Nonselective Cation Conductance Research Articles

Orexin-A Depolarizes Dissociated Rat Area Postrema Neurons through Activation of a Nonselective Cationic Conductance

The area postrema (AP) is involved in the regulation of body fluid balance, feeding behavior, and cardiovascular function. Orexin (ORX)-A is a 33 aa peptide that regulates energy metabolism and sympathetic and cardiovascular actions. ORX immunoreactive axons and their varicose terminals have been found in AP. In this study, whole-cell, current- or voltage-clamp recordings were obtained from 108 dissociated rat AP neurons. The mean resting membrane potential of these neurons (<i>n</i> = 48) was −59.24 ± 0.87 mV, the mean input resistance was 3.57 ± 0.22 GΩ, and the action potential amplitude of these cells was always &gt;90 mV. Current-clamp studies showed bath application of ORX-A depolarized the majority of AP neurons tested (68.8%; 33 of 48), whereas small proportions of cells were either hyperpolarized (16.7%; 8 of 48) or unaffected (14.6%; 7 of 48). These depolarizing effects were found to be concentration dependent from 10⁻⁸ to 10⁻¹¹m. We then examined the contributions of specific ionic conductances to the ORX-A-induced excitation of AP neurons through whole-cell, voltage-clamp studies. Our results demonstrate that in contrast to previous studies on other neuronal populations, ORX-A did not affect net whole-cell potassium currents in AP neurons. Slow depolarizing voltage ramps, however, revealed that ORX-A enhanced a nonselective cationic conductance in AP neurons, effects which would explain the depolarizing effects of the peptide. These data demonstrate that AP neurons are directly influenced by ORX-A and suggest that ORX-A may exert its effects on the central control of feeding behavior and cardiovascular function through direct actions in AP.

TRPV3 is a calcium-permeable temperature-sensitive cation channel.

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

Hypoxia activates a background conductance in freshly isolated pulmonary arterial endothelial cells of the rat

Utilising the patch-clamp recording technique we have demonstrated for the first time the effects of hypoxia on the background current in pulmonary arterial endothelial cells. Electrophysiological studies revealed the presence of a novel oxygen-sensitive, non-selective cation conductance ( I NSC) in these cells. The inward component of I NSC was significantly potentiated by hypoxia. Both the inward and outward components of I NSC were inhibited by both La 3+ and Gd 3+. Hypoxic activation of I NSC may provide an important Ca 2+ influx pathway essential for the release of a pulmonary-selective vasoconstrictor pivotal to the sustained phase of hypoxic pulmonary vasoconstriction.

Effect of reactive oxygen species on NH4+ permeation in Xenopus laevis oocytes.

To investigate the effects of reactive oxygen species (ROS) on NH4+ permeation in Xenopus laevis oocytes, we used intracellular double-barreled microelectrodes to monitor the changes in membrane potential (V(m)) and intracellular pH (pH(i)) induced by a 20 mM NH4Cl-containing solution. Under control conditions, NH4Cl exposure induced a large membrane depolarization (to V(m) = 4.0 +/- 1.5 mV; n = 21) and intracellular acidification [reaching a change in pH(i) (DeltapH(i)) of 0.59 +/- 0.06 pH units in 12 min]; the initial rate of cell acidification (dpH(i)/dt) was 0.06 +/- 0.01 pH units/min. Incubation of the oocytes in the presence of H2O2 or beta-amyloid protein had no marked effect on the NH4Cl-induced DeltapH(i). By contrast, in the presence of photoactivated rose bengal (RB), tert-butyl-hydroxyperoxide (t-BHP), or xanthine/xanthine oxidase (X/XO), the same experimental maneuver induced significantly greater DeltapH(i) and dpH(i)/dt. These increases in DeltapH(i) and dpH(i)/dt were prevented by the ROS scavengers histidine and desferrioxamine, suggesting involvement of the reactive species (1)DeltagO2 and.OH. Using the voltage-clamp technique to identify the mechanism underlying the ROS-measured effects, we found that RB induced a large increase in the oocyte membrane conductance (G(m)). This RB-induced G(m) increase was prevented by 1 mM diphenylamine-2-carboxylate (DPC) and by a low Na+ concentration in the bath. We conclude that RB, t-BHP, and X/XO enhance NH4+ influx into the oocyte via activation of a DPC-sensitive nonselective cation conductance pathway.

Nonselective cation conductance activated by muscarinic and purinergic receptors in rat spiral ganglion neurons.

The present study characterizes the ionic conductances activated by acetylcholine (ACh) and ATP, two candidate neuromodulators, in isolated spiral ganglion neurons (SGNs). Brief application (1 s) of ACh evoked in a dose-dependent manner (EC(50) = 4.1 microM) a reversible inward current with a long latency (average 1.3 s), at holding potential (V(h)) = -50 mV. This current was reversibly blocked by atropine and mimicked by muscarine. Application of ATP also evoked a reversible inward current at V(h) = -50 mV, but the current showed two components. A fast component with a short latency was largely reduced when N-methyl-D-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. The second component had a longer latency (average 1.1 s) and was presumably activated by metabotropic P2Y-like receptors. The second component of ATP-evoked current shared similar characteristics with the responses evoked by ACh: the current reversed near 0 mV, displayed inward rectification, could be carried by NMDG, and was insensitive to extracellular and intracellular calcium. This ACh-/ATP-evoked conductance was reversibly inhibited by preapplication of ionomycin. These results suggest that muscarinic receptors and purinergic metabotropic receptors activate a similar large nonselective cation conductance via a common intracellular pathway in SGNs, a candidate mechanism to regulate neuronal excitability of SGNs.

A Ca2+‐inhibited non‐selective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in some smooth muscles. The nature of the pacemaker conductance is unclear, but studies suggest that pacemaker activity is due to a voltage-independent, Ca(2+)-regulated, non-selective cation conductance. We investigated Ca(2+)-regulated conductances in murine intestinal ICC and found that reducing cytoplasmic Ca(2+) activates whole-cell inward currents and single-channel currents. Both the whole-cell currents and single-channel currents reversed at 0 mV when the equilibrium potentials of all ions present were far from 0 mV. Recordings from on-cell patches revealed oscillations in unitary currents at the frequency of pacemaker currents in ICC. Voltage-clamping cells to -60 mV did not change the oscillatory activity of channels in on-cell patches. Depolarizing cells with high external K(+) caused loss of resolvable single-channel currents, but the oscillatory single-channel currents were restored when the patches were stepped to negative potentials. Unitary currents were also resolved in excised patches. The single-channel conductance was 13 pS, and currents reversed at 0 mV. The channels responsible were strongly activated by 10(-7) M Ca(2+), and 10(-6) M Ca(2+) reduced activity. The 13 pS channels were strongly activated by the calmodulin inhibitors calmidazolium and W-7 in on-cell and excised patches. Calmidazolium and W-7 also activated a persistent inward current under whole-cell conditions. Murine ICC express Ca(2+)-inhibited, non-selective cation channels that are periodically activated at the same frequency as pacemaker currents. This conductance may contribute to the pacemaker current and generation of electrical slow waves in GI muscles.

Ca 2+-dependent and -independent Cl − secretion stimulated by the nitric oxide donor, GEA 3162, in rat colonic epithelium

The lipophilic nitric oxide-liberating drug, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), concentration-dependently induced a Cl − secretion in rat colon. At a low concentration (5×10 −5 M), the action was Ca 2+-dependent, whereas at a high concentration (5×10 −4 M), the response was independent from extracellular Ca 2+. Fura-2 experiments at isolated colonic crypts revealed that GEA 3162 induced an increase of the cytoplasmic Ca 2+ concentration due to an influx of extracellular Ca 2+, probably mediated by an activation of a nonselective cation conductance as demonstrated by whole-cell patch-clamp studies. After depolarization of the basolateral membrane, GEA 3162 (5×10 −4 M) stimulated a current, which was suppressed by glibenclamide but was resistant against blockade of protein kinases by staurosporine, suggesting an activation of apical Cl − channels directly by the nitric oxide (NO) donor. After permeabilizing the apical membrane with the ionophore, nystatin, GEA 3162 (5×10 −4 M) activated basolateral K + conductances and the Na +–K +-ATPase. Thus, the lipophilic NO donor GEA 3162 stimulates a Cl − secretion in a Ca 2+-dependent and -independent manner.

A Ca2+-inhibited non-selective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in some smooth muscles. The nature of the pacemaker conductance is unclear, but studies suggest that pacemaker activity is due to a voltage-independent, Ca2+-regulated, non-selective cation conductance. We investigated Ca2+-regulated conductances in murine intestinal ICC and found that reducing cytoplasmic Ca2+ activates whole-cell inward currents and single-channel currents. Both the whole-cell currents and single-channel currents reversed at 0 mV when the equilibrium potentials of all ions present were far from 0 mV. Recordings from on-cell patches revealed oscillations in unitary currents at the frequency of pacemaker currents in ICC. Voltage-clamping cells to −60 mV did not change the oscillatory activity of channels in on-cell patches. Depolarizing cells with high external K+ caused loss of resolvable single-channel currents, but the oscillatory single-channel currents were restored when the patches were stepped to negative potentials. Unitary currents were also resolved in excised patches. The single-channel conductance was 13 pS, and currents reversed at 0 mV. The channels responsible were strongly activated by 10−7m Ca2+, and 10−6m Ca2+ reduced activity. The 13 pS channels were strongly activated by the calmodulin inhibitors calmidazolium and W-7 in on-cell and excised patches. Calmidazolium and W-7 also activated a persistent inward current under whole-cell conditions. Murine ICC express Ca2+-inhibited, non-selective cation channels that are periodically activated at the same frequency as pacemaker currents. This conductance may contribute to the pacemaker current and generation of electrical slow waves in GI muscles.

Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2.

We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.

Mechanisms of orexin-induced depolarizations in rat dorsal motor nucleus of vagus neurones in vitro.

1. Whole-cell patch-clamp recordings were made from neurones of the dorsal motor nucleus of the vagus (DMNV), including Fluoro-gold-labelled parasympathetic preganglionic neurones (PPNs), in slices of the rat medulla. In the latter case, rats had received an I.P. injection of Fluoro-gold solution (10 microg) 2-3 days earlier. 2. Superfusion of orexin A or B (10-300 nM) caused a slow depolarization in approximately 30% of the DMNV neurones, including PPNs. Orexin-induced depolarizations, which persisted in TTX (0.5 microM)-containing Krebs solution, were reduced by 70% in a low-Na+ (26 mM) Krebs solution, indicating the involvement of Na+ ions. A significant change in orexin-induced depolarizations was not obtained in either a high-K+ (7 mM) or Cd2+ (100 microM) Krebs solution. 3. Inclusion of the hydrolysis-resistant guanine nucleotide GDP-beta-S in the patch solution significantly reduced the orexin A- or B-induced depolarizations. 4. Under whole-cell voltage-clamp conditions, the orexin-induced inward current declined with hyperpolarization, but did not reverse polarity in the potential range between -120 and 0 mV. In low-Na+ solution, the orexin-induced current was reduced, and the I-V curve reversed polarity at about -105 mV; the response was further reduced and the reversal potential shifted to -90 mV in a low-Na+, high-K+ Krebs solution. 5. It is concluded that the peptides orexin A and B, acting on orexin receptors, which are GTP-binding-protein coupled, are excitatory to DMNV neurones. In addition, more than one conductance, which may include a non-selective cation conductance and a K+ conductance, appears to be involved in the orexin-induced depolarization.

Peptidergic Excitation of Supraoptic Nucleus Neurons: Involvement of Stretch-Inactivated Cation Channels

Although the primary stimulus regulating vasopressin (VP) release is a change in systemic osmolality, other physiological parameters are known to affect VP secretion or modulate the osmotic control over its release. Neuropeptides feature prominently in afferents underlying the central regulation of the VP-releasing magnocellular neurosecretory cells (MNCs). Although little is yet known of the circumstances under which peptides are released onto MNCs, previous studies have shown that a common response profile to exogenous peptide application is a slow excitation that seems to result from the activation of a nonselective cation conductance. In this paper we review the basis for the excitatory effects of angiotensin II, cholecystokinin, and neurotensin in MNCs acutely isolated from the supraoptic nucleus of adult rats. Saturating concentrations of these three peptides evoked nonadditive increases in macroscopic cation conductance. During single-channel recordings Ang II, CCK, and NT caused kinetically identical increases in the probability of opening of 35-pS nonselective cation channels. Patches containing only one channel further revealed that the activity of single channels could be regulated by separate applications of all three peptides. Peptide-stimulated channels were also found to be inactivated by increases in membrane stretch and to be blocked by low concentrations of gadolinium (Gd3+). It is concluded that many excitatory peptides depolarize MNCs by stimulating the stretch-inactivated cation channels underlying osmoreception. Convergent regulation of these channels provides a potentially powerful mechanism for integrating signals derived from the various afferents involved in the regulation of MNCs.

ATP stimulation of P2X(7) receptors activates three different ionic conductances on cultured mouse Schwann cells.

Extracellular ATP, by acting on P2 purinergic receptors, is a potent mediator of cell-to-cell communication both within and between the nervous and the immune systems. We show here by patch-clamp recording, fluorescent dye uptake and immunocytochemistry that, in cultured mouse Schwann cells, ATP activates a P2X(7) receptor associated with three different ionic conductances. In control conditions, ATP activated an inward current (I(ATP)) with a low potency (EC(50), 7.2 mM). Replacing ATP either by the ATP analogue 2',3'-O-(4-benzoyl-4-benzoyl)-ATP (BzATP) or by the tetraacidic form ATP(4-) potentiated the inward current (ATP(4-) EC(50), 375 microM). ATP and BzATP currents were strongly reduced by periodate oxidized ATP (oATP), an antagonist of P2X(7) receptors. IATP was a mixed current composed of a nonselective cationic conductance, a cationic conductance selective for K(+) and an anionic conductance selective for Cl(-). The activation of the K(+) conductance was dependent on an influx of Ca(2+), and was blocked by charybdotoxin (ChTX) and tetraethylammonium (TEA), two potent antagonists of large conductance Ca(2+)-activated K(+) channels (BK channels). The activation of the Cl(-) conductance was insensitive to Ca(2+) but required the presence of K(+). Total removal of K(+) blocked both the Ca(2+)-activated K(+) conductance and the Cl(-) conductance, unveiling the P2X(7) nonselective cationic conductance. The P2X(7) receptor was localized by immunocytochemistry using a polyclonal antibody, anti-P2X(7), whilst its expression and functionality were both detected by the uptake of Lucifer Yellow. This receptor could regulate the synthesis and the release of cytokines by Schwann cells during pathophysiological events.

Network and intrinsic contributions to carbachol-induced oscillations in the rat subiculum.

Low-frequency network oscillations occur in several areas of the limbic system where they contribute to synaptic plasticity and mnemonic functions that are in turn modulated by cholinergic mechanisms. Here we used slices of the rat subiculum (a limbic area involved in cognitive functions) to establish how network and single neuron (intrinsic) membrane mechanisms participate to the rhythmic oscillations elicited by the cholinergic agent carbachol (CCh, 50-100 microM). We have found that CCh-induced network oscillations (intraoscillatory frequency = 5-16 Hz) are abolished by an antagonist of non-N-methyl-D-aspartate (NMDA) glutamatergic receptors (n = 6 slices) but persist during blockade of GABA receptors (n = 16). In addition, during application of glutamate and GABA receptor antagonists, single subicular cells generate burst oscillations at 2.1-6.8 Hz when depolarized with steady current injection. These intrinsic burst oscillations disappear during application of a Ca(2+) channel blocker (n = 6 cells), intracellular Ca(2+) chelation (n = 6), or replacement of extracellular Na(+) (n = 4) but persist in recordings made with electrodes containing a blocker of voltage-gated Na(+) channels (n = 7). These procedures cause similar effects on CCh-induced depolarizing plateau potentials that are contributed by a Ca(2+)-activated nonselective cationic conductance (I(CAN)). Network and intrinsic oscillations along with depolarizing plateau potentials were abolished by the muscarinic receptor antagonist atropine. In conclusion, our findings demonstrate that low-frequency oscillations in the rat subiculum rely on the muscarinic receptor-dependent activation of an intrinsic oscillatory mechanism that is presumably contributed by I(CAN) and are integrated within the network via non-NMDA receptor-mediated transmission.

Fast synaptic transmission mediated by P2X receptors in CA3 pyramidal cells of rat hippocampal slice cultures.

1. A fast ATP-mediated synaptic current was identified in CA3 pyramidal cells in organotypic hippocampal slice cultures. In the presence of inhibitors for ionotropic glutamate and GABA receptors, extracellular stimulation in the pyramidal cell layer evoked fast synaptic currents that reversed near 0 mV, reflecting an increase in a non-selective cationic conductance. This response was mimicked by focal application of ATP. Antagonists of ionotropic P2X receptors reduced both synaptic and ATP-induced currents. 2. Using a pharmacological approach, the source of synaptically released ATP was determined. Synaptic ATP responses were insensitive to presynaptic blockade of GABAergic transmission between interneurons and CA3 pyramidal cells with the mu-opioid receptor agonist D-Ala(2),MePhe(4),Met(O)(5)-ol-enkephalin (FK33-824), but were blocked by adenosine, which inhibits glutamate release from synaptic terminals in the hippocampus. However, selective inhibition of mossy fibre glutamatergic transmission with the metabotropic glutamate receptor group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) did not affect the response. This result points to the associational fibres as the source of the ATP-mediated synaptic response. 3. These results suggest that ATP, coreleased with glutamate, induces a synaptic response in CA3 pyramidal cells that is observed mainly under conditions of synchronous discharge from multiple presynaptic inputs.

The disulfonic stilbene DIDS and the marine poison maitotoxin activate the same two types of endogenous cation conductance in the cell membrane of Xenopus laevis oocytes.

In the present experiments we exposed the intra- or extracellular surface of excised giant membrane patches of Xenopus laevis oocytes bathed in 140 mmol/l Na-aspartate solution to the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS, 250 micromol/l). We observed that DIDS activated at least two cation conductances: (1) a non-selective cation (NSC) conductance that was mediated by channels of approximately 27 pS and resembled the stretch-activated cation conductance that has been observed in the oocyte cell membrane previously, and (2) a Na+-selective conductance, the single-channel events of which could not be resolved and which resembled the depolarization-induced Na+ conductance that has also been observed in the oocyte cell membrane previously. Both conductances were blocked by 1 mmol/l amiloride from the intra- and extracellular surfaces but inhibition of the NSC conductance by extracellular amiloride was less pronounced. Both conductances activated only slowly with a delay of 15-60 s after application of DIDS and remained active even after DIDS was washed off. This suggests that DIDS caused the exocytosis of preformed channels and this interpretation was supported by our additional observation that extracellular application of maitotoxin (MTX) mimicked the effects of DIDS. MTX is a marine toxin that has recently been reported to induce exocytosis in Xenopus laevis oocytes. The fact that DIDS and MTX each carry two sulfonyl groups suggests that they act on the same positively charged binding sites of an exocytosis-inducing protein. Our observations demonstrate that using DIDS to inhibit heterologously expressed anion transporters in the cell membrane of Xenopus laevis oocytes may compromise proper determination of the transporter currents. This effect can be prevented if the DIDS-activated endogenous cation conductances are suppressed by application of amiloride to the cytoplasmic surface of the cell membrane.

Participation of ionotropic and metabotropic glutamate receptors in taste cell responses to MSG

We have previously reported that monosodium glutamate (MSG) stimulation elicited three types responses (transient inward current, sustained inward current and outward current) while L-AP4 (a potent mGluR4 agonist) evoked only outward currents in C57BL/6J mouse taste cells. The outward current responses to MSG and L-AP4 appeared to be mediated by metabotropic glutamate type 4 receptors (mGluR4). In this study, we examined whether agonists of ionotropic glutamate receptor agonists (NMDA, AMPA and ibotenic acid) also elicit responses in mouse taste cells. NMDA (1 mM) elicited transient inward currents, similar to those often observed with MSG, indicating the presence of NMDA receptor/channels that are permeable to Ca2+ ions and are activated by MSG in some taste cells. The sustained inward current response to MSG appeared to result from activation of a nonselective cation conductance, but it is not known if this response is coupled to ionotropic or metabotropic receptors. AMPA (1 mM) elicited small outward currents in all responding cells. Ibotenic acid, which produces a considerably stronger umami taste than MSG in humans, elicited two types of responses in isolated cells; transient inward currents and sustained inward current, suggesting that inward, as well as outward, currents are related to umami transduction. Also, we confirmed that MSG, AP4 (2 mM) and NMDA all can increase [Ca2+]i in taste cells. These results indicate that some cells have both metabotropic and ionotropic receptors, while other taste cells have only one or the other type of receptor.

Interaction between store-operated non-selective cation channels and the Na(+)-Ca(2+) exchanger during secretion in the rat colon.

The properties of capacitative Ca(2+) influx were studied using the whole-cell patch-clamp technique in crypts isolated from rat distal colon. Store-operated cation influx was evoked by increasing the intracellular buffering capacity for Ca(2+) in the pipette solution; contamination by Cl(-) currents was reduced by the use of NMDG gluconate as the main electrolyte in the pipette solution. The permeability of the non-selective cation conductance stimulated by store depletion had the following sequence for monovalent cations: Cs(+) > Na(+) > or = Li(+). The store-operated conductance is permeable to Na(+) and Ca(2+), but in contrast to Na(+), Ca(2+) also exerts a (feedback) inhibition on its own influx. Other divalent cations shared this inhibitory action with the sequence: Ca(2+) > or = Mg(2+) > or = Ba(2+) > or = Sr(2+). Fura-2 experiments revealed that replacement of extracellular Na(+) by NMDG(+) induced an increase in the intracellular Ca(2+) concentration, which was suppressed by the Na(+)-Ca(2+) exchange inhibitor, dichlorobenzamil, indicating the presence of a Na(+)-Ca(2+) exchanger within the colonic crypt cells. In Ussing chamber experiments dichlorobenzamil induced an increase in short-circuit current (I(sc)) in the majority of tissues tested indicating that this exchanger acts as a Ca(2+)-extruding transporter under physiological conditions. When Ca(2+)-dependent anion secretion was stimulated by the acetylcholine analogue carbachol, dichlorobenzamil no longer evoked an increase in I(sc), indicating that after stimulation of the store-operated cation conductance the Na(+)-Ca(2+) exchanger is turned off. Therefore, it is concluded that the influx of Na(+) across the non-selective store-operated cation conductance serves to reduce the driving force for Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and thereby maintains the increase in the intracellular Ca(2+) concentration during induction of secretion. Experimental Physiology (2001) 86.4, 461-468.

Mechanically induced potentials in rat atrial fibroblasts depend on actin and tubulin polymerisation.

When atrial tissue contracts, mechanically induced potentials (MIPs) are generated in fibroblasts, presumably by activation of a non-selective cation conductance Gns. Non-stimulated atrial fibroblasts had a mean (+/-SD) membrane potential (Em) of -22 +/- 2 mV and an input resistance of 510 +/- 10 MS. MIP amplitude (AMIP) was 38+/-4 mV when current injection had polarised Em to Vm = -50 mV. The slope of the function relating AMIP to Vm can be regarded as a mechanosensitive factor (Xms) that describes the relative increase in Gns during a MIP. Putative involvement of cytoskeletal fibres in activation of Gns was studied by delivering drugs from the intracellular recording microelectrode. Destabilisation of F-actin by 0.2 mM cytochalasin D reduced AMIP from 38 to 16 mV and Xms from 5 to 1.8. Destabilisation of tubulin with 0.2 mM colchicine reduced AMIP to 21 mV and Xms to 2.1. The combination colchicine plus cytochalasin D reduced AMIP to 9 mV and Xms to 1.4. Promoting F-actin stability with exogenous adenosine 5'-triphosphate (ATP) increased AMIP and Xms and attenuated the effects of cytochalasin D. Similarly, facilitation of tubulin stability with guanosine 5'-triphosphate (GTP) or taxol increased AMIP and Xms and attenuated the effects of colchicine. The results suggest that transfer of mechanical energy from the deformed fibroblast surface to the Gns channel protein depends on intact F-actin and tubulin fibres.

Neurotensin excitation of serotonergic neurons in the rat nucleus raphe magnus: ionic and molecular mechanisms

To understand the cellular and molecular mechanisms by which neurotensin (NT) induces an analgesic effect in the nucleus raphe magnus (NRM), whole-cell patch-clamp recordings were performed to investigate the electrophysiological effects of NT on acutely dissociated NRM neurons. Two subtypes of neurons, primary serotonergic and secondary non-serotonergic cells, were identified from acutely isolated NRM neurons. During current-clamp recordings, NT depolarized NRM serotonergic neurons and evoked action potentials. Voltage-clamp recordings showed that NT excited serotonergic neurons by enhancing a voltage-insensitive and non-selective cationic conductance. Both SR48692, a selective antagonist of subtype 1 neurotensin receptor (NTR-1), and SR 142948A, a non-selective antagonist of NTR-1 and subtype 2 neurotensin receptor (NTR-2), failed to prevent neurotensin from exciting NRM serotonergic neurons. NT-evoked cationic current was inhibited by the intracellular administration of GDP-β-S. NT failed to induce cationic currents after dialyzing serotonergic neurons with the anti-G αq/11 antibody. Cellular Ca 2+ imaging study using fura-2 showed that NT induced the calcium release from the intracellular store. NT-evoked current was blocked after the internal perfusion of heparin, an IP 3 receptor antagonist, or BAPTA, a fast Ca 2+ chelator. It is concluded that neurotensin enhancement of the cationic conductance of NRM serotonergic neurons is mediated by a novel subtype of neurotensin receptors. The coupling mechanism via G αq/11 proteins is likely to involve the generation of IP 3, and subsequent IP 3-evoked Ca 2+ release from intracellular stores results in activating the non-selective cationic conductance.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle.

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing < 10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.