Anti-oxidant effects of estrogen reduce [Ca2+]i during metabolic inhibition.

We previously reported that 17beta-estradiol (betaE2) inhibits the rise in [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in mouse cardiomyocytes, but the mechanism has not yet been clarified. Estrogen has been reported to have anti-oxidant properties. We, therefore, have investigated whether interaction with the estrogen receptor (ER) is involved, or whether estrogen reduces free-radical-induced impairment of Na(+)-K(+) ATPase in cardiac myocytes, and whether this effect reduces [Ca(2+)](i) rise. Male mouse ventricular myocytes were studied. Flow cytometry was used with fluo-3 for [Ca(2+)](i) measurement. Dead cells were excluded from analysis by propidium iodide fluorescence. betaE2 reduced the increase in [Ca(2+)](i) during MI even in the presence of the ER blocker tamoxifen. A similar effect on [Ca(2+)](i) was produced by its non-estrogenic isomer, betaE2-estradiol. Other hormones (estrone and estriol) with a phenolic structure also inhibited Ca(2+) overload during MI, but testosterone without the structure did not. The betaE2 effect was attenuated by inhibition of Na(+)-Ca(2+) exchanger (KB-R7943) or Na(+)-K(+) ATPase (low K(+) or ouabain), but not by block of L-type Ca(2+) channel (nifedipine). Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid), a superoxide scavenger, decreased the rise in [Ca(2+)](i) and abolished the betaE2 effect during MI. We conclude that the acute cardioprotective effect of estrogen during MI may be mediated by an ER-independent anti-oxidant action, which results in improved function of Na(+)-K(+) ATPase.

Activation of connexin-43 hemichannels can elevate [Ca(2+)]i and [Na(+)]i in rabbit ventricular myocytes during metabolic inhibition.

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na(+) and Ca(2+) accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca(2+)]i and [Na(+)]i in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 microM fluo-3 AM for 30 min, or with 5 microM sodium green AM for 60 min at 37 degrees C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI+H (0.95 mM, 4.7 mM) for 60 min. Propidium iodide (PI, 25 microM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca(2+)]i and [Na(+)]i were then calculated by calibration. In some experiments, the effect of 10 microM tetrodotoxin (TTX) and 20 microM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca(2+)]i and [Na(+)]i in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca(2+)]i and [Na(+)]i in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca(2+)]i and [Na(+)]i overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na(+) and Ca(2+) channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.

Gender influences [Ca(2+)](i) during metabolic inhibition in myocytes overexpressing the Na(+)-Ca(2+) exchanger.

BACKGROUND: The Na(+)-Ca(2+) exchanger (NCX) may contribute to Ca(2+) overload and injury in ischemic cardiomyocytes. Recently, NCX overexpression was reported to increase ischemia/reperfusion injury in male and oophorectomized female but not in female mice. We therefore measured the effects of gender and estrogen on [Ca(2+)](i) and [Na(+)](i) during metabolic inhibition (MI) in myocytes from wild-type (WT) and transgenic (TG) mice overexpressing NCX. METHODS AND RESULTS: Flow cytometry was used with fluo 3 for [Ca(2+)](i) and sodium green for [Na(+)](i) measurements. Male TG mouse myocytes had higher [Ca(2+)](i) after 30 minutes of MI (1086+/-160 nmol/L, n=8) than male WT (688+/-104 nmol/L, n=9, P=0.01). The increase in [Ca(2+)](i) during MI induced by NCX overexpression in female myocytes was not significant, however (TG 552+/-62 nmol/L, n=9; WT 426+/-44 nmol/L, n=7). The magnitude of rise in [Ca(2+)](i) during MI was greater in male than female myocytes. KB-R7943, an NCX inhibitor, abolished the effect of NCX overexpression but did not totally eliminate the effect of gender on [Ca(2+)](i) during MI. NCX current density and basal Na(+) pump function were not influenced by gender. The rise in [Na(+)](i) during MI was greater in male than in female myocytes. Estrogen attenuated the increase in [Ca(2+)](i) and [Na(+)](i) in male myocytes during MI and abolished the gender difference in [Na(+)](i) during MI. CONCLUSIONS: Increased expression of NCX results in a more marked rise in [Ca(2+)](i) during MI in male than in female mouse myocytes. This gender difference appears to be mediated in part by an inhibitory effect of estrogen on the rise in [Na(+)](i), an NCX modifier, during MI.

Effects of deletion of muscle LIM protein on myocyte function.

Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (I(Ca,L)), Na/Ca exchanger current (I(Na/Ca)), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I(Ca,L)-voltage relationships, I(Na/Ca) density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 +/- 54 vs. 349 +/- 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.

The sodium pump modulates the influence of I(Na) on [Ca2+]i transients in mouse ventricular myocytes.

To investigate whether activity of the sarcolemmal Na pump modulates the influence of sodium current on excitation-contraction (E-C) coupling, we measured [Ca(2+)](i) transients (fluo-3) in single voltage-clamped mouse ventricular myocytes ([Na+](pip) = 15 or 0 mM) when the Na pump was activated (4.4 mM K(+)(o)) and during abrupt inhibition of the pump by exposure to 0 K with a rapid solution-switcher device. After induction of steady state [Ca2+](i) transients by conditioning voltage pulses (0.25 Hz), inhibition of the Na pump for 1.5 s immediately before and continuing during a voltage pulse (200 ms, -80 to 0 mV) caused a significant increase (15 +/- 2%; n = 16; p < 0.01) in peak systolic [Ca2+](i) when [Na+](pip) was 15 mM. In the absence of sodium current (I(Na), which was blocked by 60 microM tetrodotoxin (TTX)), inhibition of the Na pump immediately before and during a voltage pulse did not result in an increase in peak systolic [Ca2+](i). Abrupt blockade of I(Na) during a single test pulse with TTX caused a slight decrease in peak [Ca2+](i), whether the pump was active (9%) or inhibited (10%). With the reverse-mode Na/Ca exchange inhibited by KB-R 7943, inhibition of the Na pump failed to increase the magnitude of the peak systolic [Ca2+](i) (4 +/- 1%; p = NS) when [Na+](pip) was 15 mM. When [Na+](pip) was 0 mM, the amplitude of the peak systolic [Ca2+](i) was not altered by abrupt inhibition of the Na pump immediately before and during a voltage pulse. These findings in adult mouse ventricular myocytes indicate the Na pump can modulate the influence of I(Na) on E-C coupling in a single beat and provide additional evidence for the existence of Na fuzzy space, where [Na+] can significantly modulate Ca2+ influx via reverse Na/Ca exchange.

Optical characterization of liposomes by right angle light scattering and turbidity measurement.

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.

Expression of genes encoding vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic heart.

Vascular endothelial growth factor (VEGF) is known to play an essential role in embryonic vascular development. The heart is one of the main organs that produce VEGF, but it is still unknown how expression of VEGF gene is regulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF and its receptor (a KDR/flk-1 or Quek1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymerase chain reaction revealed that the chick VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 122 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 122 amino acids were dominant. Northern blot analysis detected an abundance of VEGF mRNA in both the embryonic heart and CEVM, even at the basal state. The levels of VEGF mRNA in CEVM were significantly augmented by forskolin (100 microM), or phorbol 12-myristate, 13-acetate (200 nM) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 microM), but not by H89 (100 microM) or bisindolylmaleimide (75 microM). Northern blot analysis also detected the chick flk-1 mRNA in abundance in the embryonic heart, and to a much lesser extent in CEVM. The expression levels of VEGF and flk-1 mRNA species were continuously high in the 6, 8 and 10-day-old chick embryonic hearts. In the 10-day-old embryonic hearts, in situ hybridization confirmed that mRNA encoding VEGF was mainly expressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected in the microvascular endothelial cells, and to a lesser extent in the ventricular myocytes. These data suggest that VEGF is produced in embryonic ventricular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by the chick embryonic heart may play important roles in embryonic cardiovascular development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves.

An adult case with an abnormal right ventricular structure causing intraventricular pressure gradient and a history of aphthous stomatitis and thrombophlebitis.

We report a 50-year-old man with a right ventricular structure causing an intraventricular pressure gradient. He had been diagnosed as vasculo-Behçet with a history of aphthous stomatitis and thrombophlebitis. He had also been suffering from atrial flutter and mild right-side heart failure. Echocardiography showed that there was an abnormal structure attached to the right ventricular free wall and protruding into the cavity, and that it caused the pressure gradient estimated to be approximately 19 mmHg. Chest X-ray computed tomography demonstrated that the structure was partially calcified. Magnetic resonance imaging depicted the structure separating the right ventricle into two chambers. Angiographic study revealed a markedly enlarged right atrium and a filling defect at the mid-portion of the right ventricle, which divided the right ventricular cavity into two parts. Hemodynamic study showed a slightly elevated right atrial pressure (mean 7 mmHg) and a peak-to-peak intraventricular pressure difference of 18 mmHg in the right ventricle. The diastolic pressure tracing of the right ventricular low pressure chamber showed a 'dip and plateau' pattern. Although the pathological features of the abnormal right ventricular structure in this case were not fully clarified, abnormal muscle bundle and/or endocardial fibrosis, which were reported to be associated with Behçet's disease, may have contributed to its generation.

Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes.

OBJECTIVES: Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. METHODS: A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique. RESULTS: PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa. CONCLUSIONS: We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.

Cellular basis for the acute inhibitory effects of IL-6 and TNF- alpha on excitation-contraction coupling.

There is controversy over whether nitric oxide (NO) mediates acute negative inotropic actions of cytokines including tumor necrosis factor-alpha (TNF- alpha). The reports from established laboratories have appeared inconsistent, which could be due to species differences. Thus, we tried to elucidate the mechanisms underlying negative inotropic actions of interleukin-6 (IL-6) and TNF- alpha in the same model. We studied the effects of cytokines on [Ca(2+)](i)transients (using indo-1), cell shortening (CS) (using a video motion detector) and the L-type Ca(2+)channel current (I(Ca)) (using the whole cell perforated patch clamp technique) in isolated guinea-pig ventricular myocytes. IL-6 (1000 U/ml) or TNF- alpha (500 U/ml) decreased both peak systolic [Ca(2+)](i)(IL-6: 0.43+/-0.01 to 0.40+/-0.01, n=5, P<0.05; TNF- alpha : 0.42+/-0.02 to 0.39+/-0.02, n=5, P<0.05) and the amplitude of CS (IL-6: 7.5+/-0.9 to 6.2+/-0.5 micrometers, n=5, P<0.05; TNF- alpha : 6.7+/-0.7 to 5.8+/-0.7 micrometers, n=5, P<0.05) without detectable reductions in I(Ca)(IL-6: 0.9+/-0.1 to 0.9+/-0.1 nA, n=4, N.S.; TNF- alpha : 1.1+/-0.3 to 1.1+/-0.2 nA, n=4, N.S.) within 5 min. The nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl- L arginine (300 micromol/l), blocked the effects of IL-6 but not of TNF- alpha. When pretreated with 20 nmol/l isoproterenol, exposure to IL-6 decreased both I(Ca)(2.8+/-0.5 to 2. 0+/-0.3 nA) and the amplitude of CS (10.4+/-2.4 to 7.5+/-1.9 micrometer) within 5 min. TNF- alpha also clearly depressed I(Ca)(2.9+/-0.9 to 2.3+/-0.7 nA) and the amplitude of CS (7.0+/-1.4 to 5.5+/-1.3 micrometer) in beta -adrenergic stimulated cells. TNF- alpha significantly increased the content of sphingosine (product of sphingomyelin pathway) in isolated heart. The effects of low dose sphingosine (5 micromol/l) mimicked those of TNF- alpha on cardiac myocytes. IL-6 produced an acute negative inotropic effect through a NO-dependent pathway while TNF- alpha did so via a sphingomyelin-dependent pathway in isolated guinea-pig ventricular myocytes.

Acquired factor VIII inhibitor in a non-hemophilic patient with chronic hepatitis C viral infection.

Production of coagulation factor VIII inhibitor is rarely encountered in non-hemophilic patients. A 63-year-old Japanese male suffered from severe bleeding tendency caused by this inhibitor. Although he did not have malignancy or collagen disease, he had chronic hepatitis C virus (HCV) infection. Although HCV is known to induce production of various autoimmune antibodies, this may be the first report of a case with both acquired factor VIII inhibitor and HCV infection.

Interactions of an antimicrobial peptide, magainin 2, with lipopolysaccharide-containing liposomes as a model for outer membranes of gram-negative bacteria.

F12W-magainin 2 preferentially interacted with lipopolysaccharide-containing bilayers, permeabilizing the membranes, compared with lipopolysaccharide-free phosphatidylcholine vesicles. Using this system, we demonstrated for the first time that the magainin peptide forms a helix upon binding to lipopolysaccharide. Incorporation of lipid A into phosphatidylcholine liposomes also enhanced interactions with the peptide. The presence of Mg2+, which nullifies the peptide's antibacterial activity against gram-negative bacteria, again weakened the interactions between the peptide and lipopolysaccharide-doped bilayers. This system seems to be useful for investigating the molecular details of peptide-lipopolysaccharide interactions.

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function. METHODS: An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 micrograms/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of L-arginine to L-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded. RESULTS: Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6 +/- 5.1-fold increases (n = 6, p < 0.05) in the iNOS mRNA signals within 6 h. The iNOS activity (+300%, P < 0.05) as well as the intracellular cGMP contents (+75%, P < 0.01) were significantly augmented in the lipopolysaccharide-stimulated cells. Both the cell contraction and [Ca2+]i were significantly reduced after the administration of a large amount (10 mM) of L-arginine in the myocytes pretreated with both lipopolysaccharide and NG-monomethyl-L-arginine (100 microM). CONCLUSION: As like as the nucleotide and amino acid sequences, the myocardial effects of the iNOS may also be evolutionary conserved.

Relationship of membrane curvature to the formation of pores by magainin 2.

Magainin 2, an antimicrobial peptide from the Xenopus skin, kills bacteria by permeabilizing the cell membranes. We have proposed that the peptide preferentially interacts with acidic phospholipids to form a peptide-lipid supramolecular complex pore, which allows mutually coupled transbilayer traffic of ions, lipids, and peptides, thus simultaneously dissipating transmembrane potential and lipid asymmetry [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In this paper, we examined the effect of membrane curvature strain on pore formation. Magainin effectively forms the pore only in phosphatidylglycerol bilayers at low peptide-to-lipid ratios, well below 1/100. In contrast, the permeabilization of phosphatidylserine, phosphatidic acid, or cardiolipin bilayers occurred at much higher peptide-to-lipid ratios (1/50 to 1/10) with some morphological change of the vesicles. The latter three classes of phospholipids are known to form hexagonal II structures under conditions of reduced interlipid electrostatic repulsions. Incorporation of phosphatidylethanolamine also inhibited the magainin-induced pore formation in the inhibitory order of dioleoylphosphatidylethanolamine > dielaidoylphosphatidylethanolamine. Addition of a small amount of palmitoyllysophosphatidylcholine enhanced the peptide-induced permeabilization of phosphatidylglycerol bilayers. Magainin greatly raised the bilayer to hexagonal II phase transition temperature of dipalmitoleoylphosphatidylethanolamine. These results suggest that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.

Chronic total occlusion of the left main coronary artery.

We report 3 patients with chronic total occlusion of the left main coronary artery, which is considered to be very rare. In all three cases, coronary arteriograms showed a total occlusion of the left main coronary artery with good collaterals from the intact right coronary arteries. All of the patients underwent successful coronary artery bypass surgery; two of the cases were followed up for more than 10 years after the surgery. The Japanese literature is reviewed, and a comparison of foreign and Japanese cases is discussed.

Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria.

Magainin peptides, isolated from Xenopus skin, have broad spectra of antimicrobial activity and low toxicities to normal eukaryotic cells, thus being good candidates for therapeutic agents. The mechanism of action is considered to be the permeabilization of bacterial membranes. A number of studies using lipid vesicles have elucidated its molecular detail. However, their interactions with bacteria are not yet well understood. In this paper, we synthesized several magainin analogs with different charges (0 to +6) and hydrophobicities, and systematically studied their interactions with the outer and inner membranes of three species of Gram-negative bacteria (Escherichia coli, Acinetobacter calcoaceticus, Proteus vulgaris). The treatment of the E. coli cells with native magainin 2 (+4) immediately induced the efflux of the intracellular K+ ions and the cell death. A number of blebs were formed on the bacterial surface and the outer membrane became leaky. An increase in the peptide's positive charge enhanced the outer membrane permeabilization and the bactericidal activity. The cationic peptides also effectively permeabilized the inner membranes rich in acidic phospholipids, indicating the importance of electrostatic interactions. Substitution of Trp for Phe simultaneously increased the bactericidal activity and the hemolytic activity. A strategy to develop potent antimicrobial peptides was discussed on the basis of these results.

Modulation of magainin 2-lipid bilayer interactions by peptide charge.

Magainin 2, an antimicrobial peptide from Xenopus skin, assumes an amphiphilic helix when bound to acidic phospholipids, forming a pore composed of a dynamic, peptide-lipid supramolecular complex [Matsuzaki et al. (1996) Biochemistry 35, 11361-11368]. Upon the disintegration of the pore, a fraction of the peptide molecules stochastically translocates across the bilayer (Matsuzaki, et al., 1995). In order to investigate the effects of peptide charge on the magainin 2-lipid bilayer interactions, we synthesized four magainin 2 analogs with different charges (0-6+). MG0: K10E, K11E, F12W-magainin 2. MG2+: K10E, F12W-magainin 2. MG4+: F12W-magainin 2. MG6+: F12W, E19Q-magainin 2 amide. An increase in charge resulted in a stronger binding of the peptide to the negatively charged membranes, suggesting that electrostatic attractions play a crucial role in the binding process. The helical stability in a trifluoroethanol/buffer mixture was decreased with increasing positive charge because of electrostatic repulsions between the closely spaced positive side chains, whereas the helicity in the lipid bilayer was much higher and appeared to be independent of the peptide charge. However, enhanced repulsions between the highly positively charged helices destabilized the pore. Therefore, the efficiency of the most basic peptide (MG6+) to translocate across the bilayer was the greatest by virtue of the short life span of its pore and the very tight membrane binding. The charge distribution of wild-type magainin 2 was found to be so designed as to exhibit the maximal lytic activity by simultaneously achieving a strong binding and a moderate pore stability.

Selectivity of felodipine for depolarized ventricular myocytes: a study at the single-cell level.

We studied the effects of felodipine (a second-generation dihydropyridine Ca2+ channel blocker) on excitation-contraction coupling (E-C coupling) in single isolated guinea-pig ventricular myocytes, using the whole-cell perforated patch-clamp technique or the Ca indicator, indo-1. Felodipine inhibited both L-type Ca2+ channel currents (ICa) and cell contractions in a concentration-dependent manner (10 pM to 100 nM) when we used a holding potential of -80 mV or -40 mV. The potency of felodipine was sharply dependent on a holding potential. Namely, use of a more depolarized holding potential markedly increased the potency of felodipine for inhibition of ICa and cell contraction. Next we current-clamped cells and obtained the resting membrane potential of -82 +/- 8 mV. When cells were current-injected at 0.1 Hz, exposure to 10 nM felodipine slightly but significantly diminished the amplitude of cell contractions (7.2 +/- 1.6 to 6.7 +/- 1.7 microns, P < 0.05) within 10 min. When cells were field stimulated, exposure of cells to 10 nM felodipine also slightly diminished the amplitude of cell shortening (5.1 +/- 2.0 to 4.6 +/- 1.9 microns, P < 0.05) and [Ca2+]i transients. We observed clear voltage-dependent blockade of E-C coupling by felodipine in ventricular myocytes. Thus, therapeutic concentrations (1-10 nM) of felodipine could inhibit E-C coupling in depolarized ventricular myocytes, which might simulate an ischemic or failing heart.

Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2.

Magainin peptides, isolated from Xenopus skin, kill bacteria by permeabilizing their cell membranes whereas they do not lyse erythrocytes. To elucidate the rationale for this membrane selectivity, we compared the effects of the membrane lipid composition and the transmembrane potential on the membrane-lytic power of magainin 2 with that of hemolytic melittin. The activity of magainin to zwitterionic phospholipids constituting the erythrocyte surface was extremely weak compared with that of melittin, and acidic phospholipids are necessary for effective action. The presence of sterols reduced the susceptibility of the membrane to magainin. The generation of an inside-negative transmembrane potential enhanced magainin-induced hemolysis. We can conclude that the absence of any acidic phospholipids on the outer monolayer and the abundant presence of cholesterol, combined with the lack of the transmembrane potential, contribute to the protection of erythrocytes from magainin's attack.

Malignant hypercalcemia due to gastric endocrine cell carcinoma.

A 62-year-old man was admitted to our Neurology Unit due to consciousness disturbance. Laboratory data showed marked hypercalcemia and azotemia. Serum parathyroid hormone-related protein (PTHrP) level was extremely high. We performed intensive hemodialysis for renal failure, but his condition deteriorated rapidly. On day 10, he died of multiple organ failure. The autopsy revealed gastric undifferentiated adenocarcinoma with systemic dissemination. Immunohistological study showed positive PTHrP staining in carcinoid-like parts of the tumor. This is the first reported case of malignant hypercalcemia due to PTHrP-producing carcinoid or endocrine cell carcinoma of the stomach.