Phasic contractions of the rat portal vein depend on intracellular Ca2+ release stimulated by depolarization

The phasic contraction to phenylephrine of the rat isolated portal vein was investigated using functional studies. Phasic contractions to phenylephrine and caffeine could be produced after several minutes in Ca(2+)-free Krebs solution, which were inhibited by cyclopiazonic acid or ryanodine. The phenylephrine and caffeine contractions were abolished, however, within 10 min in Ca(2+)-free Krebs solution and by nifedipine. This indicated the Ca(2+) stores were depleted in the absence of Ca(2+) influx through voltage-gated channels. The phasic contraction to phenylephrine was also abolished by niflumic acid even in Ca(2+)-free Krebs solution. This showed that the response depended on intracellular Ca(2+) release stimulated directly by depolarization, resulting from opening of Ca(2+)-activated Cl(-) channels, but did not require Ca(2+) influx. In support of this, K(+)-induced phasic contractions were also produced in Ca(2+)-free Krebs solution. The phenylephrine but not K(+)-induced phasic contractions in Ca(2+)-free Krebs solution were inhibited by ryanodine or cyclopiazonic acid. This would be consistent with Ca(2+) release from more superficial intracellular stores (affected most by these agents), probably by inositol 1,4,5-trisphospate, being required to stimulate the phenylephrine depolarization.     

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.     

Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom.

A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage-dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Hyperpolarization, but not depolarization, increases intracellular Ca(2+) level in cultured chick myoblasts.

Ca(2+) influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca(2+) influx rises prior to myoblast fusion. The present study examines a possible involvement of the voltage-dependent Ca(2+) influx pathways. Treatment with the L-type Ca(2+) channel blockers, diltiazem, and nifedipine did not alter cytosolic Ca(2+) levels. Depolarization with high K(+) solution and activation of Ca(2+) channel with Bay K 8644, and agonist of voltage dependent Ca(2+) channels, failed to elicit increases intracellular Ca(2+) level, indicating the absence of depolarization-operated mechanisms. In contrast, phloretin, an agonist of Ca(2+)-activated potassium (K(Ca)) channels, was able to hyperpolarize membrane potential and promoted Ca(2+) influx. These effects were completely abolished by treatment of charybdotoxin, a specific inhibitor of K(Ca) channels. In addition, gadolinium, a potent stretch-activated channel (SAC) blocker, prevented the phloretin-mediated Ca(2+) increase, indicating the involvement of SACs in Ca(2+) influx. Furthermore, phloretin stimulated precocious myoblast fusion and this effect was blocked with gadolinium or charybdotoxin. Taken together, these results suggest that induced hyperpolarization, but not depolarization increases Ca(2+) influx through stretch-activated channels, and in turn triggers myoblast fusion.     

CO2 transduction in avian intrapulmonary chemoreceptors is critically dependent on transmembrane Na+/H+ exchange

Avian intrapulmonary chemoreceptors (IPC) are vagal respiratory afferents that are inhibited by high lung Pco(2) and excited by low lung Pco(2). Previous work suggests that increased CO(2) inhibits IPC by acidifying intracellular pH (pH(i)) and that pH(i) is determined by a kinetic balance between the rate of intracellular carbonic anhydrase-catalyzed CO(2) hydration/dehydration and transmembrane extrusion of acids and/or bases by various exchangers. Here, the role of amiloride-sensitive Na(+)/H(+) exchange (NHE) in the IPC CO(2) response was tested by recording single-unit action potentials from IPC in anesthetized ducks, Anas platyrhynchos. For each of the IPC tested, blockade of the NHE using dimethyl amiloride (DMA) elicited a marked (>50%) dose-dependent decrease in mean IPC discharge (P < 0.05), suggesting that NHE is important for pH(i) regulation and CO(2) transduction in IPC. In addition, activation of the NHE using 12-O-tetradecanoylphorbol 13-acetate stimulated six of the seven IPC tested, although the overall effect was not statistically significantly (P = 0.07). Taken together, these findings suggest that CO(2) transduction in IPC is dependent on transmembrane NHE although it is likely to be much slower than carbonic anhydrase-catalyzed hydration-dehydration of CO(2).     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.     

SNAP-25 inhibits L-type Ca2+ channels in feline esophagus smooth muscle cells

We recently reported that non-secretory gastrointestinal smooth muscle cells also possessed SNARE proteins, of which SNAP-25 regulated Ca(2+)-activated (K(Ca)) and delayed rectifier K(+) channels (K(V)). Voltage-gated, long lasting (L-type) calcium channels (L(Ca)) play an important role in excitation-contraction coupling of smooth muscle. Here, we show that SNAP-25 could also directly inhibit the L-type Ca(2+) channels in feline esophageal smooth muscle cells at the SNARE complex binding synprint site. SNARE proteins could therefore regulate additional cell actions other than membrane fusion and secretion, in particular, coordinated muscle membrane excitability and contraction, through their actions on membrane Ca(2+) and K(+) channels.     

Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.     

Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca(2+)-activated K(+) channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca(2+)-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys(32), which lies in close proximity to this cluster. These analogs block IKCa1 with approximately 20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1. 6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K(Ca) channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K(+) channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K(+) channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.     

Essential role of EDHF in the initiation and maintenance of adrenergic vasomotion in rat mesenteric arteries

The possible roles of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)), nitric oxide (NO), arachidonic acid (AA) metabolites, and Ca(2+)-activated K(+) (K(Ca)) channels in adrenergically induced vasomotion were examined in pressurized rat mesenteric arteries. Removal of the endothelium or buffering [Ca(2+)](i) selectively in endothelial cells with BAPTA eliminated vasomotion in response to phenylephrine (PE; 10.0 microM). In arteries with intact endothelium, inhibition of NO synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME; 300.0 microM) or N(omega)-nitro-l-arginine (l-NNA; 300.0 microM) did not eliminate vasomotion. Neither inhibition of cGMP formation with 10.0 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor inhibition of prostanoid formation (10.0 microM indomethacin) eliminated vasomotion. Similarly, inhibition of AA cytochrome P-450 metabolism with an intraluminal application of 17-octadecynoic acid (17-ODYA) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) failed to eliminate vasomotion. In contrast, intraluminal application of the K(Ca) channel blockers apamin (250.0 nM) and charybdotoxin (100.0 nM), together, abolished vasomotion and changed synchronous Ca(2+) oscillations in smooth muscle cells to asynchronous propagating Ca(2+) waves. Apamin, charybdotoxin, or iberiotoxin (100.0 nM) alone did not eliminate vasomotion, nor did the combination of apamin and iberiotoxin. The results show that adrenergic vasomotion in rat mesenteric arteries is critically dependent on Ca(2+)-activated K(+) channels in endothelial cells. Because these channels (small- and intermediate-conductance K(Ca) channels) are a recognized component of EDHF, we conclude therefore that EDHF is essential for the development of adrenergically induced vasomotion.     

Maternal diabetes increases large conductance Ca2+-activated K+ outward currents that alter action potential properties but do not contribute to attenuated excitability of parasympathetic cardiac motoneurons in the nucleus ambiguus of neonatal mice

Previously, we demonstrated that maternal diabetes reduced the excitability and increased small-conductance Ca(2+)-activated K(+) (SK) currents of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA). In addition, blockade of SK channels with apamin completely abolished this reduction. In the present study, we examined whether maternal diabetes affects large-conductance Ca(2+)-activated K(+) (BK) channels and whether BK channels contribute to the attenuation of PCMN excitability observed in neonates of diabetic mothers. Neonatal mice from OVE26 diabetic mothers (NMDM) and normal FVB mothers (control) were used. The pericardial sac of neonatal mice at postnatal days 7-9 was injected with the tracer X-rhodamine-5 (and 6)-isothiocyanate 2 days prior to the experiment to retrogradely label PCMNs in the NA. Whole cell current- and voltage-clamps were used to measure spike frequency, action potential (AP) repolarization (half-width), afterhyperpolarization potential (AHP), transient outward currents, and afterhyperpolarization currents (I(AHP)). In whole cell voltage clamp mode, we confirmed that maternal diabetes increased transient outward currents and I(AHP) compared with normal cells. Using BK channel blockers charybdotoxin (CTx) and paxilline, we found that maternal diabetes increased CTx- and paxilline-sensitive transient outward currents but did not change CTx- and paxilline-sensitive I(AHP). In whole cell current-clamp mode, we confirmed that maternal diabetes increased AP half-width and AHP, and reduced excitability of PCMNs. Furthermore, we found that after blockade of BK channels with CTx or paxilline, maternal diabetes induced a greater increase of AP half-width but similarly decreased fast AHP without affecting medium AHP. Finally, blockade of BK channels decreased spike frequency in response to current injection in both control and NMDM without reducing the difference of spike frequency between the two groups. Therefore, we conclude that although BK transient outward currents, which may alter AP repolarization, are increased in NMDM, BK channels do not directly contribute to maternal diabetes-induced attenuation of PCMN excitability. In contrast, based on evidence from our previous and present studies, reduction of PCMN excitability in neonates of diabetic mothers is largely dependent on altered SK current associated with maternal diabetes.     

Role of potassium channels in catecholamine secretion in the rat adrenal gland

We elucidated the functional contribution of K(+) channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca(2+)-activated K(+) (SK(Ca))-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 microM) and methacholine (10-300 microM). The putative large-conductance Ca(2+)-activated K(+) channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the K(A) channel blocker mast cell degranulating peptide (100-1000 nM) nor the K(V) channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SK(Ca) channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca(2+)-activated K(+) channels may control the secretion at the presynaptic site.     

Profound alterations in the intrinsic excitability of cerebellar Purkinje neurons following neurotoxin 3-acetylpyridine (3-AP)-induced ataxia in rat: new insights into the role of small conductance K+ channels

Alterations in the intrinsic properties of Purkinje cells (PCs) may contribute to the abnormal motor performance observed in ataxic rats. To investigate whether such changes in the intrinsic neuronal excitability could be attributed to the role of Ca(2+)-activated K(+) channels (K(Ca)), whole cell current clamp recordings were made from PCs in cerebellar slices of control and ataxic rats. 3-AP induced profound alterations in the intrinsic properties of PCs, as evidenced by a significant increase in both the membrane input resistance and the initial discharge frequency, along with the disruption of the firing regularity. In control PCs, the blockade of small conductance K(Ca) channels by UCL1684 resulted in a significant increase in the membrane input resistance, action potential (AP) half-width, time to peak of the AP and initial discharge frequency. SK channel blockade also significantly decreased the neuronal discharge regularity, the peak amplitude of the AP, the amplitude of the afterhyperpolarization and the spike frequency adaptation ratio. In contrast, in ataxic rats, both the firing regularity and the initial firing frequency were significantly increased by the blockade of SK channels. In conclusion, ataxia may arise from alterations in the functional contribution of SK channels, to the intrinsic properties of PCs.     

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.     

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.     

Induction of calcium-activated potassium channel activity by hemin in human erythroleukemia cells

The agent hemin has been demonstrated to be able to initiate a coordinated differentiation program in several cell types. In the present study, we examined the ability of hemin on inducing cell differentiation and Ca(2+)-activated K(+) channel activity in erythroleukemic K562 cells. Treating undifferentiated K562 cells with hemin (0.1 mM) for five days caused these cells to display differentiation-like characteristics including chromatin aggregation, nuclear degradation, pseudopod extension of the membrane and increased hemoglobin production. However, overall cell viability was not significantly changed by the presence of hemin. After hemin treatment for different periods, the Ca(2+)-activated K(+) channel was activated by the addition of ionomycin (1 microM), and was inhibited by either clotrimazole, charybdotoxin, or EGTA. Before hemin treatment there was no significant Ca(2+)-activated K(+) channel activity present in undifferentiated K562 cells. After hemin treatment for 5 days, a significant Ca(2+)-activated K(+) channel activity was detected. This increasing Ca(2+)-activated K(+) channel activity may be contributed from a subtype of Ca(2+)-activated K(+) channel, KCNN4. These results suggest that the ability of hemin to induce increasing Ca(2+)-activated K(+) channel activity may contribute to the mechanism of hemin-induced K562 cell differentiation.     

Lysophosphatidylcholine stimulates IL-1beta release from microglia via a P2X7 receptor-independent mechanism

IL-1beta released from activated macrophages contributes significantly to tissue damage in inflammatory, degenerative, and autoimmune diseases. In the present study, we identified a novel mechanism of IL-1beta release from activated microglia (brain macrophages) that occurred independently of P2X(7) ATP receptor activation. Stimulation of LPS-preactivated microglia with lysophosphatidylcholine (LPC) caused rapid processing and secretion of mature 17-kDa IL-1beta. Neither LPC-induced IL-1beta release nor LPC-stimulated intracellular Ca(2+) increases were affected by inhibition of P2X(7) ATP receptors with oxidized ATP. Microglial LPC-induced IL-1beta release was suppressed in Ca(2+)-free medium or during inhibition of nonselective cation channels with Gd(3+) or La(3+). It was also attenuated when Ca(2+)-activated K(+) channels were blocked with charybdotoxin (CTX). The electroneutral K(+) ionophore nigericin did not reverse the suppressive effects of CTX on LPC-stimulated IL-1beta release, demonstrating the importance of membrane hyperpolarization. Furthermore, LPC-stimulated caspase activity was unaffected by Ca(2+)-free medium or CTX, suggesting that secretion but not processing of IL-1beta is Ca(2+)- and voltage-dependent. In summary, these data indicate that the activity of nonselective cation channels and Ca(2+)-activated K(+) channels is required for optimal IL-1beta release from LPC-stimulated microglia.