Javier Cuevas

Sigma-1 receptor activation prevents intracellular calcium dysregulation in cortical neurons during in vitro ischemia.

Sigma receptors are putative targets for neuroprotection following ischemia; however, little is known on their mechanism of action. One of the key components in the demise of neurons following ischemic injury is the disruption of intracellular calcium homeostasis. Fluorometric calcium imaging was used to examine the effects of sigma receptor activation on changes in intracellular calcium concentrations ([Ca2+]i) evoked by in vitro ischemia in cultured cortical neurons from embryonic rats. The sigma receptor agonist, 1,3-di-o-tolyl-guanidine (DTG), was shown to depress [Ca2+]i elevations observed in response to ischemia induced by sodium azide and glucose deprivation. Two sigma receptor antagonists, metaphit [1-(1-(3-isothiocyanatophenyl)-cyclohexyl)-piperidine] and BD-1047 (N-[2–3,4-dichlorophenyl)-ethyl]-N-methyl-2-(dimethylamino)ethylamine), were shown to blunt the ability of DTG to inhibit ischemia-evoked increases in [Ca2+]i, revealing that the effects are mediated by activation of sigma receptors and not via the actions of DTG on nonspecific targets such as N-methyl-d-aspartate receptors. DTG inhibition of ischemia-induced increases in [Ca2+]i was mimicked by the σ-1 receptor-selective agonists, carbetapentane, (+)-pentazocine and PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], but not by the sigma-2-selective agonist, ibogaine, showing that activation of sigma-1 receptors is responsible for the effects. In contrast, DTG, carbetapentane, and ibogaine blocked spontaneous, synchronous calcium transients observed in our preparation at concentrations consistent with sigma receptor-mediated effects, indicating that both sigma-1 and sigma-2 receptors regulate events that affect [Ca2+]i in cortical neurons. Our studies show that activation of sigma receptors can ameliorate [Ca2+]i dysregulation associated with ischemia in cortical neurons and, thus, identify one of the mechanisms by which these receptors may exert their neuroprotective properties.

Sigma Receptor Activation Reduces Infarct Size at 24 Hours After Permanent Middle Cerebral Artery Occlusion in Rats

The only available treatment for embolic stroke is recombinant tissue plasminogen activator, which must be administered within three hours of stroke onset. We examined the effects of 1,3-di-o-tolyguanidine (DTG), a high affinity sigma receptor agonist, as a potential treatment for decreasing infarct area at delayed time points. Rats were subjected to permanent embolic middle cerebral artery occlusion (MCAO) and allowed to recover before receiving subcutaneous injections of 15 mg/kg of DTG at 24, 48, and 72 hours. At 96 hours the rats were euthanized, and brains harvested and sectioned. Infarct areas were quantified at the level of the cortical/striatal and cortical/hippocampal regions in control (MCAO-only) and DTG treated animals using a marker for neurodegeneration, Fluoro-Jade. DTG treatment significantly reduced infarct area in both cortical/striatal and cortical/hippocampal regions by >80%, relative to control rats. These findings were confirmed by immunohistochemical experiments using the neuronal marker, mouse anti-neuronal nuclei monoclonal antibody (NeuN), which showed that application of DTG significantly increased the number of viable neurons in these regions. Furthermore, DTG blocked the inflammatory response evoked by MCAO, as indicated by decreases in the number of reactive astrocytes and activated microglia/macrophages detected by immunostaining for glial fibrillary acidic protein (GFAP) and binding of isolectin IB4, respectively. Thus, our results demonstrate that the sigma receptor-selective agonist, DTG, can enhance neuronal survival when administered 24 hr after an ischemic stroke. In addition, the efficacy of sigma receptors for stroke treatment at delayed time points is likely the result of combined neuroprotective and anti-inflammatory properties of these receptors.

Blockade of adrenoreceptors inhibits the splenic response to stroke

Recent studies have highlighted the involvement of the peripheral immune system in delayed cellular degeneration after stroke. In the permanent middle cerebral artery occlusion (MCAO) model of stroke, the spleen decreases in size. This reduction occurs through the release of splenic immune cells. Systemic treatment with human umbilical cord blood cells (HUCBC) 24 h post-stroke blocks the reduction in spleen size while significantly reducing infarct volume. Splenectomy 2 weeks prior to MCAO also reduces infarct volume, further demonstrating the detrimental role of this organ in stroke-induced neurodegeneration. Activation of the sympathetic nervous system after MCAO results in elevated catecholamine levels both at the level of the spleen, through direct splenic innervation, and throughout the systemic circulation upon release from the adrenal medulla. These catecholamines bind to splenic alpha and beta adrenoreceptors. This study examines whether catecholamines regulate the splenic response to stroke. Male Sprague-Dawley rats either underwent splenic denervation 2 weeks prior to MCAO or received injections of carvedilol, a pan adrenergic receptor blocker, prazosin, an alpha1 receptor blocker, or propranolol, a beta receptor blocker. Denervation was confirmed by reduced splenic expression of tyrosine hydroxylase. Denervation prior to MCAO did not alter infarct volume or spleen size. Propranolol treatment also had no effects on these outcomes. Treatment with either prazosin or carvedilol prevented the reduction in spleen size, yet only carvedilol significantly reduced infarct volume (p < 0.05). These results demonstrate that circulating blood borne catecholamines regulate the splenic response to stroke through the activation of both alpha and beta adrenergic receptors.

Two distinct classes of functional α7-containing nicotinic receptor on rat superior cervical ganglion neurons

1 Nicotinic acetylcholine receptors (nAChRs) that bind α‐bungarotoxin (αBgt) were studied on isolated rat superior cervical ganglion (SCG) neurons using whole‐cell patch clamp recording techniques. 2 Rapid application of ACh onto the soma of voltage clamped neurons evoked a slowly desensitizing current that was reversibly blocked by αBgt (50 nm). The toxin‐sensitive current constituted on average about half of the peak whole‐cell response evoked by ACh. 3 Nanomolar concentrations of methyllycaconitine blocked the αBgt‐sensitive component of the ACh‐evoked current as did intracellular dialysis with an anti‐α7 monoclonal antibody. The results indicate that the slowly reversible toxin‐sensitive response elicited by ACh arises from activation of an unusual class of α7‐containing receptor (α7‐nAChR) similar to that reported previously for rat intracardiac ganglion neurons. 4 A second class of functional α7‐nAChR was identified on some SCG neurons by using rapid application of choline to elicit responses. In these cases a biphasic response was obtained, which included a rapidly desensitizing component that was blocked by αBgt in a pseudo‐irreversible manner. The pharmacology and kinetics of the responses resembled those previously attributed to α7‐nAChRs in a number of other neuronal cell types. 5 Experiments measuring the dissociation rate of 125I‐labelled αBgt from SCG neurons revealed two classes of toxin‐binding site. The times for toxin dissociation were consistent with those required to reverse blockade of the two kinds of αBgt‐sensitive response. 6 These results indicate that rat SCG neurons express two types of functional α7‐nAChR, differing in pharmacology, desensitization and reversibility of αBgt blockade.

Sigma Receptors Suppress Multiple Aspects of Microglial Activation

During brain injury, microglia become activated and migrate to areas of degenerating neurons. These microglia release proinflammatory cytokines and reactive oxygen species causing additional neuronal death. Microglia express high levels of sigma receptors, however, the function of these receptors in microglia and how they may affect the activation of these cells remain poorly understood. Using primary rat microglial cultures, it was found that sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines in response to the activators adenosine triphosphate (ATP), monocyte chemoattractant protein 1 (MCP‐1), and lipopolysaccharide (LPS). Next, the role of sigma receptors in the regulation of calcium signaling during microglial activation was explored. Calcium fluorometry experiments in vitro show that stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with the microglial response to these activators. Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. In addition, sigma receptor activation also prevented membrane ruffling in a calcium‐independent manner, indicating that sigma receptors regulate the function of microglia via multiple mechanisms.

sigma-1 receptor modulation of acid-sensing ion channel a (ASIC1a) and ASIC1a-induced Ca2+ influx in rat cortical neurons.

Acid-sensing ion channels (ASICs) are proton-gated cation channels found in peripheral and central nervous system neurons. The ASIC1a subtype, which has high Ca2+ permeability, is activated by ischemia-induced acidosis and contributes to the neuronal loss that accompanies ischemic stroke. Our laboratory has shown that activation of σ receptors depresses ion channel activity and [Ca2+]i dysregulation during ischemia, which enhances neuronal survival. Whole-cell patch-clamp electrophysiology and fluorometric Ca2+ imaging were used to determine whether σ receptors regulate the function of ASIC in cultured rat cortical neurons. Bath application of the selective ASIC1a blocker, psalmotoxin1, decreased proton-evoked [Ca2+]i transients and peak membrane currents, suggesting the presence of homomeric ASIC1a channels. The pan-selective σ-1/σ-2 receptor agonists, 1,3-di-o-tolyl-guanidine (100 μM) and opipramol (10 μM), reversibly decreased acid-induced elevations in [Ca2+]i and membrane currents. Pharmacological experiments using σ receptor-subtype-specific agonists demonstrated that σ-1, but not σ-2, receptors inhibit ASIC1a-induced Ca2+ elevations. These results were confirmed using the irreversible σ receptor antagonist metaphit (50 μM) and the selective σ-1 antagonist BD1063 (10 nM), which obtunded the inhibitory effects of the σ-1 agonist, carbetapentane. Activation of ASIC1a was shown to stimulate downstream Ca2+ influx pathways, specifically N-methyl-d-aspartate and (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptors and voltage-gated Ca2+ channels. These subsequent Ca2+ influxes were also inhibited upon activation of σ-1 receptors. These findings demonstrate that σ-1 receptor stimulation inhibits ASIC1a-mediated membrane currents and consequent intracellular Ca2+ accumulation. The ability to control ionic imbalances and Ca2+ dysregulation evoked by ASIC1a activation makes σ receptors an attractive target for ischemic stroke therapy.

VIP and PACAP potentiation of nicotinic ACh-evoked currents in rat parasympathetic neurons is mediated by G-protein activation

The effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP27 and PACAP38) on isolated parasympathetic neurons of rat intracardiac and submandibular ganglia were examined under voltage clamp using whole‐cell patch‐clamp recording techniques. VIP and PACAP (≤ 10 n m) selectively and reversibly increased the affinity of nicotinic acetylcholine receptor channels (nAChRs) for their agonists resulting in a potentiation of acetylcholine (ACh)‐evoked whole‐cell currents at low agonist concentrations. VIP‐induced potentiation was observed with either ACh or nicotine as the cholinergic agonist. The VIP‐ but not the PACAP‐induced potentiation of ACh‐evoked currents was inhibited by [Ac‐Tyr1, D‐Phe2]‐GRF 1–29, amide (100 nm), a selective antagonist of VPAC1 and VPAC2 receptors; whereas the PACAP38‐ but not the VIP‐induced potentiation was inhibited by 100 nm PACAP6–38, a PAC1 and VPAC2 receptor antagonist. The signal transduction pathway mediating VIP‐ and PACAP‐induced potentiation of nicotinic ACh‐evoked currents involves a pertussis toxin (PTX)‐sensitive G‐protein. Intracellular application of 200 μm GTPγS or GDPβS inhibited VIP‐induced potentiation of ACh‐evoked whole‐cell currents. GTPγS alone potentiated ACh‐ and nicotine‐evoked currents and the magnitude of these currents was not further increased by VIP or PACAP. The G‐protein subtype modulating the neuronal nAChRs was examined by intracellular dialysis with antibodies directed against αo, αi‐1,2, αi‐3 or β G‐protein subunits. Only the anti‐Gαo and anti‐Gβ antibodies significantly inhibited the effect of VIP and PACAP on ACh‐evoked currents. The potentiation of ACh‐evoked currents by VIP and PACAP may be mediated by a membrane‐delimited signal transduction cascade involving the PTX‐sensitive Go protein.

Electrophysiological properties and responses to simulated ischemia in cat ventricular myocytes of endocardial and epicardial origin.

In multicellular preparations, there are differences in action potential configuration between endocardium and epicardium, and electrophysiological alterations induced by ischemia are more drastic in epicardium than in endocardium. The present study was designed to examine electrophysiological properties of single cardiac myocytes enzymatically isolated from the endocardial and epicardial surfaces of the cat left ventricle and to determine whether the differential responses to ischemia of intact tissue occur in single cells. Action potentials recorded from the isolated single cells of epicardial surface had lower action potential amplitude and a prominent notch between phase 1 and phase 2, compared with those of the cells isolated from the endocardial surface; these findings are similar to those in intact endocardial and epicardial preparations. Resting membrane potentials recorded from both endocardial and epicardial single cells were sensitive to the change in extracellular K+ concentration and had properties of a K+ electrode. Action potential duration was frequency dependent in both cell types and was shorter in epicardial cells than in endocardial cells at a stimulation rate of 3 Hz. When the cells were superfused with Tyrodes solution that was altered to mimic an ischemic environment in vivo (PO2, 30-40 mm Hg; pH 6.8; [K+], 10 mM; and glucose free), resting membrane potential, action potential amplitude, and action potential duration were reduced, and the refractory period was shortened in both endocardial and epicardial single cells, but there were no differences in the degree of changes in action potentials and refractory periods induced between the two cell types. Action potential changes induced by L-alpha-lysophosphatidylcholine (5-40 mg/l) were also similar in endocardial and epicardial single cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Local anaesthetic blockade of neuronal nicotinic ACh receptor-channels in rat parasympathetic ganglion cells

1 The effects of the local anaesthetics QX‐222 and procaine on nicotinic acetylcholine (ACh)‐evoked currents in cultured parasympathetic cardiac neurones of the rat were investigated by use of the whole‐cell, perforated‐patch, and outside‐out recording configurations of the patch clamp method. 2 QX‐222 and procaine, applied to the extracellular surface, reversibly inhibited the peak amplitude of the whole‐cell nicotinic ACh‐evoked current in a concentration‐dependent manner, with half‐maximal inhibitory concentrations (IC50) of 28 μm and 2.8 μm, respectively, at − 80 mV. In these neurones, the sustained inward current mediated by M1 muscarinic receptor activation was unaltered by QX‐222, and neither local anaesthetic affected the adenosine 5′‐triphosphate (ATP)‐evoked current. 3 QX‐222 and procaine block of nicotinic ACh‐evoked inward current was voltage‐dependent and enhanced by hyperpolarization. An e‐fold change in their dissociation equilibrium constants (Kd) resulted from a 62 mV and a 122 mV change in membrane potential, respectively. 4 Both local anaesthetics produce a concentration‐dependent increase in the half‐time of decay of the nicotinic ACh‐evoked inward current. 5 Measurements of unitary currents in outside‐out patches showed that QX‐222 reversibly increased the mean burst duration and closed time and reduced the mean channel open time and open‐state probability of the nicotinic ACh receptor‐channel (AChR) in a concentration‐dependent manner. 6 The Kd and voltage sensitivity of local anaesthetic block of the nicotinic AChR in rat intracardiac neurones suggests that the pore‐forming region of this channel differs from that of the AChR in frog and rat skeletal muscle and from the neuronal α4β2 ACh receptor‐channel.

MicroRNA-301a Mediated Regulation of Kv4.2 in Diabetes: Identification of Key Modulators

Diabetes is a metabolic disorder that ultimately results in major pathophysiological complications in the cardiovascular system. Diabetics are predisposed to higher incidences of sudden cardiac deaths (SCD). Several studies have associated diabetes as a major underlying risk for heart diseases and its complications. The diabetic heart undergoes remodeling to cope up with the underlying changes, however ultimately fails. In the present study we investigated the changes associated with a key ion channel and transcriptional factors in a diabetic heart model. In the mouse db/db model, we identified key transcriptional regulators and mediators that play important roles in the regulation of ion channel expression. Voltage-gated potassium channel (Kv4.2) is modulated in diabetes and is down regulated. We hypothesized that Kv4.2 expression is altered by potassium channel interacting protein-2 (KChIP2) which is regulated upstream by NFkB and miR-301a. We utilized qRT-PCR analysis and identified the genes that are affected in diabetes in a regional specific manner in the heart. At protein level we identified and validated differential expression of Kv4.2 and KChIP2 along with NFkB in both ventricles of diabetic hearts. In addition, we identified up-regulation of miR-301a in diabetic ventricles. We utilized loss and gain of function approaches to identify and validate the role of miR-301a in regulating Kv4.2. Based on in vivo and in vitro studies we conclude that miR-301a may be a central regulator for the expression of Kv4.2 in diabetes. This miR-301 mediated regulation of Kv4.2 is independent of NFkB and Irx5 and modulates Kv4.2 by direct binding on Kv4.2 3′untranslated region (3′-UTR). Therefore targeting miR-301a may offer new potential for developing therapeutic approaches.