EAD and DAD mechanisms analyzed by developing a new human ventricular cell model.

It has long been suggested that the Ca(2+)-mechanisms are largely involved in generating the early afterdepolarization (EAD) as well as the delayed afterdepolarization (DAD). This view was examined in a quantitative manner by applying the lead potential analysis to a new human ventricular cell model. In this ventricular cell model, the tight coupled LCC-RyR model (CaRU) based on local control theory (Hinch et al. 2004) and ion channel models mostly based on human electrophysiological data were included to reproduce realistic Ca(2+) dynamics as well as the membrane excitation. Simultaneously, the Ca(2+) accumulation near the Ca(2+) releasing site was incorporated as observed in real cardiac myocytes. The maximum rate of ventricular repolarization (-1.02 mV/ms) is due to IK1 (-0.55 mV/ms) and the rest is provided nearly equally by INCX (-0.20 mV/ms), INaL (-0.16 mV/ms) and INaT (-0.13 mV/ms). These INaL and INaT components are due to closure of the voltage gate, which remains partially open during the plateau potential. DADs could be evoked by applying high-frequency stimulations supplemented by a partial Na(+)/K(+) pump inhibition, or by a microinjection of Ca(2+). EADs was evoked by retarding the inactivation of INaL. The lead potential (VL) analysis revealed that IK1 and IKr played the primary role to reverse the AP repolarization to depolarizing limb of EAD. ICaL and INCX amplified EAD, while the remaining currents partially antagonized dVL/dt. The maximum rate of rise of EAD was attributable to the rapid activation of both ICaL (45.5%) and INCX (54.5%).

The nonlinear elastic and viscoelastic passive properties of left ventricular papillary muscle of a guinea pig heart.

The mechanical behavior of the heart muscle tissues is the central problem in finite element simulation of the heart contraction, excitation propagation and development of an artificial heart. Nonlinear elastic and viscoelastic passive material properties of the left ventricular papillary muscle of a guinea pig heart were determined based on in-vitro precise uniaxial and relaxation tests. The nonlinear elastic behavior was modeled by a hypoelastic model and different hyperelastic strain energy functions such as Ogden and Mooney-Rivlin. Nonlinear least square fitting and constrained optimization were conducted under MATLAB and MSC.MARC in order to obtain the model material parameters. The experimental tensile data was used to get the nonlinear elastic mechanical behavior of the heart muscle. However, stress relaxation data was used to determine the relaxation behavior as well as viscosity of the tissues. Viscohyperelastic behavior was constructed by a multiplicative decomposition of a standard Ogden strain energy function, W, for instantaneous deformation and a relaxation function, R(t), in a Prony series form. The study reveals that hypoelastic and hyperelastic (Ogden) models fit the tissue mechanical behaviors well and can be safely used for heart mechanics simulation. Since the characteristic relaxation time (900 s) of heart muscle tissues is very large compared with the actual time of heart beating cycle (800 ms), the effect of viscosity can be reasonably ignored. The amount and type of experimental data has a strong effect on the Ogden parameters. The in vitro passive mechanical properties are good initial values to start running the biosimulation codes for heart mechanics. However, an optimization algorithm is developed, based on clinical intact heart measurements, to estimate and re-correct the material parameters in order to get the in vivo mechanical properties, needed for very accurate bio-simulation and for the development of new materials for the artificial heart.

Role of Ca2+ transporters and channels in the cardiac cell volume regulation.

Na+/K+ pump is one of key mechanisms to maintain cell volume. When it is inhibited, cells are at risk of swelling. However, in guinea pig ventricular myocytes, the cell area as an index of cell volume was almost constant during 90 min Na+/K+ pump blockade with 40 microM ouabain despite the marked membrane depolarization. In this study, involvements of Ca2+ transporters and channels in the cardiac cell volume regulation were proposed by conducting the computer simulation in parallel with the experimental validation.

Modelling Cl- homeostasis and volume regulation of the cardiac cell.

We aim at introducing a Cl- homeostasis to the cardiac ventricular cell model (Kyoto model), which includes the sarcomere shortening and the mitochondria oxidative phosphorylation. First, we examined mechanisms underlying the cell volume regulation in a simple model consisting of Na+/K+ pump, Na+-K+-2Cl- cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator, volume-regulated Cl- channel and background Na+, K+ and Cl- currents. The high intracellular Cl- concentration of approximately 30 mM was achieved by the balance between the secondary active transport via NKCC1 and passive currents. Simulating responses to Na+/K+ pump inhibition revealed the essential role of Na+/K+ pump in maintaining the cellular osmolarity through creating the negative membrane potential, which extrudes Cl- from a cell, confirming the previous model study in the skeletal muscle. In addition, this model well reproduced the experimental data such as the responses to hypotonic shock in the presence or absence of beta-adrenergic stimulation. Finally, the volume regulation via Cl- homeostasis was successfully incorporated to the Kyoto model. The steady state was well established in the comprehensive cell model in respect to both the intracellular ion concentrations and the shape of the action potential, which are all in the physiological range. The source code of the model, which can reproduce every result, is available from http://www.sim-bio.org/.

Intracellular Ca(2+) dynamics and sarcomere length in single ventricular myocytes.

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastructural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca(2+)] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca(2+) overload. The [Ca(2+)] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.

Involvement of Ca2+ buffering and Na+/Ca2+ exchange in the positive staircase of contraction in guinea-pig ventricular myocytes.

The mean sarcomere length (SL) of guinea-pig cardiac myocytes was recorded simultaneously with the whole-cell current under voltage-clamp conditions. After blocking both sarcoplasmic reticulum (SR) and L-type Ca(2+) channels with ryanodine, cyclopiazonic acid and nicardipine, strong depolarizing pulses induced only the tonic component of SL shortening through the reverse mode of Na(+)/Ca(2+) exchange (NCX). A positive staircase of SL shortening was observed on applying a train pulses to +60~+100 mV at 2 Hz and trans-membrane Ca(2+) flux was calculated from the time integral of the Na(+)/Ca(2+) exchange current ( I(NCX)). Changes in cytosolic [Ca(2+)] ([Ca(2+)](i)) were determined indirectly using the experimental [Ca(2+)](i)/SL relationship. Cellular Ca(2+) buffering was characterized by a lumped single-component system with a maximum binding capacity of 200 micro M and a dissociation constant of 613 nM. Despite the decrease in driving force, the amplitude of the outwards I(NCX) at +60 mV gradually increased along with the positive staircase. The model simulation suggested that this increase of outwards I(NCX) is caused by a dramatic increase in Ca(2+)-mediated activation of NCX.

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes.

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.

Lipopolysaccharide induction of indoleamine 2,3-dioxygenase is mediated dominantly by an IFN-gamma-independent mechanism.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in the L-tryptophan-kynurenine pathway, which converts an essential amino acid, L-tryptophan, to N-formylkynurenine. It has been speculated that IFN-gamma is a dominant IDO inducer in vivo. The present study used IFN-gamma or TNF-alpha gene-disrupted mice and IFN-gamma antibody-treated mice to demonstrate that lipopolysaccharide (LPS)-induced systemic IDO is largely dependent on TNF-alpha rather than IFN-gamma. IFN-gamma-independent IDO induction was also demonstrated in vitro with LPS-stimulated monocytic THP-1 cells. These findings clearly indicate that there is an IFN-gamma-independent mechanism of IDO induction in addition to the IFN-gamma-dependent mechanism.

3-Hydroxyanthranilic acid, an L-tryptophan metabolite, induces apoptosis in monocyte-derived cells stimulated by interferon-gamma.

3-Hydroxyanthranilic acid (3-HAA), a metabolite of L-tryptophan, accumulates in monocyte-derived cells (THP-1), but not in other cell lines tested (MRC-9, H4, U373MG, Wil-NS), following immune stimulation that induces indoleamine-2,3-dioxygenase (IDO), a rate-limiting enzyme in the L-tryptophan kynurenine pathway. We examined whether metabolites of the L-tryptophan-kynurenine pathway act to induce apoptosis in monocytes/macrophages. Of the L-tryptophan metabolites tested, only 3-HAA at a concentration of 200 micromol/L was found to induce apoptosis in THP-1 and U937 cells. The addition of ferrous or manganese ions further enhanced apoptosis and free radical formation by 3-HAA in these two types of cells. The apoptotic response induced by 3-HAA was significantly attenuated by the addition of antioxidant, alpha-tocopherol or Trolox (a water-soluble analogue of vitamin E), and the xanthine oxidase inhibitor, allopurinol. In addition, the 3-HAA-induced apoptotic response was slightly attenuated by catalase, but not by superoxide dismutase (SOD), indicating that generation of hydrogen peroxide is involved in this response. Interferon-gamma (IFN-gamma), an inducer of IDO, potently induced apoptosis in THP-1 cells, but not in U937 cells, in the presence of ferrous or manganese ions. This different susceptibility to apoptosis inducer between THP-1 and U937 cells may depend on the capacity of the cells for 3-HAA synthesis following IDO induction by IFN-gamma. Furthermore, apoptosis was suppressed by cycloheximide in THP-1 cells, suggesting that newly synthesized proteins may be essential for apoptotic events. These results suggest that 3-HAA induces apoptosis in monocytes/macrophages under inflammatory or other pathophysiological conditions.

Mechanism of increases in L-kynurenine and quinolinic acid in renal insufficiency.

Marked increases in metabolites of the L-tryptophan-kynurenine pathway, L-kynurenine and quinolinic acid (Quin), were observed in serum and cerebrospinal fluid (CSF) of both the rat and human with renal insufficiency. The mechanisms responsible for their accumulation after renal insufficiency were investigated. In patients with chronic renal insufficiency, elevated levels of serum L-kynurenine and Quin were reduced by hemodialysis. In renal-insufficient rats, Quin and L-kynurenine levels in serum, brain, and CSF were also increased parallel to the severity of renal insufficiency. Urinary excretion of Quin (3.5-fold) and L-kynurenine (2.8-fold) was also increased. Liver L-tryptophan 2,3-dioxygenase activity (TDO), a rate-limiting enzyme of the kynurenine pathway, was increased in proportion to blood urea nitrogen and creatinine levels. Kynurenine 3-hydroxylase and quinolinic acid phosphoribosyltransferase were unchanged, but the activities of kynureninase, 3-hydroxyanthranilate dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) were significantly decreased. Systemic administrations of pyrazinamide (ACMSDase inhibitor) increased serum Quin concentrations in control rats, demonstrating that changes in body ACMSDase activities in response to renal insufficiency are important factors for the determination of serum Quin concentrations. We hypothesize the following ideas: that increased serum L-kynurenine concentrations are mainly due to the increased TDO and decreased kynureninase activities in the liver and increased serum Quin concentrations are due to the decreased ACMSDase activities in the body after renal insufficiency. The accumulation of CSF L-kynurenine is caused by the entry of increased serum L-kynurenine, and the accumulation of CSF Quin is secondary to Quin from plasma and/or Quin precursor into the brain.

Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells.

Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na(+) current, L- and T-type Ca(2+) currents, hyperpolarization-activated cation current, and various types of delayed rectifier K(+) currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (I(st)). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na(+) current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na(+) or L-type Ca(2+) channels both in unitary conductance and gating kinetics. Because I(st) was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I(st) for generation of intrinsic cardiac automaticity was suggested.

The sustained inward current and inward rectifier K+ current in pacemaker cells dissociated from rat sinoatrial node.

1. Myocytes were dissociated from the sinoatrial (SA) node of rat heart using a new enzymatic dissociation technique. Only a small number of isolated SA node myocytes showed regular rhythmic contractions and spontaneous action potentials, and these were used in the present study. 2. The spontaneous action potential was resistant to TTX, and the action potential parameters were similar to those of rabbit and guinea-pig pacemaker cells. Major time- and voltage-dependent currents were the delayed rectifier K+ current IKr, the L-type Ca2+ current ICa,L and the sodium current INa. The hyperpolarization-activated cation current (If) was recorded from approximately 50 % of the cells with hyperpolarization beyond -90 mV. 3. The instantaneous current jump at the onset of a hyperpolarizing pulse showed inward rectification and was largely blocked by Ba2+. This Ba2+-sensitive current corresponded well to the inward rectifier K+ current (IK1), although it was much smaller in amplitude than in the ventricle. 4. A sustained inward current was activated on depolarization from -80 mV to the voltage range of slow diastolic depolarization. The current was blocked by nicardipine, enlarged by isoprenaline and was insensitive to removal of external Ca2+. These characteristics were similar to the sustained inward current, Ist, previously described in the rabbit and guinea-pig SA node cells. 5. The role of Ist was considered by constructing empirical equations, which were applied to the experimental record of the action potential. It is demonstrated that the voltage-dependent activation of Ist constitutes a positive feedback loop with the depolarization of the membrane.

Nicardipine-sensitive Na+-mediated single channel currents in guinea-pig sinoatrial node pacemaker cells.

1. The Na+-dependent inward currents underlying slow diastolic depolarization of sinoatrial (SA) node cells were examined. Using a Na+-rich, Ca2+-free pipette solution a novel single channel current was recorded in addition to the conventional Na+ and L-type Ca2+ currents. The current (termed ist, as it reflects the whole-cell sustained inward current, Ist) does not show obvious inactivation during a 700 ms depolarization and is unique in having a smaller amplitude (1.1 +/- 0.18 pA at -60 mV, n = 12) than the Na+ current through conventional Na+ ( approximately 3.3 pA) and Ca2+ channels (9.6 +/- 0.32 pA at -60 mV, n = 8). The mean unitary conductance of ist channels was 13.3 pS. 2. The recording of ist was infrequent, was observed only in spontaneously beating SA node cells, and was facilitated by adding Bay-K 8644 to the pipette solution. Overlapping of ist events was observed and ist was abolished by bath application of nicardipine. 3. In the ensemble average, the activation of ist was evident by depolarization beyond -70 mV, and the dynamic voltage range of activation (-70 to -30 mV) encompassed the extent of the slow diastolic depolarization. The current density of ist was 0.33 pA pF-1 at -60 mV, as estimated from the number of channels per membrane patch, the open probability and the unitary amplitude. 4. Cumulative histograms for both open and closed times were fitted with a sum of two exponential components. The slow time constants decreased with depolarization, while the fast time constants and the fraction of the fast component were voltage independent. The number of bursts per sweep increased with depolarization. The time constant of the first latency histogram was about two orders of magnitude larger than those in cardiac L-type Ca2+ channels and decreased with depolarization. 5. It is suggested that the ist channels might be responsible for the whole-cell Ist.

Changes in cell volume induced by ion channel flux in guinea-pig cardiac myocytes.

1. The cell width of guinea-pig ventricular myocytes was measured using an optic device during patch-clamp experiments and the relationship between the ion channel flux and changes in cell volume was examined. 2. On superfusing myocytes with 50, 70, 150 and 200% osmotic solutions, the relative cell width changed to 121.1 (n = 4), 110.8 (n = 27), 87.1 (n = 6) and 82.6% (n = 6) of control, respectively. Changes in cell length were less than 2% in these test solutions. 3. The application of 300 nmol/L isoprenaline to myocytes swollen in the 70% hypotonic solution induced a decrease in cell width from 111.2 to 106.2% (n = 13). The application of isoprenaline in the isotonic solution also induced a decrease in cell width to 96.5% in eight of 13 cells. A membrane depolarization of 2-4 mV accompanied the isoprenaline-induced decrease in volume. In the remaining five cells, neither an obvious isoprenaline-induced decrease in volume nor membrane depolarization was observed. Under ruptured whole-cell voltage clamp conditions, the activation of inward isoprenaline-induced Cl- current decreased cell width. 4. Cell width was seen to either decrease or increase when a large outward or inward K+ current, respectively, was induced by shifting the holding potential or by applying 200 mumol/L pinacidil. Under gramicidin-perforated whole-cell clamp conditions, the cell width did not change, even when a large inward K+ current was induced. 5. When the test solution was applied to half of an elongated myocyte by using a micropipette, the cell width increased or decreased in the part exposed to the hypotonic or hypertonic test solutions, respectively. In contrast, in the other half of the elongated myocyte, the cell width responded in the opposite direction. 6. It is concluded that a continuous ionic flux through ion channels is capable of inducing changes in cell volume by generating a localized osmotic gradient across the cardiac sarcolemma.

Sarcomere length during contraction of isolated guinea-pig ventricular myocytes.

An improved method was developed for measuring sarcomere length (SML) during twitch contractions of single cardiac ventricular myocytes, using a charge-coupled photodiode array self-scanning at a rate of 1.5 ms/element. The average resting SML of 111 cells was 1.88+/-0.04 microm (mean +/-SD). When contractions were triggered by action potentials under perforated-patch conditions, the time course of SML shortening closely followed changes in cell length. A large variation was observed in contraction time course between myocytes, some cells having a phasic component with a duration at 50% shortening (full-width at half-maximum; FWHM) of approximately 40 ms, while others shortened more slowly (FWHM of phasic component @100 ms). FWHM was highly correlated with relaxation half-time, but with neither action potential duration nor resting SML. The kinetics of slowly contracting cells could not be converted to the rapid type by using conditioning trains or applying isoprenaline. The steady-state SML/pCa relation in ventricular myocytes was measured by applying solutions of various pCa immediately after localized punctures of the surface membrane using a focal laser beam. The Hill coefficient, nH, was @4-5 and K1/2@400-500 nM, but there was no evidence of two populations of cells with different Ca2+ sensitivities.

Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node.

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (If or Ih) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brain If cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1-3. The transmembrane region of HAC4 was most homologous to partially cloned mouse If BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at -87.2 +/- 2.8 mV under control conditions and at -64.4 +/- 2.6 mV in the presence of intracellular 0.3 mM cAMP. The reversal potential was -34.2 +/- 0.9 mV in 140 mM Na+o and 5 mM K+o versus 10 mM Na+i and 145 mM K+i. These results indicate that HAC4 forms If in rabbit heart SA node.

4F2 (CD98) heavy chain is associated covalently with an amino acid transporter and controls intracellular trafficking and membrane topology of 4F2 heterodimer.

4F2, also termed CD98, is an integral membrane protein consisting of a heavy chain linked to a light chain by disulfide bond. We have generated a monoclonal antibody to the mouse 4F2 light chain and cloned the cDNA. It encodes a mouse counterpart of rat L-type amino acid transporter-1, and induces system L amino acid transport in Xenopus oocytes in the presence of 4F2 heavy chain. Transfection studies in mammalian cells have indicated that the 4F2 heavy chain is expressed on the plasma membrane on its own, whereas the 4F2 light chain can be transported to the surface only in the presence of 4F2 heavy chain. 4F2 heavy chain is expressed diffusely on the surface of fibroblastic L cells, whereas it is localized selectively to the cell-cell adhesion sites in L cells expressing cadherins. These results indicate that the 4F2 heavy chain is associated covalently with an amino acid transporter and controls the cell surface expression as well as the membrane topology of the 4F2 heterodimer. Although 4F2 heavy and light chains are expressed coordinately in most tissues, the light chain is barely detected by the antibody in kidney and intestine, despite the presence of heavy chain in a complex form. The results predict the presence of multiple 4F2 light chains.

Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels.

1. In order to investigate the mechanism underlying MgATP-dependent recovery of ATP-sensitive potassium (KATP) channels, we expressed Kir6.2/SUR2A (inwardly rectifying K+ channel subunit/sulfonylurea receptor) or C-terminal-truncated Kir6.2 (Kir6.2DeltaC26) in COS7 cells (Green monkey kidney cells), and carried out inside-out patch clamp experiments. 2. After patch excision in ATP-free internal solution, the activity of Kir6.2/SUR2A channels could be maximally recovered by the application of 5 mM MgATP. Subsequent application of 100 microM Ca2+ induced a rapid decay of Kir6.2/SUR2A activity to 11.6 +/- 1.1 % (mean +/- s.e.m.) of the control level (Ca2+-induced run-down; n = 64). 3. MgATP (5 mM) recovered 99.4 +/- 4.2 % (n = 13) of the Ca2+-induced run-down. Protein kinase inhibitors such as W-7, H-7, H-8 and genistein did not inhibit this reaction. However, wortmannin, an inhibitor of phosphatidylinositol 3- and 4-kinases, blocked the MgATP-dependent recovery in a concentration-dependent manner; the magnitudes of recovery were 35.7 +/- 7.2 % (10 microM) and 4.3 +/- 2.5 % (100 microM) of the Ca2+-induced run-down. 4. MgUDP (10 mM) reversed the Ca2+-induced run-down of Kir6.2/SUR2A channels by 60.4 +/- 7.6 % (n = 5). Wortmannin failed to modify this reaction. 5. Kir6.2DeltaC26 channels, which opened in the absence of SUR2A, were less sensitive to Ca2+; Kir6.2DeltaC26 channels were inactivated to 44.8 +/- 4.4 % (n = 14) by 100 microM Ca2+. MgATP recovered the Ca2+-induced run-down of Kir6.2DeltaC26 by 89.8 +/- 7. 7 % (n = 9), and 100 microM wortmannin inhibited this reaction (1.8 +/- 2 %, n = 7). 6. Application of 10 microM phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) recovered the activity of Kir6.2/SUR2A channels after Ca2+-induced run-down (104.3 +/- 6.4 %, n = 10). Even after the MgATP-dependent recovery was blocked by 100 microM wortmannin, PI-4,5-P2 reactivated the channels (102.3 +/- 8.6 %, n = 5). Similar results were obtained with Kir6.2DeltaC26. 7. These results suggest that the entity of MgATP-dependent recovery may be membrane lipid phosphorylation rather than protein phosphorylation, and that synthesis of PI-4,5-P2 or phosphatidylinositol-3,4, 5-trisphosphate may upregulate Kir6.2 channels.

L-tryptophan-kynurenine pathway metabolite 3-hydroxyanthranilic acid induces apoptosis in macrophage-derived cells under pathophysiological conditions.

Accumulation of L-kynurenine and 3-hydroxyanthranilic acid (3HAA) occurs in the monocyte-derived cells following immune stimulation, and may derive from L-tryptophan following induction of indoleamine-2,3-dioxygenase. In the present study, we evaluate the possibility that 3HAA acts as an endogenous inducer of monocyte/macrophage apoptosis. Supplementation with 200 microM of 3HAA, but not other L-tryptophan metabolites tested, significantly increased the number of apoptotic cells in both THP-1 and U937 cells. Catalase, superoxide dismutase and manganese ions markedly enhanced apoptosis in the presence of 3HAA in these cells. The present results suggest that 3HAA induces the macrophage/monocyte apoptosis under certain conditions, which may be relevant to pathophysiology of inflammatory conditions.