Selective induction of DeltaFosB in the brain after transient forebrain ischemia accompanied by an increased expression of galectin-1, and the implication of DeltaFosB and galectin-1 in neuroprotection and neurogenesis.

Transient forebrain ischemia causes selective induction of DeltaFosB, an AP-1 (activator protein-1) subunit, in cells within the ventricle wall or those in the dentate gyrus in the rat brain prior to neurogenesis, followed by induction of nestin, a marker for neuronal precursor cells, or galectin-1, a beta-galactoside sugar-binding lectin. The adenovirus-mediated expression of FosB or DeltaFosB induced expression of nestin, glial fibrillary acidic protein and galectin-1 in rat embryonic cortical cells. DeltaFosB-expressing cells exhibited a significantly higher survival and proliferation after the withdrawal of B27 supplement than the control or FosB-expressing cells. The decline in the DeltaFosB expression in the survivors enhanced the MAP2 expression. The expression of DeltaFosB in cells within the ventricle wall of the rat brain also resulted in an elevated expression of nestin. We therefore conclude that DeltaFosB can promote the proliferation of quiescent neuronal precursor cells, thus enhancing neurogenesis after transient forebrain ischemia.

A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents.

Frequenin, a Ca(2+)-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K(+) currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca(2+)-dependent manner. It also accelerates recovery from inactivation. Closely related Ca(2+)-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca(2+)-sensitive regulatory component of native A-type K(+) currents.

Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters.

Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex, causes cardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Using cultured myotubes prepared from skeletal muscle of normal and Bio14.6 hamsters (J2N-k strain), we investigated the possibility that the delta-SG deficiency may lead to alterations in ionic conductances, which may ultimately lead to myocyte damage. In cell-attached patches (with Ba(2+) as the charge carrier), an approximately 20-pS channel was observed in both control and Bio14.6 myotubes. This channel is also permeable to K(+) and Na(+) but not to Cl(-). Channel activity was increased by pressure-induced stretch and was reduced by GdCl(3) (>5 microM). The basal open probability of this channel was fourfold higher in Bio14.6 myotubes, with longer open and shorter closed times. This was mimicked by depolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes, the unidirectional basal Ca(2+) influx was enhanced compared with control. This Ca(2+) influx was sensitive to GdCl(3), signifying that stretch-activated cation channels may have been responsible for Ca(2+) influx in Bio14.6 hamster myotubes. These results suggest a possible mechanism by which cell damage might occur in this animal model of muscular dystrophy.

Is the molecular composition of K(ATP) channels more complex than originally thought?

ATP-sensitive K+ (K(ATP)) channels are abundantly expressed in the heart and may be involved in the pathogenesis of myocardial ischemia. These channels are heteromultimeric, consisting of four pore-forming subunits (Kir6.1, Kir6.2) and four sulfonylurea receptor (SUR) subunits in an octameric assembly. Conventionally, the molecular composition of K(ATP) channels in cardiomyocytes and pancreatic beta -cells is thought to include the Kir6.2 subunit and either the SUR2A or SUR1 subunits, respectively. However, Kir6.1 mRNA is abundantly expressed in the heart, suggesting that Kir6.1 and Kir6.2 subunits may co-assemble to form functional heteromeric channel complexes. Here we provide two independent lines of evidence that heteromultimerization between Kir6.1 and Kir6.2 subunits is possible in the presence of SUR2A. We generated dominant negative Kir6 subunits by mutating the GFG residues in the channel pore to a series of alanine residues. The Kir6.1-AAA pore mutant subunit suppressed both wt-Kir6.1/SUR2A and wt-Kir6.2/SUR2A currents in transfected HEK293 cells. Similarly, the dominant negative action of Kir6.2-AAA does not discriminate between either of the wild-type subunits, suggesting an interaction between Kir6.1 and Kir6.2 subunits within the same channel complex. Biochemical data support this concept: immunoprecipitation with Kir6.1 antibodies also co-precipitates Kir6.2 subunits and conversely, immunoprecipitation with Kir6.2 antibodies co-precipitates Kir6.1 subunits. Collectively, our data provide direct electrophysiological and biochemical evidence for heteromultimeric assembly between Kir6.1 and Kir6.2. This paradigm has profound implications for understanding the properties of native K(ATP)channels in the heart and other tissues.

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2.

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels.

Thyroid hormone increases pacemaker activity in rat neonatal atrial myocytes.

The effects of thyroid hormone (3,3',5-triiodo- L -thyronine, T3) on pacemaker activity were studied with electrophysiological and pharmacological approaches using spontaneously beating neonatal atrial myocytes cultured from 2-day-old rats. Treatment with T3 (10(-8)m) for 24-48 h led to a positive chronotropic effect. The beating rate of T3-treated cells was 244+/-19 beats/min and for control cells it was 122+/-10 beats/min (P<0.05). Action potentials were recorded and showed that the predominant effect of T3 was to increase the diastolic depolarization rate (99.5+/-9.8 in T3-treated group v 44.0+/-7.8 mV/s in untreated group). Some cells that exhibited pacemaker activity lacked a pacemaker current (I(f)) under voltage clamp conditions I(f)was recorded in 5 of 12 spontaneously active control cells and in 6 of 10 T3-treated cells. In those cells exhibiting the pacemaker current, the I(f)density was significantly larger in T3-treated cells (-7.9+/-2.6 pA/pF v-1.8+/-0.5 pA/pF in control). The L-type Ca2+ current density was similar in the two groups (at -7 mV, -7.5+/-1.5 in treated group v-8.6+/-1.0 pA/pF in control). In the presence of T3, the Na+-Ca2+ exchanger current (I(Na/Ca)) density was larger (e.g. at +60 mV, it was 4.8+/-0.5 v 3.5+/-0.2 pA/pF in control cells, P<0.05). As intracellular Ca2+ is extruded from the cell, the electrogenic Na+-Ca2+ exchanger causes a declining inward current, which may contribute to the pacemaker potential-this declining inward current was demonstrated using the action potential voltage clamp technique and was shown to be larger in T3-treated myocytes. Our data demonstrate that thyroid hormone enhances pacemaker activity and that this may be due in part to an increased Na+-Ca2+ exchanger activity.

Physiological functions of the regulatory domains of the cardiac Na(+)/Ca(2+) exchanger NCX1.

Physiological functions of the intracellular regulatory domains of the Na(+)/Ca(2+) exchanger NCX1 were studied by examining Ca(2+) handling in CCL39 cells expressing a low-affinity Ca(2+) regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na(+) inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Delta246-672). We found that D447V/D498I was unable to efficiently extrude Ca(2+) from the cytoplasm, particularly during a small rise in intracellular Ca(2+) concentration induced by the physiological agonist alpha-thrombin or thapsigargin. The same mutant took up Ca(2+) much less efficiently than the wild-type NCX1 in Na(+)-free medium when transfectants were not loaded with Na(+), although it appeared to take up Ca(2+) normally in transfectants preloaded with Na(+). XIP-4YW and, to a lesser extent, Delta246-672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na(+)-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca(2+) handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.

Chimeric analysis of Na(+)/Ca(2+) exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni(2+) or Li(+).

Externally applied Ni(2+), which apparently competes with Ca(2+) in all three isoforms of Na(+)/Ca(2+) exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li(+) is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423-C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni(2+) or Li(+) by measuring intracellular Na(+)-dependent (45)Ca(2+) uptake in CCL39 cells stably expressing NCX1/NCX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal alpha-1 and alpha-2 repeats are individually responsible for the alteration of Ni(2+) sensitivity, both together accounting for approximately 80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the alpha-2 repeat fully accounts for the differential Li(+) sensitivity between the isoforms. The Ni(2+) sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the alpha-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the alpha-2 repeat (V820A in NCX1 and A809V in NCX3). On the other hand, the Li(+) sensitivity was mimicked by double substitution mutation in the alpha-2 repeat (V820A/Q826V in NCX1 and A809V/V815Q in NCX3). Single substitution mutations at Asn(125) and Val(820) of NCX1 caused significant alterations in the interactions of the exchanger with Ca(2+) and Ni(2+), and Ni(2+) and Li(+), respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the alpha repeats, these regions might form an ion interaction domain in the exchanger.

The role of Kir2.1 in the genesis of native cardiac inward-rectifier K+ currents during pre- and postnatal development.

Our results demonstrate that (a) the Kir2.1 gene encodes a native K+ channel protein with a 21-pS conductance; (b) this channel has an important role in the genesis of adult ventricular 1K1; and (c) the contribution of Kir2.1 channel proteins to 1K1 changes during development. The lack of contribution of Kir2.1 to fetal 1K1 channels is interesting from the point of view of possible future generation of knockout mice lacking Kir2.1, since cardiac abnormalities would not be expected to result in fetal lethality. These observations provide further support for a generalized hypothesis that different genes may code for 1K1 channel proteins at various developmental stages. However, the effects of these AS-oligos must first be examined on native 1K1 channels in cardiac myocytes before definite conclusions can be reached.

Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal alpha-1 and alpha-2 repeats. Unexpectedly, we found that the connecting loop in the alpha-1 repeat forms a re-entrant membrane loop with both ends facing the extracellular side and one residue (Asn-125) being accessible from the inside and that the region containing the alpha-2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation.

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.

Inhibition of rat ventricular IK1 with antisense oligonucleotides targeted to Kir2.1 mRNA.

The cardiac inward rectifying K+ current (IK1) is important in maintaining the maximum diastolic potential. We used antisense oligonucleotides to determine the role of Kir2.1 channel proteins in the genesis of native rat ventricular IK1. A combination of two antisense phosphorothioate oligonucleotides inhibited heterologously expressed Kir2.1 currents in Xenopus oocytes, either when coinjected with Kir2.1 cRNA or when applied in the incubation medium. Specificity was demonstrated by the lack of inhibition of Kir2.2 and Kir2.3 currents in oocytes. In rat ventricular myocytes (4-5 days culture), these oligonucleotides caused a significant reduction of whole cell IK1 (without reducing the transient outward K+ current or the L-type Ca2+ current). Cell-attached patches demonstrated the occurrence of multiple channel events in control myocytes (8, 14, 21, 35, 43, and 80 pS). The 21-pS channel was specifically knocked down in antisense-treated myocytes (fewer patches contained this channel, and its open frequency was reduced). These results demonstrate that the Kir2.1 gene encodes a specific native 21-pS K(+)-channel protein and that this channel has an essential role in the genesis of cardiac IK1.

Modulation of Kv4 channels, key components of rat ventricular transient outward K+ current, by PKC.

Current evidence suggests that members of the Kv4 subfamily may encode native cardiac transient outward current (I(to)). Antisense hybrid-arrest with oligonucleotides targeted to Kv4 mRNAs specifically inhibited rat ventricular I(to), supporting this hypothesis. To determine whether protein kinase C (PKC) affects I(to) by an action on these molecular components, we compared the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and rat ventricular I(to). Phorbol 12-myristate 13-acetate (PMA) suppressed both Kv4.2 and Kv4.3 currents as well as native I(to), but not after preincubation with PKC inhibitors (e.g., chelerythrine). An inactive stereoisomer of PMA had no effect. Phenylephrine or carbachol inhibited Kv4 currents only when coexpressed, respectively, with alpha1C-adrenergic or M1 muscarinic receptors (this inhibition was also prevented by chelerythrine). The voltage dependence and inactivation kinetics of Kv4.2 were unchanged by PKC, but small effects on the rates of inactivation and recovery from inactivation of native I(to) were observed. Thus Kv4.2 and Kv4.3 proteins are important subunits of native rat ventricular I(to), and PKC appears to reduce this current by affecting the molecular components of the channels mediating I(to).

Effects of thiol-modifying agents on KATP channels in guinea pig ventricular cells.

ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)

Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration.

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.

Melittin cardiotoxicity in cultured mouse cardiac myocytes and its correlation with calcium overload.

Venom from the honey bee Apis mellifera induces cardiovascular dysfunction. We studied which constituent(s) of the venom induces cardiotoxicity and how, using cultured cardiac myocytes from mouse fetuses. Among the venom constituents, only melittin caused contractile and morphological effects; other peptides, such as apamin and mastparan; enzymes, such as phospholipase A2; and low-molecular-weight compounds, such as histamine and dopamine, did not. Treatment with 4.5 micrograms/ml melittin, which accounts for about half the dry weight of the venom, induced the same cardiotoxic effects as treatment with 9.0 micrograms/ml whole venom; these effects were a transient increase in the spontaneous beating rate, then a decrease, then cessation of beating, and finally, morphological degeneration. The cardiotoxicity of whole bee venom was completely destroyed by pretreatment of the venom with antimelittin antibody. These results suggest that bee venom cardiotoxicity is attributable to melittin. When spontaneous beating ceased following the addition of melittin or whole venom, an increase in systolic [Ca2+]i, was observed. On further incubation with melittin or bee venom, morphological injury, such as balloon degeneration, occurred concomitant with a further increase in the [Ca2+]i. An extracellular Ca2+ concentration of more than 10(-6) M was necessary for morphological injury. Melittin depolarized the maximum diastolic potentials, inhibited the generation of action potentials, and induced an increase in [Na+]i. Cells were protected against the melittin-induced increase in [Ca2+]i by pretreatment with bepridil, an inhibitor of Na(+)-Ca2+ exchange, but not by Ca2+ channel blockers such as verapamil. These observations suggest that the melittin-induced increase in [Ca2+]i was due to entry of extracellular Ca2+ via the sarcolemmal Na(+)-Ca+ exchange system.

Metabolic coupling of glutathione between mouse and quail cardiac myocytes and its protective role against oxidative stress.

Cultured quail myocytes were much more resistant to H2O2 toxicity than cultured mouse myocytes. The intracellular concentration of glutathione ([GSH]i) and the activity of gamma-glutamylcysteine synthetase (gamma-GCS) in quail heart cells were about five and three times higher, respectively, than in mouse heart cells, although catalase and glutathione peroxidase (GSHpx) activity was similar in both. Preloading of gamma-glutamylcysteine monoethyl ester (gamma-GCE), a membrane-permeating GSH precursor, increased the H2O2 resistance of cultured mouse myocytes. These observations suggest that the high [GSH]i and the high activity of gamma-GCS in quail myocytes are responsible for their high resistance to H2O2. Both H2O2 sensitivity and [GSH]i of mosaic sheets composed of equal amounts of mouse and quail myocytes approximated those of sheets composed entirely of quail myocytes. From these observations, it is hypothesized that GSH was transferred from quail myocytes to mouse myocytes, probably through gap junctions between them, and that quail myocytes resynthesized GSH by a feedback mechanism, thus maintaining their intracellular GSH levels. When the fluorescent dye lucifer yellow was injected into a beating quail myocyte in a mosaic sheet, it spread to neighboring mouse myocytes but not to neighboring L cells (a cell line derived from mouse connective tissue). These observations indicate that existence of gap junctions in the region of cell contact between mouse and quail myocytes but not between quail myocytes and L cells. When quail myocytes preloaded with [3H]gamma-GCE were cocultured with mouse myocytes and L cells, the radioactivity was transmitted to neighboring mouse myocytes but not L cells. These observations show that GSH and/or its precursors can be transmitted from quail myocytes to mouse myocytes through gap junctions and that this can protect mouse myocytes from H2O2 toxicity. Mouse myocyte sheets composed of 10(4) cells or more showed higher resistance to H2O2 toxicity than single isolated mouse myocytes. Metabolic coupling of GSH between myocytes may contribute at least in part to this high resistance of the cell sheets.

Contractile and morphological impairment of cultured fetal mouse myocytes induced by oxygen radicals and oxidants. Correlation with intracellular Ca2+ concentration.

There is evidence that reperfusion injury of cardiac tissue may be caused by the generation of oxygen-derived free radicals and oxidants and by the induction of intracellular calcium overload, although the relation between these two mechanisms of injury is uncertain. In addition, the relation between the types of cellular injury and specific active species is unclear. In an attempt to resolve these problems, we investigated the effects of oxygen radicals and oxidants, which are purportedly generated during reperfusion after prolonged ischemia, and various antioxidants on contractility and morphology of cultured fetal mouse cardiac myocytes. Xanthine oxidase in the presence of xanthine, H2O2, HOCl, and NH2Cl induced cessation of spontaneous beating followed by cessation of electrical stimulation-elicited beating but did not induce an increase in [Ca2+]i. After prolonged incubation with xanthine oxidase + xanthine and H2O2, the cardiac myocytes showed morphological degeneration (at least 80% of the cells developed hypercontraction) with a concomitant increase in [Ca2+]i. These observations suggest that contractile impairment does not result in an increase of [Ca2+]i, but hypercontraction does. Catalase, but not superoxide dismutase, protected the cultured cardiac myocytes against xanthine oxidase + xanthine- and H2O2-induced contractile and morphological impairment. In the light of this observation, we hypothesize that the superoxide anion is not responsible for these types of impairment. Addition of dimethylthiourea (an .OH scavenger) and intracellular preloading with deferoxamine (an iron chelator) protected the myocytes against H2O2-induced contractile and morphological damage, but intracellular preloading with iron enhanced it. These observations led us to hypothesize that intracellularly generated .OH may be a mediator of H2O2-induced injury to cultured cardiac myocytes. In addition, we observed that H2O2 itself induced cessation of spontaneous but not electrical stimulation-elicited beating.