Nonselective Cation Conductance Research Articles

Postsynaptic actions of substance P on rat periaqueductal grey neurons in vitro

The postsynaptic actions of substance P on rat midbrain periaqueductal grey (PAG) neurons were examined using whole-cell patch-clamp recordings in brain slices. Substance P produced an inward current in a subpopulation (60%) of PAG neurons. The substance P induced current was concentration dependent (EC 50=27 nM) and was reduced by the NK1, NK2 and NK3 antagonists L-732,138 (20 μM), GR 159897 (3 μM) and SB 218795 (3 μM). The selective NK1, NK2 and NK3 agonists [Sar 9,Met(O 2) 11]-Substance P (100 nM), GR 64349 (300–500 nM) and senktide (300 nM) also produced inward currents in subpopulations of neurons. A greater proportion of substance P-sensitive neurons (70%) than substance P-insensitive neurons (31%) responded to the μ/δ opioid agonist met-enkephalin (10 μM). Substance P reduced the outward current produced by met-enkephalin. The reversal potential of the substance P induced current varied from −5 mV to below −140 mV in the absence of met-enkephalin, and was −105 mV in the presence of met-enkephalin. These results indicate that substance P acts via NK1, NK2 and NK3 receptors to excite subpopulations of opioid-sensitive and insensitive PAG neurons by increasing a non-selective cation conductance and by reducing a K + current. In addition, substance P has anti-opioid actions that are largely mediated by a reduction in the opioid induced K + current.

Essential Role of a Ca 2+ -Selective, Store-Operated Current ( I SOC ) in Endothelial Cell Permeability

Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca 2+ ] i ) in microvascular endothelial cells are unknown. Depletion of stored Ca 2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca 2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca 2+ entry current, I SOC . Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated I SOC in PAECs, and revealed I SOC in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate I SOC provides a [Ca 2+ ] i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling I SOC activation coordinate the site-specific vascular response to inflammation.

Angiotensin depolarizes parvocellular neurons in paraventricular nucleus through modulation of putative nonselective cationic and potassium conductances

Neurosecretory parvocellular neurons in the hypothalamic paraventricular nucleus (PVN) exercise considerable influence over the adenohypophysis and thus play a critical role in neuroendocrine regulation. ANG II has been demonstrated to act as a neurotransmitter in PVN, exerting significant impact on neuronal excitability and also influencing corticotrophin-releasing hormone secretion from the median eminence and, therefore, release of ACTH from the pituitary. We have used whole cell patch-clamp techniques in hypothalamic slices to examine the effects of ANG II on the excitability of neurosecretory parvocellular neurons. ANG II application resulted in a dose-dependent depolarization of neurosecretory neurons, a response that was maintained in tetrodotoxin (TTX), suggesting a direct mechanism of action. The depolarizing actions of this peptide were abolished by losartan, demonstrating these effects are AT(1) receptor mediated. Voltage-clamp analysis using slow voltage ramps revealed that ANG II activates a voltage-independent conductance with a reversal potential of -37.8 +/- 3.8 mV, suggesting ANG II effects on a nonselective cationic current. Further, a sustained potassium current characteristic of I(K) was significantly reduced (29.1 +/- 4.7%) by ANG II. These studies identify multiple postsynaptic modulatory sites through which ANG II can influence the excitability of neurosecretory parvocellular PVN neurons and, as a consequence of such actions, control hormonal secretion from the anterior pituitary.

Bisphosphonates stimulate an endogenous nonselective cation channel in Xenopus oocytes: potential mechanism of action

The mechanisms of action of bisphosphonates (BPs) have been poorly determined. Besides their actions on osteoclasts, these agents exhibit gastrointestinal complications. They have also recently been described as affecting various preparations that express an epithelial Na(+) channel (ENaC). To understand the effects of BP on ion channels and the ENaC in particular, we used the Xenopus oocyte expression system. Alendronate, and similarly risedronate, two aminobisphosphonates, caused a large stimulation of an endogenous nonselective cation conductance (NSCC). This stimulation averaged 63 +/- 12 muS (n = 18) 60 min after the addition of 2 mM alendronate. The effects on the endogenous NSCC were blocked by extracellular acidification to pH 6.4. On the other hand, alendronate caused a small inhibition of ENaC conductance at pH 7.4 and 6.4, but the effects at pH 6.4 were more readily observed in the absence of changes of the endogenous conductance. The effects on membrane capacitance were also markedly different, with a clear decrease at pH 6.4 and no consistent changes at pH 7.4. The effects on the endogenous channel were further augmented by genistein and were inhibited by a tyrosine phosphatase inhibitor, indicating the involvement of the tyrosine kinase pathway. Stimulation of NSCC with BP is expected to cause membrane depolarization and may explain, in part, its mechanisms of action in inhibiting osteoclasts.

Interleukin-1β depolarizes magnocellular neurons in the paraventricular nucleus of the hypothalamus through prostaglandin-mediated activation of a non selective cationic conductance

Interleukin-1β (IL-1β) is involved in hypothalamic regulation of the neuroimmune response by influencing the synthesis and secretion of corticotropin releasing hormone (CRH), vasopressin (VP) and other stress-related mediators. VP secretion from magnocellular (MNC) neurons of the paraventricular nucleus (PVN) of the hypothalamus at the posterior pituitary and/or median eminence contributes to increasing adrenocorticotropin hormone (ACTH) output and ultimately glucocorticoid release, which then contributes to the stress response. In this study, using whole-cell patch clamp recordings from neurons in a slice preparation of the rat PVN, we show that MNC neurons are also influenced by IL-1β. In response to 1 nM IL-1β, 62% of MNC neurons tested depolarized (mean depolarization=10.9±1.4 mV); effects which were maintained in the presence of a sodium channel blocker, tetrodotoxin (TTX). The effects of IL-1β on MNC neurons were blocked in the presence of a specific cyclooxygenase (COX)-2 inhibitor, NS-398, indicating a dependence on prostaglandins (PG) in mediating these effects. In response to direct application of 1 μM PGE 2, 57% of MNC neurons depolarized, exhibiting a membrane potential change similar to that induced by IL-1β (mean depolarization=7.8±1.1 mV). Voltage clamp experiments examining the effects of PGE 2 on the currents evoked by slow voltage ramps revealed activation of a conductance characteristic of a non-selective cationic conductance (NSCC) (voltage-independent, with a reversal potential of −41.8±7.6 mV), suggesting that this prostanoid directly modifies cationic currents in MNC neurons. These data provide evidence that IL-1β depolarizes MNC neurons in the PVN as a result of prostaglandin-mediated activation of a NSCC.

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.

Orexins cause depolarization via nonselective cationic and K+ channels in isolated locus coeruleus neurons

The locus coeruleus (LC) contains noradrenergic neurons that are innervated by orexin (ORX)-like immunoreactive axons and express both orexin receptor-1 and -2. We studied effects of ORX-A and -B (ORX-A/B) on dissociated LC neurons by using whole-cell patch clamp techniques. In current-clamp mode, LC neurons were depolarized by application of ORX-A (10(-7) M) [53% of neurons tested; 9.0+/-0.2 mV (n=5)], or ORX-B (10(-7) M) [38% of neurons tested; 4.0+/-0.1 mV (n=5)]. Firing frequencies of action potentials increased during application [1.1+/-0.2 Hz (n=5) in ORX-A; 0.8+/-0.2 Hz (n=5) in ORX-B] and returned to the control level [0.2+/-0.1 Hz (n=5)] after removal. The ORX-A/B-induced depolarization was well maintained in the presence of TTX (3x10(-7) M), CNQX (10(-6) M) and AP5 (10(-5) M). In voltage-clamp mode, removal of external Na+ suppressed both ORX-A/B-induced currents and shifted their reversal potentials from approximately -45 mV to -60 mV. In addition, ORX-A/B inhibited sustained K+ currents. These results suggest that ORX-A/B increase the firing frequency of LC neurons through the depolarization probably produced by both augmentation of the nonselective cationic conductance and inhibition of the sustained K+ conductance.

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes.

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-permeable non-selective cation conductance (I(cat)) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I(cat) was recorded in cell-attached and inside-out patches with similar unitary conductance values. In inside-out patches with relatively high constitutive activity the G-protein activator GTPgammaS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G-protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G-protein inhibitor GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1-oeoyl-2-acetyl-sn-glycerol (OAG) decreased the probability of channel opening (NP(o)) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP(o). These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G(alphaq)/G(alpha11)-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP(o) and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) protein activity leading to channel opening via a DAG-mediated but PKC-independent mechanism. Finally, with whole-cell recording it is shown that noradrenaline increases I(cat) and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I(cat) in these arterial myocytes.

ATP Excites Interneurons and Astrocytes to Increase Synaptic Inhibition in Neuronal Networks

We investigated the role of extracellular ATP at astrocytes and inhibitory GABAergic interneurons in the stratum radiatum area of the mouse hippocampus. We show that exogenously applied ATP increased astrocyte intracellular Ca2+ levels and depolarized all calbindinand calretinin-positive interneurons in the stratum radiatum region of mouse hippocampus, leading to action potential firing and enhanced synaptic inhibition onto the postsynaptic targets of interneurons. Electrophysiological, pharmacological, and immunostaining studies suggested that the effect of ATP on interneurons was mediated by P2Y1 receptors, and that the depolarization of interneurons was caused by the concomitant reduction and activation of potassium and nonselective cationic conductances, respectively. Electrical stimulation of the Schaffer collaterals and perforant path, as well as local stimulation within the stratum radiatum, evoked increases in intracellular Ca2+ in astrocytes. Facilitation of GABAergic IPSCs onto interneurons also occurred during electrical stimulation. Both the stimulation-evoked increases in astrocyte Ca2+ levels and facilitation of GABAergic IPSCs were sensitive to antagonists of P2Y1 receptors and mimicked by exogenous P2Y1 receptor agonists, suggesting that endogenously released ATP can activate P2Y receptors on both astrocytes and interneurons. Overall, our data are consistent with the hypothesis that ATP released from neurons and astrocytes acts on P2Y1 receptors to excite interneurons, resulting in increased synaptic inhibition within intact hippocampal circuits.

Glucagon-like peptide 1 excites hypocretin/orexin neurons by direct and indirect mechanisms: implications for viscera-mediated arousal.

Glucagon-like peptide 1 (GLP-1) is produced by neurons in the caudal brainstem that receive sensory information from the gut and project to several hypothalamic regions involved in arousal, interoceptive stress, and energy homeostasis. GLP-1 axons and receptors have been detected in the lateral hypothalamus, where hypocretin neurons are found. The electrophysiological actions of GLP-1 in the CNS have not been studied. Here, we explored the GLP-1 effects on GFP (green fluorescent protein)-expressing hypocretin neurons in mouse hypothalamic slices. GLP-1 receptor agonists depolarized hypocretin neurons and increased their spike frequency; the antagonist exendin (9-39) blocked this depolarization. Direct GLP-1 agonist actions on membrane potential were abolished by choline substitution for extracellular Na+, and dependent on intracellular GDP, suggesting that they were mediated by sodium-dependent conductances in a G-protein-dependent manner. In voltage clamp, the GLP-1 agonist Exn4 (exendin-4) induced an inward current that reversed near -28 mV and persisted in nominally Ca2+-free extracellular solution, consistent with a nonselective cationic conductance. GLP-1 decreased afterhyperpolarization currents. GLP-1 agonists enhanced the frequency of miniature and spontaneous EPSCs with no effect on their amplitude, suggesting presynaptic modulation of glutamate axons innervating hypocretin neurons. Paraventricular hypothalamic neurons were also directly excited by GLP-1 agonists. In contrast, GLP-1 agonists had no detectable effect on neurons that synthesize melanin-concentrating hormone (MCH). Together, our results show that GLP-1 agonists modulate the activity of hypocretin, but not MCH, neurons in the lateral hypothalamus, suggesting a role for GLP-1 in the excitation of the hypothalamic arousal system possibly initiated by activation by viscera sensory input.

Evidence for Activation of Endogenous Transporters in Xenopus laevis Oocytes Expressing the Plasmodium falciparum Chloroquine Resistance Transporter, PfCRT

A large body of genetic, reverse genetic, and epidemiological data has linked chloroquine-resistant malaria to polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum. To investigate the biological function of the chloroquine resistance transporter, PfCRT, as well as its role in chloroquine resistance, we functionally expressed this protein in Xenopus laevis oocytes. Our data show that PfCRT-expressing oocytes exhibit a depolarized resting membrane potential and a higher intracellular pH compared with control oocytes. Pharmacological and electrophysiological studies link the higher intracellular pH to an enhanced amiloride-sensitive H(+) extrusion and the low membrane potential to an activated nonselective cation conductance. The finding that both properties are independent of each other, together with the fact that they are endogenously present in X. laevis oocytes, supports a model in which PfCRT activates transport systems. Our data suggest that PfCRT plays a role as a direct or indirect activator or modulator of other transporters.

Actions of subtype-specific purinergic ligands on rat spiral ganglion neurons

In a previous study we showed that, in rat spiral ganglion neurons (SGNs), the adenosine 5'-triphosphate (ATP)-evoked currents were a combination of the activation of ionotropic receptors (the first fast current) and the activation of metabotropic receptors which secondarily opened non-selective cation channels. These two conductances imply the involvement of different receptor subtypes. In the present study, we tested three subtype-specific purinergic ligands: alpha,beta-methylene ATP (a;pha,beta-meATP) for P2X receptors, uridine 5'-triphosphate (UTP) for P2Y receptors and 2'-3'-O-(4-benzoylbenzoyl) ATP (Bz-ATP) for P2Z (P2X(7)) receptors. Application of 100 microM alpha,beta-meATP did not trigger any significant change in membrane conductance, while the SGNs were responsive to ATP. Pressure application of UTP (100 microM, 1 s) evoked an inward current averaging 344+/-169 pA at a holding potential of -50 mV. The conductance developed after a latency averaging 1.5+/-0.6 s, took 4-6 s to peak and reversed slowly within 15-30 s. The current-voltage curve reversed near 0 mV, suggesting a non-selective cation conductance, like the second component of the ATP conductance. Bz-ATP evoked an inward current which developed without latency, was sustained during ligand application and was rapidly inactivated at the end of application: the same characteristics as the first component of the ATP-evoked current. The Bz-ATP conductance reversed around -10 mV, indicating also a non-selective cation conductance. These results suggest that, in SGNs, ATP acts via two different receptor subtypes, ionotropic P2Z receptors and metabotropic P2Y receptors, and that these two receptor subtypes can assume different physiological roles.

Pituitary adenylate cyclase-activating polypeptide enhances the hyperpolarization-activated nonselective cationic conductance, Ih, in dissociated guinea pig intracardiac neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) peptides, which are co-localized with acetylcholine in preganglionic parasympathetic fibers innervating guinea pig intracardiac ganglia, depolarize and increase excitability of intracardiac neurons. Perforated patch whole cell recordings were used to test whether PACAP27-enhanced activation of Ih contributed to the increase in excitability. In current clamp, 100 nM PACAP27 increased rectification during 500-ms hyperpolarizations and increased the number of anodal break action potentials (APs). PACAP27 also increased the number of APs produced by 500-ms depolarizing currents. In voltage clamp, the effects of 100 nM PACAP27 were determined during hyperpolarizing steps from −50 mV to voltages between −60 and −120 mV. PACAP27 increased the amplitude and rate of activation of Ih. PACAP27 shifted the voltage dependence of activation of Ih by 6.6 mV. The effect of PACAP27 was eliminated by pretreatment with the Ih inhibitor ZD7288 (100 μM). The adenylyl cyclase activator forskolin (10 μM) produced a similar shift in the voltage dependence of Ih activation. We conclude that PACAP27 enhances Ih by shifting the voltage dependence of activation and propose that this effect is mediated primarily by PAC1 receptor activation of adenylyl cyclase and generation of cAMP. Furthermore, we propose that the peptide-enhanced Ih contributes to the PACAP27-induced increase in membrane excitability.

Ionic conductances involved in generation and propagation of electrical slow waves in phasic gastrointestinal muscles.

Considerable work has led many to conclude that interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. These cells form electrically coupled networks within the pacemaker regions of the GI tract, and ICC are electrically coupled to smooth muscle cells. ICC express unique ion channels that periodically produce inward (pacemaker) currents. Recent work has suggested that the inward current is produced by a calcium (Ca2+)-regulated, nonselective cation conductance. Channels responsible for this conductance oscillate in open probability in response to the periodic drop in intracellular Ca2+ concentration during the slow wave cycle. Pacemaker activity generates slow waves that are propagated actively through ICC networks. Depolarization coordinates the pacemaker activity through the ICC network by activating a dihydropyridine-resistant Ca2+ conductance. Entry of small amounts of Ca2+ into ICC entrains spontaneous pacemaker activity and produces cell-to-cell propagation of slow waves. This review discusses the mechanisms and conductances involved in generation and propagation of electrical slow waves in ICC.

Role of calcium in neurotensin-evoked enhancement in firing in mesencephalic dopamine neurons.

Neurotensin (NT) increases neurotransmission within the mesolimbic dopamine system by enhancing the firing rate of dopaminergic (DAergic) neurons and by acting at the nerve terminal level. The signal transduction pathways involved in these effects have not been characterized, but NT receptors are coupled to the phospholipase C pathway and Ca(2+) mobilization. However, an enhancement of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by NT in DAergic neurons has yet to be demonstrated. Furthermore, the hypothesis that the excitatory effects of NT in DAergic neurons are Ca(2+) dependent is currently untested. In whole-cell recording experiments, DAergic neurons in culture were identified by their selective ability to express a cell-specific green fluorescent protein reporter construct. These experiments confirmed that NT increases firing rate in cultured DAergic neurons. This effect was Ca(2+) dependent because it was blocked by intracellular dialysis with BAPTA. Using Ca(2+) imaging, we showed that NT caused a rapid increase in [Ca(2+)](i) in DAergic neurons. Most of the Ca(2+) originated from the extracellular medium. NT-induced excitation and Ca(2+) influx were blocked by SR48692, an antagonist of the type 1 NT receptor. Blocking IP(3) receptors using heparin prevented the excitatory effect of NT. Moreover, Zn(2+) and SKF96365 both blocked the excitatory effect of NT, suggesting that nonselective cationic conductances are involved. Finally, although NT can also induce a rise in [Ca(2+)](i) in astrocytes, we find that NT-evoked excitation of DAergic neurons can occur independently of astrocyte activation.

NK-3 receptor activation depolarizes and induces an after-depolarization in pyramidal neurons in gerbil cingulate cortex

The involvement of tachykinins in cortical function is poorly understood. To study the actions of neurokinin-3 (NK3) receptor activation in frontal cortex, whole cell patch clamp recordings were performed from pyramidal neurons in slices of cingulate cortex from juvenile gerbils. Senktide (500 nM), a selective NK3 receptor agonist, induced a transient increase in spontaneous EPSPs in layer V pyramidal neurons, accompanied by a small depolarization (∼4 mV). EPSPs during senktide had a larger amplitude and faster 10–90% rise time than during control. Senktide induced a transient depolarization in layer II/III pyramidal neurons, which often reached threshold for spikes. The depolarization (∼6 mV) persisted in TTX, and was accompanied by an increase in input resistance. Senktide also transiently induced a slow after-depolarization, which appeared following a depolarizing pulse. The slow after-depolarization persisted in TTX. These data suggest that activation of NK3 receptors on layer II/III pyramidal neurons induce post-synaptic depolarization and an after-depolarization, which could be mediated by blockade of a leak potassium conductance and a non-selective cation conductance, respectively.

Secretin depolarizes nucleus tractus solitarius neurons through activation of a nonselective cationic conductance.

The recent suggestion that secretin may be useful in treating autism and schizophrenia has begun to focus attention on the mechanisms underlying this gut-brain peptide's actions in the central nervous system (CNS). In vitro autoradiographic localization of (125)I-secretin binding sites in rat brain shows the highest binding density in the nucleus tractus solitarius (NTS). Recent evidence suggests that intravenous infusion of secretin causes fos activation in NTS, a relay station playing important roles in the central regulation of autonomic functions. In this study, whole cell patch-clamp recordings were obtained from 127 NTS neurons in rat medullary slices. The mean resting membrane potential of these neurons was -54.7 +/- 0.3 mV, the mean input resistance was 3.7 +/- 0.2 GOmega, and the action potential amplitude of these neurons was always >70 mV. Current-clamp studies showed that bath application of secretin depolarized the majority (80.8%; 42/52) of NTS neurons tested, whereas the remaining cells were either unaffected (17.3%; 9/52) or hyperpolarized (1.9%; 1/52). These depolarizing effects were maintained in the presence of 5 microM TTX and found to be concentration dependent from 10(-12) to 10(-7) M. Using voltage-clamp techniques, we also identified modulatory actions of secretin on specific ion channels. Our results demonstrate that while secretin is without effect on net whole cell potassium currents, it activates a nonselective cationic conductance (NSCC). These results show that NTS neurons are activated by secretin as a consequence of activation of a NSCC and support the emerging view that secretin can act as a neuropeptide within the CNS.

Effect of hypertonic saline on rat hypothalamic paraventricular nucleus magnocellular neurons in vitro

The effect of hypertonic saline on rat hypothalamic paraventricular nucleus (PVN) magnocellular neurons was examined using a whole-cell patch-clamp technique. Under a current-clamp, 58/68 of magnocellular neurons were depolarized by hypertonic stimulation. Under a voltage-clamp, hypertonic saline produced an inward current via increased non-selective cationic conductance and shifting of the reversal potential to more positive values. Furthermore, hypertonic saline even without a change in osmolality increased spontaneous excitatory postsynaptic currents (sEPSCs). A bath application of CNQX almost completely blocked EPSCs. Extracellular application of gadolinium blocked the hypertonic saline- and mannitol-induced response. These results suggest that PVN magnocellular neurons are responsive to osmolality and Na + concentrations. Hypertonic saline excited PVN magnocellular neurons via osmo-reception, Na +-detection, and excitatory glutamatergic synaptic input.

A Key Role for TRPM7 Channels in Anoxic Neuronal Death

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. Here, we show that in neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD was activated by reactive oxygen/nitrogen species (ROS), and permitted neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminated the need for AET to rescue anoxic neurons and permitted the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role.

Mechanism of active repolarization of inhibitory junction potential in murine colon.

Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with micro-electrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. Ba2+ (500 muM), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of Ba2+, Ni2+, and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that Ni2+ decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both Ni2+ and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells.