ATP and nitric oxide modulate a Ca(2+)-activated non-selective cation current in macrovascular endothelial cells

We have studied the properties of a non-selective cation current (NSC(Ca)) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca(2+) concentration, [Ca(2+)](i). Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca(2+)](i) relationships were fitted to a Ca(2+) binding site model with a concentration for half-maximal activation of 417 +/- 76 nM, a Hill coefficient of 2.3 +/- 0.8 and a maximum current of -23.9 +/- 2.7 pA/pF at -50 mV. The Ca(2+)-activated channel is more permeable to Na(+) than for Cs(+) ( P(Cs)/ P(Na)=0.58, n=7), but virtually impermeable to Ca(2+). Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca(2+)](i) were smaller than in the absence of ATP, but were not significantly different at 2 microM. The intracellular Ca(2+) concentration for half-maximal activation of the Ca(2+)-activated current was shifted to 811 +/- 12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSC(Ca) in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso- N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSC(Ca). In contrast, nitro- L-arginine (NLA), which inhibits all NO-synthases, potentiated NSC(Ca), whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSC(Ca). It is concluded that the Ca(2+)-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO.     

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.     

Intracellular Mg(2+) hyperpolarizes rabbit coronary artery smooth muscle cells by differential modulation of Ca(2+)(i)-dependent ion channels

We investigated the role of intracellular Mg(2+) ([Mg(2+)](i)) in the regulation of membrane potential ( V(m)) in rabbit coronary artery smooth muscle cells. V(m), membrane currents and intracellular Ca(2+) ([Ca(2+)](i)) were measured using standard patch-clamp and microfluorometry techniques. When [Ca(2+)](i) was increased by caffeine, V(m) depolarized at low [Mg(2+)](i) (0.1 mM), but hyperpolarized at high [Mg(2+)](i) (> or =1.2 mM). Effects of [Mg(2+)](i) on caffeine-induced currents were investigated. [Mg(2+)](i) selectively facilitated the activation of Ca(2+)-activated K(+) currents ( I(K,Ca)), while Ca(2+)-activated Cl(-) currents ( I(Cl,Ca)) were unaffected. Simultaneous recording of [Ca(2+)](i) and I(K,Ca) at different [Mg(2+)](i) showed that [Mg(2+)](i) increased the Ca(2+) sensitivity of I(K,Ca). [Ca(2+)](i) also inhibited voltage-dependent K(+) (K(V)) currents, although this effect was significant only at low [Mg(2+)](i). These results imply that the relative contributions of I(K,Ca), I(Cl,Ca) and K(V) currents to V(m) during an increase in [Ca(2+)](i) are affected by [Mg(2+)](i): at low [Mg(2+)](i), activation of I(Cl,Ca) and inhibition of K(V) currents depolarized V(m); at high [Mg(2+)](i) the activation of I(K,Ca) predominated, resulting in hyperpolarization of V(m). In conclusion, [Mg(2+)](i) hyperpolarizes V(m) by selective facilitation of I(K,Ca) and may thus possibly contributes to the relaxation of the coronary artery.     

Arachidonic acid-induced activation of large-conductance potassium channels and membrane hyperpolarization in mouse B cells

Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we found that WEHI-231, an immature B cell line, expresses voltage-independent K(+) channels called large-conductance background K( + ) channels (LK(bg)). Arachidonic acid (AA) has attracted attention because of its potential regulatory roles in the apoptosis of immature B cells. To elucidate the functional targets of AA, we investigated the effects of AA on membrane currents, voltages, and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) of WEHI-231 and Bal-17 cells that represent immature and mature mouse B cells, respectively. In whole-cell patch clamp, both Kv and IKCa1 were inhibited by AA. On the other hand, AA activated LK(bg) current and non-selective cationic (NSC) current in WEHI-231 while only NSC current in Bal-17. Inside-out patch clamp study showed that AA directly activates LK(bg). AA induced hyperpolarization of WEHI-231 and depolarization of Bal-17 cells, respectively. The selective functional expression of LK(bg) and their activation by AA were also confirmed in the immature B cells (B220(+)/AA4.1(+)) freshly isolated from mouse spleen. In fura-2 spectrofluorimetry, AA induced persistent increase in [Ca(2+)](c) of WEHI-231 cells, which was attenuated by KCl-induced depolarization. In Bal-17 cell, however, AA induced only a transient increase of [Ca(2+)](c). In summary, the novel type of background K(+) channels (LK(bg)) in immature B cells is strongly activated while the other K(+) channels (Kv and IKCa1) commonly expressed in lymphocytes are inhibited by AA. The hyperpolarization and augmentation of Ca(2+) influx by LK(bg) activation might play a role in the response of immature B cells to AA.     

Contribution of different Ca-activated K channels to the first phase of the response to TRH in clonal rat anterior pituitary cells

AIMS: Thyrotropin-releasing hormone (TRH) induces biphasic changes in electrical activity, cytosolic free Ca(2+) level ([Ca(2+)](i)), and prolactin secretion from both clonal GH cells and native lactotrophs. The first phase of the TRH response is characterized by hyperpolarization because of activation of Ca(2+)-activated K(+) channels (K(Ca)). In the present study, the relative contribution of BK, SK, and IK channels to the first phase of the TRH response in GH(4) cells was assessed. METHODS: The expression of IK channels was confirmed by PCR with specific primers for SK4 (IK). The response to TRH was studied using the perforated patch technique and Ca(2+) microfluoromety (fura-2). The involvement of different K(Ca) channels was estimated by employing the specific channel blockers iberiotoxin (BK), apamin (SK) and clotrimazole (IK). RESULTS: Application of 100 nM iberiotoxin, 1 microM apamin, and 10 microM clotrimazole reduced the peak value of the outward K(+) current during the first phase of the TRH response by 33, 26, and 33%, respectively. Clotrimazole also shortened the duration of the outward current response by 60%, causing a reduction of total charge movement by 73%. All these toxin-induced reductions were significant (P < 0.05). A combination of all three toxins abolished the current response almost completely. CONCLUSION: All the three main types of K(Ca) channels are involved in the first phase of the TRH response, with IK as the major contributor. This is the first demonstration of a dominant role of IK compared with BK and SK channels in excitable cells.     

Chemical anoxia activates ATP-sensitive and blocks Ca(2+)-dependent K(+) channels in rat dorsal vagal neurons in situ

The contribution of subclasses of K(+) channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca(2+) measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the K(ATP) channel blocker tolbutamide, but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K(+) channel blockers also revealed that ongoing activation of K(ATP) and SK channels counteracts a tonic, spike-related rise in intracellular Ca(2+) ([Ca(2+)](i)) under normoxic conditions, but did not modify the rise of [Ca(2+)](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca(2+)](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca(2+)](i) rise were abolished by Ca(2+)-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca(2+)](i) increase. Intracellular BAPTA for Ca(2+) chelation blocked the afterhyperpolarizing current and the accompanying [Ca(2+)](i) increase, but had no effect on the cyanide-induced outward current although the associated [Ca(2+)](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca(2+)](i) transient with the Ca(2+) pump blocker cyclopiazonic acid did not evoke an outward current.Our results show that anoxia mediates a persistent hyperpolarization due to activation of K(ATP) channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca(2+)](i) does not interfere with the activity of these K(+) channels.     

Effects of extracellular ATP on freshly isolated mouse skeletal muscle cells during pre-natal and post-natal development

Extracellular adenosine 5'-triphosphate (ATP) has profound effects on membrane conductance and on the intracellular free [Ca(2+)] ([Ca(2+)](i)) in cultured skeletal muscle cells. The aim of the present study was to examine the occurrence and to characterize the properties of such responses during mammalian muscle development in vivo. The effect of ATP (0.2 mM) was tested on membrane current and [Ca(2+)](i) in freshly isolated pre- and post-natal mouse skeletal muscle cells. Pre-natal cells were from 14- to 19-day-old fetuses. In pre- and early post-natal cells, very small elevations of [Ca(2+)](i) (<50 nM) following ATP application could be detected with the fluorescent indicator fura-2. A clear subsarcolemmal rise in [Ca(2+)] was however associated to the presence of ATP, as demonstrated by increased activity of plasma membrane Ca(2+)-activated K(+) channels in cells bathed in a depolarizing, high-calcium-containing solution. In cells voltage-clamped at -80 mV in external Tyrode, ATP induced an inward current associated with an increased membrane conductance. The mean maximal amplitude of the ATP-induced current was -0.84 +/- 0.07 A/F ( n=39). The response to ATP was still present after birth, although its amplitude tended to decrease with post-natal development and was completely absent in muscle cells from 3- to 6-month-old mice. The ATP-induced current could be abolished reversibly by suramin. Our results suggest that, over the range of developmental stages examined, skeletal muscle cells display an ionotropic purinergic signalling pathway with functional properties qualitatively consistent with what is observed in cultured myotubes.     

Interleukin-10 modulates [Ca2+]i response induced by repeated NMDA receptor activation with brief hypoxia through inhibition of InsP(3)-sensitive internal stores in hippocampal neurons

The goal of this study was to evaluate an effect of interleukin-10 (IL-10) on the Ca(2+) response induced by repeated NMDA receptor activation with brief hypoxia in cultured hippocampal neurons. We focused on the importance of internal Ca(2+) stores in the modulation of this Ca(2+) response by IL-10. To test this, we compared roles of InsP(3)- and ryanodine-sensitive internal stores in the effects of IL-10. Measurements of intracellular cytosolic calcium concentration ([Ca(2+)](i)) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. Repeated episodes of NMDA receptor activation with brief hypoxia induced the spontaneous (s) [Ca(2+)](i) increases about 3 min after each hypoxic episode. The amplitude of the s[Ca(2+)](i) increases was progressively enhanced from the first hypoxic episode to the third one. IL-10 (1 ng/ml) abolished these s[Ca(2+)](i) increases. Exposure of cultured hippocampal neurons with thapsigargin (1 μM) or an inhibitor of phospholipase C (U73122, 1 μM) for 10 min also abolished the s[Ca(2+)](i) increases. On the other hand, antagonist of ryanodine receptors (ryanodine, 1 μM) did not affect this Ca(2+) response. These studies appear to provide the first evidence that Ca(2+) release from internal stores is affected by anti-inflammatory cytokine IL-10 in brain neurons. It is suggested that these data increase our understanding of the neuroprotective mechanisms of IL-10 in the early phase of hypoxia.     

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.     

Properties of large conductance calcium-activated potassium channels in pyramidal neurons from the hippocampal CA1 region of adult rats

The properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels were studied in rat hippocampal CA1 pyramidal neurons by using the patch-clamp technique in the excised-inside-out-patch configuration. The lowest [Ca(2+)](i) in which BK(Ca) channel activities were observed was 0.01 microM with the membrane potential of +20 mV and the [Ca(2+)](i) at which P(O) of the channel is equal to 0.5 was 2 microM. The unitary conductance of the single BK(Ca) channel was 245.4 pS with symmetrical 140 mM K(+) on both sides of the excised membrane. With a fixed [Ca(2+)](i) of 2 microM, P(O) increased e-fold with a 17.0 mV positive change in the membrane potential. Two exponentials, with time constants of 2.8 ms and 19.2 ms at the membrane potential of +120 mV with 2 microM [Ca(2+)](i), were required to describe the observed open time distribution of BK(Ca) channel, suggesting the existence of two distinct open channel states with apparently normal conductance. A BK(Ca) channel occasionally entered an apparent third open channel state with the single channel current amplitude about 45% of the normal amplitude. The properties of BK(Ca) channel, which were found in this study to be more steeply dependent on voltage and more sensitive to [Ca(2+)](i) in adult hippocampal neurons than in cultured or immature hippocampal neurons, may be responsible for the shortened duration of action potential in hippocampal CA1 pyramidal neurons of adult rat.     

Properties of a calcium-activated K(+) current on interneurons in the developing rat hippocampus

Calcium-activated potassium currents have an essential role in regulating excitability in a variety of neurons. Although it is well established that mature CA1 pyramidal neurons possess a Ca(2+)-activated K(+) conductance (I(K(Ca))) with early and late components, modulation by various endogenous neurotransmitters, and sensitivity to K(+) channel toxins, the properties of I(K(Ca)) on hippocampal interneurons (or immature CA1 pyramidal neurons) are relatively unknown. To address this problem, whole-cell voltage-clamp recordings were made from visually identified interneurons in stratum lacunosum-moleculare (L-M) and CA1 pyramidal cells in hippocampal slices from immature rats (P3-P25). A biphasic calcium-activated K(+) tail current was elicited following a brief depolarization from the holding potential (-50 mV). Analysis of the kinetic properties of I(K(Ca)) suggests that an early current component differs between these two cell types. An early I(K(Ca)) with a large peak current amplitude (200.8 +/- 13.2 pA, mean +/- SE), slow time constant of decay (70.9 +/- 3.3 ms), and relatively rapid time to peak (within 15 ms) was observed on L-M interneurons (n = 88), whereas an early I(K(Ca)) with a small peak current amplitude (112.5 +/- 7.3 pA), a fast time constant of decay (39.4 +/- 1.6 ms), and a slower time-to-peak (within 26 ms) was observed on CA1 pyramidal neurons (n = 85). Removal of extracellular calcium or addition of inorganic Ca(2+) channel blockers (cadmium, nickel, or cobalt) was used to demonstrate the calcium dependence of these currents. Addition of norepinephrine, carbachol, and a variety of channel toxins (apamin, iberiotoxin, verruculogen, paxilline, penitrem A, and charybdotoxin) were used to further distinguish between I(K(Ca)) on these two hippocampal cell types. Verruculogen (100 nM), carbachol (100 microM), apamin (100 nM), TEA (1 mM), and iberiotoxin (50 nM) significantly reduced early I(K(Ca)) on CA1 pyramidal neurons; early I(K(Ca)) on L-M interneurons was inhibited by apamin and TEA. Combined with previous work showing that the firing properties of hippocampal interneurons and pyramidal cells differ, our kinetic and pharmacological data provide strong support for the hypothesis that different types of Ca(2+)-activated K(+) current are present on these two cell types.     

Ca2+-activated K+-current density is correlated with soma size in rat vestibular-afferent neurons in culture

Vestibular-afferent neurons (VANs) transmit information about linear and angular accelerations during head movements from vestibular end organs to vestibular nuclei. In situ, these neurons show heterogeneous discharge patterns that may be produced by differences in their intrinsic properties. However, little is known about the ionic currents underlying their different firing patterns. Using the whole cell patch-clamp technique, we analyzed the expression of Ca(2+) and Ca(2+)-activated K(+) currents (I(KCa)) in primary cultured neurons isolated from young rats (p7-p10). We found two overlapping subpopulations of VANs classified according to low-threshold Ca(2+)-current [low-voltage-activated (LVA)] expression; LVA (-) neurons, formed by small cells, and LVA (+) neurons composed of medium to large cells. The I(KCa) in both cell-groups was carried through channels of high (BK), intermediate (IK), and low conductance (SK), besides a resistant channel to classical blockers (IR). BK was expressed preferentially in LVA (+) cells, whereas IR expression was preferentially in LVA (-) cells. No correlation between SK and IK expression with the soma size was found. Current-clamp experiments showed that BK participates in the adaptation of discharge and in the duration of the action potential, whereas SK and IK did not show a significant contribution to electrical discharge of cultured VANs. However, because of the low number of VANs in culture with repetitive firing it is difficult to interpret our results in terms of discharge patterns. Our results demonstrate that vestibular-afferent neurons possess different Ca(2+)-activated K(+) (K(Ca)) channels and that their expression, heterogeneous among the cells, would contribute to explain some of the differences in the electrical-firing properties of these neurons.     

Mobilization of intracellular Ca(2+) by thromboxane A(2) does not affect Ca(2+)-activated K(+) channels in rat colonic crypt cells

The effects of 9,11-epithio-11,12-methano-thromboxane A(2) (STA(2)), a stable thromboxane A(2) analogue, and carbachol on colonic Ca(2+)-activated K(+) channels were studied. In indo-1-loaded single cells in isolated rat colonic crypts, both STA(2) (0.1 microM) and carbachol (10 microM) transiently increased intracellular free Ca(2+) concentration ([Ca(2+)](i)) by 136 and 155 nm, respectively. In whole-cell current-clamp experiments of the colonic crypt cells with Cl(-)-free solutions, carbachol (10 microM) hyperpolarized the cell by 19.7 mV, while STA(2) (0.1 microM) did not affect the membrane potential. In the isolated colonic mucosa that was permeabilized mucosally by a monovalent ionophore nystatin in the presence of a serosally directed K(+) gradient, carbachol (10 microM) transiently elicited K(+) current, but STA(2) (0.1 microM) did not. These results indicate that STA(2) elevates [Ca(2+)](i) in rat colonic crypt cells but does not activate basolateral Ca(2+)-activated K(+) channels.     

Inhibition effect of arachidonic acid on hypoxia-induced [Ca(2+)](i) elevation in PC12 cells and human pulmonary artery smooth muscle cells

[Ca(2+)](i) elevation is a key event when O(2) sensitive cells, e.g. PC12 cells and pulmonary artery smooth muscle cells, face hypoxia. Ca(2+) entry pathways in mediating hypoxia-induced [Ca(2+)](i) elevation include: voltage-gated Ca(2+) channels (VGCCs), transient receptor potential (TRP) channel and Na(+)-Ca(2+) ex-changer (NCX). In the pulmonary artery, accumulated evidence strongly suggests that prostaglandins (PGs) are involved in pulmonary inflammation and cause vasoconstriction during hypoxia. In this study, we investigated the effect of arachidonic acid (AA), the upstream substrate for PGs, on hypoxia response in O(2) sensitive cells. Exogenous application of AA significantly inhibited hypoxia-induced [Ca(2+)](i) elevation. This effect was due to AA itself rather than its degenerative products. The pharmacological modulation of endogenous AA showed that the prevention of AA generation by blockage of cPLA2, diacylglycerol (DAG) lipase and fatty acid hydrolysis (FAAH), augments hypoxia-induced [Ca(2+)](i) elevation, whereas prevention of AA degeneration attenuates hypoxia-induced [Ca(2+)](i) elevation. Over-expression of COX2 enhances hypoxia-induced [Ca(2+)](i) elevation and this enhancement is reversed by exogenous AA. Our results suggest that AA inhibits hypoxia response. The dynamic alterations in cellular lipids might determine cell response to hypoxia.     

Localized IP3-evoked Ca2+ release activates a K+ current in primary vagal sensory neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP(3), and the consequent release of Ca(2+), could trigger a Ca(2+)-activated K(+) current (I(IP3)). Photorelease of caged IP(3) evoked an I(IP3) that averaged 2.36 +/- 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at -50 mV. I(IP3) was abolished by intracellular BAPTA (2 mM), a Ca(2+) chelator. Changing the K(+) equilibrium potential by increasing extracellular K(+) ion concentration caused a predicted Nernstian shift in the reversal potential of I(IP3). These results indicated that I(IP3) was a Ca(2+)-dependent K(+) current. I(IP3) was unaffected by three common antagonists of Ca(2+)-activated K(+) currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 microM) included in the patch pipette. We have previously demonstrated that both IP(3)-evoked Ca(2+) release and Ca(2+)-induced Ca(2+) release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca(2+)-activated K(+) current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca(2+) stores, we showed that IP(3)-evoked Ca(2+) release occurs independently of CICR, but interestingly, that a component of I(IP3) requires CICR. These data suggest that IP(3)-evoked Ca(2+) release activates a K(+) current that is pharmacologically distinct from other Ca(2+)-activated K(+) currents in NGNs. We describe several models that explain our results based on Ca(2+) signaling microdomains in NGNs.     

Effects of increasing Ca2+ channel-vesicle separation on facilitation at the crayfish inhibitory neuromuscular junction

We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca(2+) channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg(2+)](o), but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM omega-Aga IVA, which reduced total Ca(2+) influx and release to levels comparable to that recorded in 40 mM [Mg(2+)](o), did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca(2+) channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with omega-Aga IVA were probed with broad action potentials in the presence of K(+) channel blockers. After Ca(2+) channel-vesicle separation was increased by omega-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg(2+)](o). EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in omega-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca(2+) influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca(2+) channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after omega-Aga IVA poisoning. Finally, we sought correlation between residual Ca(2+) and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca(2+) indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca(2+)) to that evoked during action potential (local Ca(2+)). This ratio provides an estimate of relative changes between residual Ca(2+) and local Ca(2+) important for release. There is a significant increase in the ratio when Ca(2+) influx is reduced by 40 mM [Mg(2+)](o). The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca(2+) channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca(2+) and lends support to the residual Ca(2+) hypothesis of facilitation.     

Acute effects of glucocorticoids on ATP-induced Ca2+ mobilization and nitric oxide production in cochlear spiral ganglion neurons

Rapid, non-genomic effects of glucocorticoids on extracellular adenosine 5'-triphosphate (ATP)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) changes and nitric oxide (NO) production were investigated in type I spiral ganglion neurons (SGNs) of the guinea-pig cochlea using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye 4,5-diaminofluorescein (DAF-2). Pretreatment of SGNs with 1 microM dexamethasone for 10 min, a synthetic glucocorticoid hormone, enhanced the ATP-induced [Ca(2+)](i) increase in SGNs. RU 38486, a competitive glucocorticoid receptor antagonist eliminated the effects of dexamethasone on the ATP-induced [Ca(2+)](i) increase in SGNs. These acute effects of dexamethasone were dependent on the presence of extracellular Ca(2+), thereby suggesting that dexamethasone may rapidly enhance the Ca(2+) influx through the activation of ionotropic P2X receptors which may interact with glucocorticoid-mediated membrane receptors. Extracellular ATP increased the intensity of DAF-2 fluorescence, indicating NO production in SGNs. The ATP-induced NO production was mainly due to the Ca(2+) influx through the activation of P2 receptors. S-nitroso-N-acetylpenicillamine, a NO donor, enhanced the ATP-induced [Ca(2+)](i) increase in SGNs while L-N(G)-nitroarginine methyl ester (L-NAME), a NO synthesis inhibitor, inhibited it. Dexamethasone enhanced the ATP-induced NO production in SGNs. The augmentation of dexamethasone on ATP-induced NO production was abolished in the presence of l-NAME. It is concluded that the ATP-induced [Ca(2+)](i) increase induces NO production which enhances a [Ca(2+)](i) increase in SGNs by a positive-feedback mechanism. Dexamethasone enhances the ATP-induced [Ca(2+)](i) increase in SGNs which results in the augmentation of NO production. The present study suggests that NO may play an important role in auditory signal transduction. Our results also indicate that glucocorticoids may rapidly affect auditory neurotransmission due to a novel non-genomic mechanism.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.