These tardbp-/- larvae, however, mature normally due to the upregulated expression of an alternative splice variant of the tardbp paralog tardbp-like, or tardbpl. We generated a mutant line with a CRISPR/Cas9-mediated 5-base pair deletion encompassing the ATG start codon of tardbpl and in-crossed these with tardbp-/- mutants to obtain tardbp-/- and tardbpl-/- double mutants, herein referred to as hom/hom. We subsequently characterized morphological, coiling, locomotor, synaptic, and NMJ structural abnormalities in the hom/hom mutants and in their genotypic controls. We observed that hom/hom mutants displayed gross morphological defects, early lethality, reduced locomotor function, aberrant quantal transmission, and perturbed synapse architecture at the NMJ. We further employed pharmacological manipulations in an effort to rescue phenotypic defects and observed that tardbp+/-; tardbpl-/- (herein referred to as het/hom) mutants, but not hom/hom mutants, were sensitive to chronic treatments of BAY K 8644, an L-type calcium channel agonist. This result highlights the importance of partial vs. complete loss of allelic functions of TDP-43. NEW & NOTEWORTHY This study highlights the importance of partial vs. complete loss of allelic functions of TDP-43 in a zebrafish loss of function model, thus making it an attractive tool for drug screening approaches.

Effect of reperfusion on vascular smooth muscle reactivity in three contraction models.

BACKGROUND: Ischemia and reperfusion remain inseparable elements of numerous medical procedures such as by-pass surgery, organ transplantation or other cardiology and intervention radiology. The contraction of the smooth muscle of the vessel is considered to be one of the basic components leading to impaired perfusion, an increase in the oxygen deficit of the endothelium of the vessel, and subsequently also to tissues vascularized by the vessel. Main aim of this study was to evaluate the effect of ischemia and reperfusion on vascular smooth muscle cells stimulated pharmacologically with mastoparan-7 (direct G-protein activator) in comparison to stimulation of G-protein coupled receptor agonist - phenylephrine, and direct calcium channel activator - Bay K8644. MATERIAL AND METHODS: Experiments were performed on isolated and perfused tail artery of Wistar rats. Contraction force in our model was measured by increased level of perfusion pressure with a constant flow. RESULTS: Concentration-response curves obtained for phenylephrine, mastoparan-7 and Bay K8644 presented a sigmoidal relation. Ischemia induced hyporreactivity of vessels, whereas during reperfusion the significant time related hyperreactivity for phenylephrine and mastoparan-7 only but not for Bay K8644. These reactions were secondary to the modulation of calcium influx from intra- and extracellular calcium stores. CONCLUSIONS: Results of our experiments suggest that mastoparan-7 significantly induces contraction of vascular smooth muscle cells not only for controls but in the presence of ischemia and reperfusion too. Potential therapeutic applications of the observed reactions are important. They may include regenerative processes within the nervous system, studies on the improvement of blood flow within the microcirculation, or antimicrobial activity. Modulation of the G protein-phospholipase C response may also be an interesting point of action of future drugs modifying the response to stimulation during ischemia in particular, such activities could take place during the transport of organs for transplantation.

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons.

CONTEXT: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities. OBJECTIVE: The effects of fucoidan on Ca2+ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined. MATERIALS AND METHODS: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca2+ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5 mg/mL or 1.5 mg/mL was applied for 3 min to determine its effects on Ca2+ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5 mg/mL for 3 h. RESULTS: The Ca2+ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca2+ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca2+ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca2+ channels, PR1/PR2. DISCUSSION AND CONCLUSIONS: Our data indicate that fucoidan suppresses the intracellular Ca2+ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca2+ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.

Differences in L-type Ca2+ channel activity partially underlie the regional dichotomy in pumping behavior by murine peripheral and visceral lymphatic vessels.

We identified a regional dichotomy in murine lymphatic contractile function with regard to vessel location within the periphery or visceral cavity. All vessels isolated from peripheral regions [cervical, popliteal, inguinal, axillary, and internodal inguinal axillary (Ing-Ax)] developed robust contractions with maximal ejection fractions (EFs) of 50-80% in our ex vivo isobaric myograph experiments. Conversely, vessels isolated from the visceral cavity (mesenteric, thoracic duct, and iliac) demonstrated maximal EFs of ≤10%. Using pressure myography, sharp electrode membrane potential recordings, and Ca2+ imaging, we assessed the role of L-type Ca2+ channels in this contractile dichotomy. Ing-Ax membrane potential revealed a ~2-s action potential (AP) cycle (resting -35 mV, spike -5 mV, and plateau -11 mV) with a plateau phase that was significantly lengthened by the L-type Ca2+ channel agonist Bay K8644 (BayK; 100 nM). APs recorded from mesenteric vessels, however, displayed a slower upstroke and an elongated time over threshold. BayK (100 nM) increased the mesenteric AP upstroke velocity and plateau duration but also significantly hyperpolarized the vessel. Contractions of vessels from both regions were preceded by Ca2+ flashes, detected with a smooth muscle-specific endogenous Ca2+ reporter, that typically were coordinated over the length of the vessel. Similar to the membrane potential recordings, Ca2+ flashes in mesenteric vessels were weaker and had a slower rise time but were longer lasting than those in Ing-Ax vessels. BayK (100 nM) significantly increased the Ca2+ transient amplitude and duration in both vessels and decreased time to peak Ca2+ in mesenteric vessels. However, a higher concentration (1 µM) of BayK was required to produce even a modest increase in EF in visceral lymphatics, which remained at <20%. NEW & NOTEWORTHY Lymphatic collecting vessels isolated from murine peripheral tissues, but not from the visceral cavities, display robust contractile behavior similar to lymphatic vessels from other animal models and humans. These differences are partially explained by L-type Ca2+ channel activity as revealed by the first measurements of murine lymphatic action potentials and contraction-associated Ca2+ transients.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

Hydroxyl Group and Vasorelaxant Effects of Perillyl Alcohol, Carveol, Limonene on Aorta Smooth Muscle of Rats.

The present study used isometric tension recording to investigate the vasorelaxant effect of limonene (LM), carveol (CV), and perillyl alcohol (POH) on contractility parameters of the rat aorta, focusing in particular on the structure-activity relationship. LM, CV, and POH showed a reversible inhibitory effect on the contraction induced by electromechanical and pharmacomechanical coupling. In the case of LM, but not CV and POH, this effect was influenced by preservation of the endothelium. POH and CV but not LM exhibited greater pharmacological potency on BayK-8644-induced contraction and on electromechanical coupling than on pharmacomechanical coupling. In endothelium-denuded preparations, the order of pharmacological potency on electrochemical coupling was LM < CV < POH. These compounds inhibited also, with grossly similar pharmacological potency, the contraction induced by phorbol ester dibutyrate. The present results suggest that LM, CV and POH induced relaxant effect on vascular smooth muscle by means of different mechanisms likely to include inhibition of PKC and IP3 pathway. For CV and POH, hydroxylated compounds, it was in electromechanical coupling that the greater pharmacological potency was observed, thus suggesting a relative specificity for a mechanism likely to be important in electromechanical coupling, for example, blockade of voltage-dependent calcium channel.