Blockade Of Na Research Articles

Abstract 1071: Efficacy of targeted osmotic lysis using pulsed electric field stimulation compared to paclitaxel for treating murine, triple-negative breast carcinoma

Abstract Targeted osmotic lysis (TOL), the concurrent stimulation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps, kills up to 100% of highly malignant, human and murine breast cancer cells in vitro and slows the growth of murine breast cancer homografts and increases survival of the hosts in vivo when compared to homografts treated with negative controls. In this study we compared the efficacy of TOL and paclitaxel, a current standard chemotherapeutic agent for treating advanced murine breast cancer, as a positive control. For this, BALB/c mice with 4T1 homografts were treated with TOL alone, paclitaxel alone or with a combination regimen of paclitaxel and TOL. For TOL, mice received 8 hourly doses (3 mg/kg) of digoxin on 2 successive days. Upon reaching steady-state levels (dose 5) the mice were exposed to 4 X 30 minutes of pulsed electric field stimulation (18 V/m, 10 ms pulses, 15 interstimulus intervals) hourly for a total of 2 hours of stimulation. Alternatively, mice received paclitaxel (20 mg/kg) by i.p. injection. Mice that were treated concurrently with TOL and paclitaxel received paclitaxel 2 days prior to treatment with TOL. Groups of mice that received digoxin alone, stimulation alone, paclitaxel + digoxin, paclitaxel + stimulation or vehicle alone served as negative controls. Initially, the homografts were measured daily and then every other day with a digital caliper until criteria for humane endpoint euthanasia were met. The mean time to meeting criteria for humane endpoint euthanasia in mice treated with paclitaxel alone (2 days) was identical to that observed in the negative controls. Mice treated with TOL alone, on average, met criteria at post-treatment day 5, whereas administration of paclitaxel two days before TOL reduced the effectiveness of TOL, decreasing mean survival to day 3 post-treatment. Immunohistochemical evidence indicated that paclitaxel’s effect on tubulin may impede the transport and insertion of channels into the cell membrane, thereby reducing the channel density and the effectiveness of TOL. By contrast, the administration of TOL either before or after treatment with paclitaxel improved host survival. Normal tissues were unaffected. These results are evidence that TOL is equal to or better than paclitaxel for increasing survival of murine hosts with advanced breast cancer when TOL is administered before or between paclitaxel dosing cycles. Sequential use of TOL may well improve survival, but simultaneous administration of paclitaxel and TOL will likely reduce TOL’s adjunctive benefit. Because of the conserved nature of the sodium channel/sodium pump mechanism, the comparable efficacy to TOL to a currently approved treatment and the favorable adverse effect profile, we submit that TOL warrants further study as a promising option for treating individuals with advanced neoplastic disease. Citation Format: Harry J. Gould, Kelly J. Sherman, Dennis Paul. Efficacy of targeted osmotic lysis using pulsed electric field stimulation compared to paclitaxel for treating murine, triple-negative breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1071.

Targeted Osmotic Lysis of Highly Invasive Breast Carcinomas Using Pulsed Magnetic Field Stimulation of Voltage-Gated Sodium Channels and Pharmacological Blockade of Sodium Pumps.

Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0–80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na+-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60–100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+ /K+ ATPase.

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca2+ /calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein-expressing astrocytes (astroglial calcineurin KO [ACN-KO]). Here, we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K+ homeostasis. Indeed, blockade of Na+ /K+ -ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca2+ -sensitive switch.

H2S-Mediated Changes in Erythrocyte Volume: Role of Gardos Channels, Na+,K+,2Cl- Cotransport and Anion Exchanger.

The effect of H2S on changes in erythrocyte volume was studied by spectrophotometrical and potentiometric methods. It was found that H2S donor NaHS (2.5, 10, and 100 μM) induced an increase in erythrocyte volume in heterosmotic media. Activation of Gardos channels with A23187 or ascorbate-phenazine methosulfate system causes erythrocyte shrinkage and hyperpolarization of their membrane, while addition of NaHS restored erythrocyte volume. The decrease in erythrocyte volume upon blockade of Na+,K+,2Cl- cotransporter (bumetanide) or anion exchanger (SITS) was abolished by H2S donor NaHS, which attested to an important role of these transporters and chlorine conductivity of the membrane in the maintenance of the homeostasis of blood cells.

Modeling Analysis of Axonal After Potential at Hippocampal Mossy Fibers.

Action potentials reliably propagate along the axons, and after potential often follows the axonal action potentials. After potential lasts for several tens of millisecond and plays a crucial role in regulating excitability during repetitive firings of the axon. Several mechanisms underlying the generation of after potential have been suggested, including activation of ionotropic autoreceptors, accumulation of K+ ions in the surrounding extracellular space, the opening of slow voltage-dependent currents, and capacitive discharge of upstream action potentials passively propagated through axon cable. Among them, capacitive discharge is difficult to examine experimentally, since the quantitative evaluation of a capacitive component requires simultaneous recordings from at least two different sites on the connecting axon. In this study, a series of numerical simulation of the axonal action potential was performed using a proposed model of the hippocampal mossy fiber where morphological as well as electrophysiological data are accumulated. To evaluate the relative contribution of the capacitive discharge in axonal after potential, voltage-dependent Na+ current as well as voltage-dependent K+ current was omitted from a distal part of mossy fiber axons. Slow depolarization with a similar time course with the recorded after potential in the previous study was left after blockade of Na+ and K+ currents, suggesting that a capacitive component contributes substantially in axonal after potential following propagating action potentials. On the other hand, it has been shown that experimentally recorded after potential often showed clear voltage-dependency upon changes in the initial membrane potential, obviously deviating from voltage-independent nature of the capacitive component. The simulation revealed that activation of voltage-dependent K+ current also contributes to shape a characteristic waveform of axonal after potential and reconstitute similar voltage-dependency with that reported for the after potential recorded from mossy fiber terminals. These findings suggest that the capacitive component reflecting passive propagation of upstream action potential substantially contributes to the slow time course of axonal after potential, although voltage-dependent K+ current provided a characteristic voltage dependency of after potential waveform.

Deletion of TRPC6 Attenuates NMDA Receptor-Mediated Ca2+ Entry and Ca2+-Induced Neurotoxicity Following Cerebral Ischemia and Oxygen-Glucose Deprivation.

Transient receptor potential canonical 6 (TRPC6) channels are permeable to Na+ and Ca2+ and are widely expressed in the brain. In this study, the role of TRPC6 was investigated following ischemia/reperfusion (I/R) and oxygen-glucose deprivation (OGD). We found that TRPC6 expression was increased in wild-type (WT) mice cortical neurons following I/R and in primary neurons with OGD, and that deletion of TRPC6 reduced the I/R-induced brain infarct in mice and the OGD- /neurotoxin-induced neuronal death. Using live-cell imaging to examine intracellular Ca2+ levels ([Ca2+]i), we found that OGD induced a significant higher increase in glutamate-evoked Ca2+ influx compared to untreated control and such an increase was reduced by TRPC6 deletion. Enhancement of TRPC6 expression using AdCMV-TRPC6-GFP infection in WT neurons increased [Ca2+]i in response to glutamate application compared to AdCMV-GFP control. Inhibition of N-methyl-d-aspartic acid receptor (NMDAR) with MK801 decreased TRPC6-dependent increase of [Ca2+]i in TRPC6 infected cells, indicating that such a Ca2+ influx was NMDAR dependent. Furthermore, TRPC6-dependent Ca2+ influx was blunted by blockade of Na+ entry in TRPC6 infected cells. Finally, OGD-enhanced Ca2+ influx was reduced, but not completely blocked, in the presence of voltage-dependent Na+ channel blocker tetrodotoxin (TTX) and dl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) blocker CNQX. Altogether, we concluded that I/R-induced brain damage was, in part, due to upregulation of TRPC6 in cortical neurons. We postulate that overexpression of TRPC6 following I/R may induce neuronal death partially through TRPC6-dependent Na+ entry which activated NMDAR, thus leading to a damaging Ca2+ overload. These findings may provide a potential target for future intervention in stroke-induced brain damage.

Traumatic brain injury induces rapid enhancement of cortical excitability in juvenile rats.

Following a traumatic brain injury (TBI), 5-50% of patients will develop posttraumatic epilepsy (PTE) with children being particularly susceptible. Currently, PTE cannot be prevented and there is limited understanding of the underlying epileptogenic mechanisms. We hypothesize that early after TBI the brain undergoes distinct cellular and synaptic reorganization that facilitates cortical excitability and promotes the development of epilepsy. To examine the effect of pediatric TBI on cortical excitability, we performed controlled cortical impact (CCI) on juvenile rats (postnatal day 17). Following CCI, animals were monitored for the presence of epileptiform activity by continuous in vivo electroencephalography (EEG) and/or sacrificed for in vitro whole-cell patch-clamp recordings. Following a short latent period, all animals subjected to CCI developed spontaneous recurrent epileptiform activity within 14 days. Whole-cell patch-clamp recordings of layer V pyramidal neurons showed no changes in intrinsic excitability or spontaneous excitatory postsynaptic currents (sEPSCs) properties. However, the decay of spontaneous inhibitory postsynaptic currents (sIPSCs) was significantly increased. In addition, CCI induced over a 300% increase in excitatory and inhibitory synaptic bursting. Synaptic bursting was prevented by blockade of Na(+)-dependent action potentials or select antagonism of glutamate or GABA-A receptors, respectively. Our results demonstrate that CCI in juvenile rats rapidly induces epileptiform activity and enhanced cortical synaptic bursting. Detection of epileptiform activity early after injury suggests it may be an important pathophysiological component and potential indicator of developing PTE.

Blockade of Na+ channels in pancreatic α-cells has antidiabetic effects.

Pancreatic α-cells express voltage-gated Na(+) channels (NaChs), which support the generation of electrical activity leading to an increase in intracellular calcium, and cause exocytosis of glucagon. Ranolazine, a NaCh blocker, is approved for treatment of angina. In addition to its antianginal effects, ranolazine has been shown to reduce HbA1c levels in patients with type 2 diabetes mellitus and coronary artery disease; however, the mechanism behind its antidiabetic effect has been unclear. We tested the hypothesis that ranolazine exerts its antidiabetic effects by inhibiting glucagon release via blockade of NaChs in the pancreatic α-cells. Our data show that ranolazine, via blockade of NaChs in pancreatic α-cells, inhibits their electrical activity and reduces glucagon release. We found that glucagon release in human pancreatic islets is mediated by the Nav1.3 isoform. In animal models of diabetes, ranolazine and a more selective NaCh blocker (GS-458967) lowered postprandial and basal glucagon levels, which were associated with a reduction in hyperglycemia, confirming that glucose-lowering effects of ranolazine are due to the blockade of NaChs. This mechanism of action is unique in that no other approved antidiabetic drugs act via this mechanism, and raises the prospect that selective Nav1.3 blockers may constitute a novel approach for the treatment of diabetes.

Protein kinase G regulates β-synuclein in response to repeated exposure to cocaine in the rat dorsal striatum in a Ca2+-dependent manner

Protein kinase G (PKG) activation plays a crucial role in neuronal plasticity after repeated exposure to cocaine in the dorsal striatum. The present study investigated the characteristics of β-synuclein expression by PKG activation after repeated cocaine administration in the rat dorsal striatum. The results demonstrated that repeated, but not acute, exposure to cocaine (20mg/kg) once a day for 7 consecutive days significantly upregulated expression of β-synuclein. Furthermore, this upregulation was decreased by the depletion of Ca2+, but not blockade of Na+ influx. Blockade of N-methyl-d-aspartate (NMDA) receptors and ryanodine-sensitive Ca2+ channels also decreased the elevation of β-synuclein expression by repeated cocaine administration. Inhibition of neuronal nitric oxide synthase, which can activate PKG, did not alter the expression of β-synuclein elevated by repeated cocaine administration. These findings suggest that the expression of β-synuclein can be regulated by Ca2+-dependent PKG activation via stimulation of NMDA receptors and voltage-operated Ca2+ channels in the endoplasmic reticulum in the dorsal striatum.

Delayed phospholamban phosphorylation in post-conditioned heart favours Ca2+ normalization and contributes to protection.

It has been shown that sarcoplasmic reticulum calcium ATPase (SERCA) plays a critical role in reperfusion injury. Moreover, ischaemic post-conditioning (PoCo) results in protein kinase G (PKG) activation which has been proposed to modulate phospholamban (PLB) and SERCA. We assessed whether PLB phosphorylation contributes to the cardioprotective effects of PoCo. Isolated Sprague-Dawley rat hearts were submitted to 40 min of ischaemia and reperfusion with and without a PoCo protocol that reduced infarct size by 48%. Reperfusion caused a rapid phosphorylation in PLB at Ser16 and Thr17 that was delayed by PoCo. NO-independent activation of soluble guanylate cyclase (sGC) (ataciguat) and cAMP-dependent protein kinase (PKA) inhibition (KT5720) mimicked the reduction in Ser16 phosphorylation in reperfused control hearts, while in PoCo hearts the inhibitors of PKG (KT5823) and phosphodiesterase 2 (BAY-60-7550) reverted it. CaMKII activity measured by Thr287 phosphorylation was reduced in PoCo. In reperfused control hearts, inhibition of PLB phosphorylation or SERCA (thapsigargin) simulated the cardioprotective effects of PoCo. Ataciguat reduced cytosolic Ca(2+) oscillations and improved Ca(2+) recovery in cardiomyocytes subjected to anoxia-reoxygenation and infarct size by 32% in rats with 30 min of the left anterior descending coronary artery occlusion and 2 h of reperfusion. Blockade of Na(+)/Ca(2+)-exchanger (NCX; KB-R7943) impaired Ca(2+) control in cardiomyocytes and abolished cardioprotection in PoCo hearts. PoCo reduces SERCA activity at the onset of reperfusion by delaying PLB phosphorylation through activation of PKG and inhibition of PKA and CaMKII. This effect contributes to PoCo protection by favouring cytosolic Ca(2+) extrusion through NCX, and it may be mimicked by pharmacological stimulation of sGC.

Chapter 4 - Cytoarchitecture and CO2 Sensitivity of Phox2b-Positive Parafacial Neurons in the Newborn Rat Medulla

Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) compose one of the respiratory rhythm generators in the medulla of the newborn rat. It has been shown that a subgroup of pFRG/Pre-I neurons could also work as central chemoreceptor neurons, because the CO(2) sensitivity of these Pre-I neurons was preserved even after blockade of Na(+) channels and Ca(2+) channels, and the membrane depolarization induced by hypercapnic stimulation was mainly because of the closing of K(+) channels. These neurons, some of which were identified to be glutamatergic, express the transcription factor Phox2b. Phox2b expression was one of the most noticeable characteristics of pFRG/Pre-I neurons. We also found that Phox2b-expressing neurons in the parafacial region of the rostral ventral medulla tended to assemble around capillary blood vessels. In contrast, another subclass of the pFRG/Pre-I neurons was Phox2b-negative and CO(2)-insensitive. Some of these neurons were identified to be glycinergic or GABAergic. Thus, Phox2b expression is a key genetic marker that can be used to more clearly establish the cell architecture of the pFRG, which consists of heterogeneous neuronal subtypes. In this chapter, we elaborate on the CO(2) sensitivity of Phox2b-positive/negative parafacial neurons and the cytoarchitecture in the newborn rat medulla, and discuss ionic mechanisms of CO(2) responsiveness.

Abstract 211: Blockade of Na+-Sensitive K+ Channels Alleviates Frequency-Dependent Electrical Failure in an Ex-Vivo Model of VF-Induced Cardiac Arrest

Ventricular fibrillation (VF), global ischemia and beta-adrenergic stimulation form the common context of sudden cardiac arrest. Regional and global loss of excitability under these conditions sets the stage of resuscitation failure due to either asystole or post-reperfusion VF recurrence. We hypothesized that Na+-sensitive K+ current (IKNa) significantly contributes to electrical failure under combined conditions of ischemia, high excitation frequency, and beta-adrenergic stimulation. We performed optical mapping of voltage-sensitive dye RH237 in the anterior right and left ventricle (RV and LV) of Langendorff-perfused rabbit hearts subjected to normothermic global no-flow ischemia, isoproterenol (30 nM) and pacing at the cycle length (CL) of either 300 ms or 200 ms. 2,3-butanedione monoxime (20 mM) was used to reduce motion artifact. Excitable area was measured in optical maps based on the criterion of the minimum time-derivative of the upstroke of the optical action potential. Decrease of the pacing CL from 300 to 200 ms dramatically accelerated loss of excitability in both RV and LV (Figure, p<0.0001). Treatment with IKNa blocker R56865 significantly postponed frequency-dependent electrical failure (p<0.0001), the protective effect being larger in RV than in LV (see Figure). After 5 min of reperfusion, R56865 treatment group also showed better recovery of excitability, effect being predominant in the LV in comparison to 200 ms CL group. This is the first study to show that IKNa activation can promote electrical failure under conditions mimicking VF-induced cardiac arrest.

Blockade of Na+/H+ exchanger type 3 causes intracellular acidification and hyperexcitability via inhibition of pH-sensitive K+ channels in chemosensitive respiratory neurons of the dorsal vagal nucleus in rats

Extracellular pH (pHe) and intracellular pH (pHi) are important factors for the excitability of chemosensitive central respiratory neurons that play an important role in respiration and obstructive sleep apnea. It has been proposed that inhibition of central Na(+)/H(+) exchanger 3 (NHE-3), a key pHi regulator in the brainstem, decreases the pHi, leading to membrane depolarization for the maintenance of respiration. However, how intracellular pH affects the neuronal excitability of respiratory neurons remains largely unknown. In this study, we showed that NHE-3 mRNA is widely distributed in respiration-related neurons of the rat brainstem, including the dorsal vagal nucleus (DVN). Whole-cell patch clamp recordings from DVN neurons in brain slices revealed that the standing outward current (Iso) through pH-sensitive K(+) channels was inhibited in the presence of the specific NHE-3 inhibitor AVE0657 that decreased the pHi. Exposure of DVN neurons to an acidified pHe and AVE0657 (5 μmol/L) resulted in a stronger effect on firing rate and Iso than acidified pHe alone. Taken together, our results showed that intracellular acidification by blocking NHE-3 results in inhibition of a pH-sensitive K(+) current, leading to synergistic excitation of chemosensitive DVN neurons for the regulation of respiration.

Single KATP channel opening in response to stimulation of AMPA/kainate receptors is mediated by Na+ accumulation and submembrane ATP and ADP changes

Excessive stimulation of glutamatergic receptors (GluRs) can overexcite neurons. This can be dampened by KATP channels linking metabolic and neuronal activities, but the cross-talk has not yet been examined on the single channel level. In the brainstem and hippocampal neurons, GluR agonists augmented the open state probability (Popen) of KATP channels with relative efficacy: kainate AMPA > NMDA > t-ACPD. Inhibition of calcium influx and chelation of intracellular calcium did not modify the effects. Kainate did not augment production of reactive oxygen species measured with roGFP1. H2O2 slightly increased Popen, but GluR effects were not modified. GluR actions were abolished in Na(+)-free solutions and after blockade of Na(+)-K(+)-ATPase. KATP channels in open-cell patch-clamp measurements were inhibited by ATP, stimulated by ADP, and kainate was effective only in the presence of ATP. GluR stimulation enhanced ATP consumption that decreased submembrane ATP levels, whereas metabolic poisoning diminished bulk ATP. Modelling showed strong ATP depletion and ADP accumulation near the membrane, and both effects contributed to Popen increases after GluR stimulation. Kainate and hypoxia activated KATP channels in the functional brainstem slices. Inhibition of aerobic ATP production and GluR stimulation were about equally effective in KATP channel opening during hypoxia. Induction of seizure-like activity in hippocampal slices with Mg(2+)-free solutions was accompanied by ATP decrease and KATP channel opening. We propose that KATP channels and GluRs are functionally coupled that can regulate long-lasting changes of neuronal activity in the CNS neurons.

SKF83959 Is a Potent Allosteric Modulator of Sigma-1 Receptor

SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 (D(1) receptor) agonist, has shown many D(1) receptor-independent effects, such as neuroprotection, blockade of Na(+) channel, and promotion of spontaneous glutamate release, which resemble the effects of the sigma-1 receptor activation. In the present work, we explored the potential modulation of SKF83959 on the sigma-1 receptor. The results indicated that SKF83959 dramatically promoted the binding of (3)H(+)-pentazocine (a selective sigma-1 receptor agonist) to the sigma-1 receptor in brain and liver tissues but produced no effect on (3)H-progesterone binding (a sigma-1 receptor antagonist). The saturation assay and the dissociation kinetics assay confirmed the allosteric effect. We further demonstrated that the SKF83959 analogs, such as SCH22390 [(R)-(1)-7-chloro-8- hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] and SKF38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide], also showed the similar allosteric effect on the sigma-1 receptor in the liver tissue but not in the brain tissue. Moreover, all three tested chemicals elicited no significant effect on (3)H-1,3-di(2-tolyl)-guanidine ((3)H-DTG) binding to the sigma-2 receptor. The present data uncovered a new role of SKF83959 and its analogs on the sigma-1 receptor, which, in turn, may reveal the underlying mechanism for the D(1) receptor-independent effect of the drug.

A study of the mechanism of the antiarrhythmic action of Allapinin

Allapinine (lappaconitine hydrobromide) is a drug for the treatment of cardiac arrhythmias, it shows IC class antiarrhythmics properties. Its action mechanism is associated with blockade of Na(+)-channels with subsequent inhibition of the depolarization rate and, consequently, of the slowing and reducing the excitability of the cardiac conduction system. At the moment, it is not established, what factors are associated with side effects of Allapinine, and therefore it seems important to study the molecular mechanisms of its action. The target genes were identified in a rat model of aconitine-induced arrhythmia using a commercial kit "Rat Neuroscience Ion Channels & Transporters RT2 Profiler PCR Array" (SABioscienses). Comparison of the expression of 84 genes in the experimental (aconitine arrhythmias/Allapinine) and control (aconitine arrhythmias/saline) animals revealed changes in the mRNA level of 18 genes. It has been shown an increase in mRNA levels of genes encoding various types of K(+)-channels (kcna6, kcnj1, kcnj4, kcnq2, kcnq4), Ca(2+)-channel (cacna 1g), vesicular acetylcholine transporter (slc 18a3). Decrease in the mRNA level was observed for genes encoding the Na(+)-channel (scn8a), K(+)-channels (kcne 1, kcns 1), membrane transporters (atp4a, slc6a9). Taken together, it appears that the effect of Allapinine on aconitine--induced arrhythmias is due to modulation of genes encoding Na(+)-, K(+)-, Ca(2+)-channels, conducting ionic currents (I(Na), I(to), I(Ks), I(K1), I(CaT)), which are involved in the formation of different phases of the action potential. The effect of the drug on the mRNA levels of genes encoding the acetylcholine and glycine transporters, suggesting the participation of these neurotransmitters in the mechanisms of anti-arrhythmic properties of the Allapinine.

Effects of leptin on pedunculopontine nucleus (PPN) neurons.

Leptin, a hormone that regulates appetite and energy expenditure, is increased in obese individuals, although these individuals often exhibit leptin resistance. Obesity is characterized by sleep/wake disturbances, such as excessive daytime sleepiness, increased REM sleep, increased nighttime arousals, and decreased percentage of total sleep time. Several studies have shown that short sleep duration is highly correlated with decreased leptin levels in both animal and human models. Arousal and rapid eye movement (REM) sleep are regulated by the cholinergic arm of the reticular activating system, the pedunculopontine nucleus (PPN). The goal of this project was to determine the role of leptin in the PPN, and thus in obesity-related sleep disorders. Whole-cell patch-clamp recordings were conducted on PPN neurons in 9- to 17-day-old rat brainstem slices. Leptin decreased action potential (AP) amplitude, AP frequency, and h-current (I(H)). These findings suggest that leptin causes a blockade of Na⁺ channels. Therefore, we conducted an experiment to test the effects of leptin on Na⁺ conductance. To determine the average voltage dependence of this conductance, results from each cell were equally weighted by expressing conductance as a fraction of the maximum conductance in each cell. I Na amplitude was decreased in a dose-dependent manner, suggesting a direct effect of leptin on these channels. The average decrease in Na⁺ conductance by leptin was ~40 %. We hypothesize that leptin normally decreases activity in the PPN by reducing I(H) and I(Na) currents, and that in states of leptin dysregulation (i.e., leptin resistance) this effect may be blunted, therefore causing increased arousal and REM sleep drive, and ultimately leading to sleep-related disorders.

Effects of antiepileptic drugs on GABA release from rat and human neocortical synaptosomes

In epilepsy, allegedly, a neurotransmitter imbalance between the inhibitory GABA and the excitatory glutamate prevails. Therefore, some antiepileptic drugs (AEDs) are thought to increase GABA release. Because little is known about corresponding presynaptic effects of AEDs in the human brain, this study investigated the effects of carbamazepine, lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and valproate on (3)H-GABA release from human neocortical synaptosomes preincubated with (3)H-GABA. To obtain information on possible species differences, rat neocortical synaptosomes were investigated concomitantly. Release was evoked by either veratridine (1, 3.2, or 10 μM), which prevents activated voltage-dependent Na(+) channels from closing, or elevation of extracellular [K(+)] from 3 to 15 mM. The exocytosis inhibitor tetanus toxin (TeT) or withdrawal of buffer Ca(2+) (Ca (e) (2+) ) reduced K(+)-evoked release in both species, while blockade of Na(+) channels with tetrodotoxin had no effect. K(+)-evoked release was characterized as predominant, Ca(2+)-dependent and Na(+)-independent, exocytosis. Carbamazepine and phenytoin in the rat and carbamazepine, phenytoin, lamotrigine, and valproate in human tissue reduced K(+)-evoked (3)H-GABA release. With respect to veratridine-evoked release, Ca (e) (2+) withdrawal did not reduce release in the rat; it even increased the release in human tissue. TeT was slightly inhibitory in the rat. Blockade of GABA transport diminished veratridine-evoked (3)H-GABA release in either species. This release was characterized as mediated mainly by transporter reversal. Carbamazepine, lamotrigine, and phenytoin in rat tissue and carbamazepine and phenytoin in human decreased veratridine-induced (3)H-GABA release. Interestingly, no AED increased (3)H-GABA release. The reduction by AEDs of veratridine-evoked release was more intense than that of K(+)-evoked release. In conclusion, reduction of GABA release by AEDs may be the actual objective in a pathologically altered neuronal network where GABA acts in a depolarizing fashion.

Mechanisms involved in the nitric oxide independent inhibitory neurotransmission to the pig urinary bladder neck

The current study investigates the mechanisms involved in nitric oxide (NO)-independent, nonadrenergic, noncholinergic (NANC) inhibitory neurotransmission to the pig urinary bladder neck. Urothelium-denuded strips were mounted in organ baths containing physiological saline solution (PSS) at 37°C for isometric force recordings. The relaxations to electrical field stimulation (EFS) were carried out on strips treated with guanethidine, atropine and N(G) -nitro-L-arginine, to block noradrenergic neurotransmission, muscarinic receptors and NO synthase, respectively, and precontracted with phenylephrine. EFS (1-16 Hz) produced frequency-dependent relaxations which were abolished by the blockade of neuronal voltage-activated Na(+) channels. Nonselective and selective inhibition of COX and COX-1, respectively, and blockade of Na(+) -K(+) ATPase reduced the EFS-induced relaxations. However, blockade of COX-2, soluble guanylyl cyclase, large-, intermediate- and small-conductance Ca(2+) -activated K(+) channels, ATP-dependent K(+) channels, voltage-gated K(+) channels, cAMPc-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) failed to modify the nerve-mediated relaxations. The NO-independent inhibitory neurotransmission to the pig urinary bladder neck is mediated, in part, through prostanoids release from a COX-1 pathway, and through activation of the Na(+) -K(+) ATPase. PKA and PKG pathways and postjunctional K(+) channels do not appear to be involved in the NO-independent nerve-mediated relaxations.

Diphenidol inhibited sodium currents and produced spinal anesthesia

The aim of this study was to evaluate the effect of diphenidol on blocking Na(+) currents and spinal anesthesia. We used the patch-clamp method to examine if diphenidol blocked Na(+) currents. Lidocaine, a common used local anesthesia, was used as control. We also evaluated the potencies and durations of diphenidol and lidocaine on spinal blockades of motor function, proprioception, and nociception in rats. Lidocaine exhibited a concentration- and state-dependent effect on tonic blockade of voltage-gated Na(+) currents in mouse neuroblastoma N2A cells (IC(50) of 8.1 and 138.9 microM at holding potentials of -70 and -100 mV, respectively). Diphenidol was more potent (IC(50) of 0.77 and 62.6 microM at holding potentials of -70 and -100 mV, respectively). However, unlike lidocaine, block of Na(+) currents by diphenidol lacked use-dependence. We also found that diphenidol acted like lidocaine and produced dose-related spinal blockades of motor function, proprioception and nociception. Although diphenidol had similar potencies of spinal anesthesia compared with lidocaine it produced a much longer duration of spinal blockades than lidocaine. Our results demonstrated that intrathecal diphenidol produced a long duration and similar potency on spinal anesthesia compared with lidocaine in rats. The anesthetic effect of diphenidol could be in part due to its blockade of Na(+) currents.