1-alpha-calcidol modulates major human monocyte antigens and toll-like receptors TLR 2 and TLR4 in vitro.

Since vitamin D derivatives are known to interfere with the cellular immune response, we analysed the possible effect of 1-alpha-calcidol (AC) on major monocyte antigens CD14 (an endotoxin receptor), HLA-DR, and toll-like recptors 2 and 4 (TLR2, TLR4). Peripheral blood monocytes were isolated from healthy donors and cultured by standard protocol followed by incubation with various concentrations of AC in unstimulated and LPS-activated cells. After 24, 48 and 72 hours cells were harvested and analysed for the expression of antigens by flowcytometry. Compared to the controls AC increased the expression of CD14 in a dose and time dependent manner (after 72 hours culture time p < 0.01). AC was capable of further stimulating CD14 expression in LPS activated monocytes (p < 0.05). Both LPS and AC downmodulated HLA-DR dramatically after 24 (p < 0.05), 48 (p < 0.01) and 72 hours (p < 0.0001). The expression of TLR2 but not of TLR4 was inhibited by 10-7M AC. The data reveal that AC significantly modulates the expression of CD14, HLA-DR as well as of TLR2, all involved as targets and effector molecules in antigen recognition and processing, relevant to overcome infections and organ lesions.

Mutations that Affect Agrobacterium vitis-Induced Grape Necrosis also Alter Its Ability to Cause a Hypersensitive Response on Tobacco.

ABSTRACT Tn5-induced mutations in Agrobacterium vitis F2/5 resulted in both altered grape necrosis and tobacco leaf panel collapse phenotypes, suggesting that the underlying mechanisms of the reactions are related. The reaction on tobacco resembles the classical hypersensitive response (HR) caused by several plant pathogenic bacteria in that it is observable within 14 h, is inhibited by treatment of plants with metabolic inhibitors, and results in the inability to recover the pathogen from the necrotic zone. Strains of A. vitis differ with regard to their efficiency of causing the reaction on tobacco. An EcoRI fragment from one mutant, M6, which is necrosis-altered and HR-minus, was cloned and sequenced. Sequence analysis revealed that the Tn5 insertion occurred in a region that shares significant homology with genes involved in long chain fatty acid production by the marine bacteria Shewanella spp. and Moritella marina. Complementation of M6 with a cosmid clone from an F2/5 DNA library restored the tobacco HR and grape necrosis phenotypes.

Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging.

OBJECTIVE: The observation of local 'elementary' Ca2+ release events (Ca2+ sparks) through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) has changed our understanding of excitation-contraction (EC) coupling in cardiac and smooth muscle. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation by activating co-localized Ca2+ activated K+ (K(Ca)) channels (STOCs). However, all prior studies on Ca2+ sparks have been performed in non-human tissues. METHODS: In order to understand the possible significance of Ca2+ sparks to human cardiovascular function, we used high spatial resolution confocal imaging to record Ca2+ sparks in freshly-isolated, individual myocytes of human coronary arteries loaded with the Ca2+ indicator fluo-3. RESULTS: Local SR Ca2+ release events recorded in human myocytes were similar to 'Ca2- sparks' recorded previously from non-human smooth muscle cells. In human myocytes, the peak [Ca2+]i amplitudes of Ca2+ sparks (measured as F/F0) and width at half-maximal amplitude were 2.3 and 2.27 microm, respectively. The duration of Ca2+ sparks was 62 ms. Ca2+ sparks were completely inhibited by ryanodine (10 micromol/l). Ryanodine-sensitive STOCs could be identified with typical properties of K(Ca) channels activated by Ca2+ sparks. CONCLUSION: Our data implies that modern concepts suggesting an essential role of Ca2+ spark generation in EC coupling recently derived from non-human muscle are applicable to human cardiovascular tissue. Although the basic properties of Ca2+ sparks are similar, our results demonstrate that Ca2+ sparks in coronary arteries in humans, have features distinct from non-arterial smooth muscle cells of other species.

Structural insights into phosphoinositide 3-kinase catalysis and signalling.

Phosphoinositide 3-kinases (PI3Ks) are ubiquitous lipid kinases that function both as signal transducers downstream of cell-surface receptors and in constitutive intracellular membrane and protein trafficking pathways. All PI3Ks are dual-specificity enzymes with a lipid kinase activity which phosphorylates phosphoinositides at the 3-hydroxyl, and a protein kinase activity. The products of PI3K-catalysed reactions, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), PtdIns(3,4)P2 and PtdIns(3)P, are second messengers in a variety of signal transduction pathways, including those essential to cell proliferation, adhesion, survival, cytoskeletal rearrangement and vesicle trafficking. Here we report the 2.2 A X-ray crystallographic structure of the catalytic subunit of PI3Kgamma, the class I enzyme that is activated by heterotrimeric G-protein betagamma subunits and Ras. PI3Kgamma has a modular organization centred around a helical-domain spine, with C2 and catalytic domains positioned to interact with phospholipid membranes, and a Ras-binding domain placed against the catalytic domain where it could drive allosteric activation of the enzyme.

Farnesol blocks the L-type Ca2+ channel by targeting the alpha 1C subunit.

We recently demonstrated that farnesol, a 15-carbon isoprenoid, blocks L-type Ca2+ channels in vascular smooth muscle cells. To elucidate farnesol's mechanism of action, we performed whole-cell and perforated-patch clamp experiments in rat aortic A7r5 cells and in Chinese hamster ovary (CHO) C9 cells expressing smooth muscle Ca2+ channel alpha 1C subunits. Farnesol dose-dependently and voltage-independently inhibited Ba2+ currents in both A7r5 and CHOC9 cells, with similar half-maximal inhibitions at 2.6 and 4.3 micromol/L, [corrected] respectively (P=NS). In both cell lines, current inhibition by farnesol was prominent over the whole voltage range without changes in the current-voltage relationship peaks. Neither intracellular infusion of the stable GDP analogue guanosine-5'-O-(2-thiodiphosphate) (100 micromol/L) [corrected] via the patch pipette nor strong conditioning membrane depolarization prevented the inhibitory effect of farnesol, which indicates G protein-independent inhibition of Ca2+ channels. In an analysis of the steady-state inactivation curve for voltage dependence, farnesol induced a significant, negative shift ( approximately 10 mV) of the potential causing 50% channel inactivation in both cell lines (P<0. 001). In contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unlike pharmacological Ca2+ channel blockers, farnesol blocked Ca2+ currents in the resting state: initial block was 63+/-8% in A7r5 cells and 50+/-9% in CHOC9 cells at a holding potential of -80 mV. We then gave 500 mg/kg body weight farnesol by gavage to Sabra hypertensive and normotensive rats and found that farnesol reduced blood pressure significantly in the hypertensive strain for at least 48 hours. We conclude that farnesol may represent an endogenous smooth muscle L-type Ca2+ channel antagonist. Because farnesol is active in cells expressing only the pore-forming alpha1 subunit, the data further suggest that this subunit represents the molecular target for farnesol binding and principal action. Finally, farnesol has a blood pressure-lowering action that may be relevant in vivo.

Hydrogen peroxide, potassium currents, and membrane potential in human endothelial cells.

BACKGROUND: Hydrogen peroxide (H2O2) and reactive oxygen species are implicated in inflammation, ischemia-reperfusion injury, and atherosclerosis. The role of ion channels has not been previously explored. METHODS AND RESULTS: K+ currents and membrane potential were recorded in endothelial cells by voltage- and current-clamp techniques. H2O2 elicited both hyperpolarization and depolarization of the membrane potential in a concentration-dependent manner. Low H2O2 concentrations (0.01 to 0.25 micromol/L) inhibited the inward-rectifying K+ current (KIR). Whole-cell K+ current analysis revealed that H2O2 (1 mmol/L) applied to the bath solution increased the Ca2+-dependent K+ current (KCa) amplitude. H2O2 increased KCa current in outside-out patches in a Ca2+-free solution. When catalase (5000 micro/mL) was added to the bath solution, the outward-rectifying K+ current amplitude was restored. In contrast, superoxide dismutase (1000 u/mL) had only a small effect on the H2O2-induced K+ current changes. Next, we measured whole-cell K+ currents and redox potentials simultaneously with a novel redox potential-sensitive electrode. The H2O2-mediated KCa current increase was accompanied by a whole-cell redox potential decrease. CONCLUSIONS: H2O2 elicited both hyperpolarization and depolarization of the membrane potential through 2 different mechanisms. Low H2O2 concentrations inhibited inward-rectifying K+ currents, whereas higher H2O2 concentrations increased the amplitude of the outward K+ current. We suggest that reactive oxygen species generated locally increases the KCa current amplitude, whereas low H2O2 concentrations inhibit KIR via intracellular messengers.

L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells.

Despite intensive interest in understanding the differentiation of vascular smooth muscle cells (VSMC), no information is available about differential regulation of ion channels in these cells. Since expression of the L-type Ca2+ channel can be influenced by differentiation in other cell types, we tested the hypothesis that the L-type (C class) channel is a specific differentiation marker of VSMC and that expression of these channels depends on the state of cell differentiation. We used rat aortic (A7r5) VSMC, which express functional L-type Ca2+ channels, and induced dedifferentiation by cell culture in different media. Treatment with retinoic acid was used to redifferentiate the VSMC. We characterized the differentiated state of the cells by using immunohistochemistry and Western blot analysis for smooth muscle (SM) alpha-actin and SM-myosin heavy chain (MHC). The number of functional Ca2+ channels was significantly decreased in dedifferentiated VSMC and increased upon differentiation with retinoic acid. Ca2+ channel function was assessed by whole-cell voltage clamp techniques. Using Western blot and dihydropyridine binding analysis, we found that the expression of the Ca2+ channel alpha1 subunit, and to a lesser extent the beta2 subunit, was directly correlated with the expression of SM alpha-actin and SM-MHC. We conclude that expression of L-type Ca2+ channel alpha1 subunits, and thus a functional Ca2+ channel, is highly coordinated with expression of the SM-specific proteins required for specialized smooth muscle cell functions. Furthermore, our results demonstrate that the L-type Ca2+ channel is a novel marker for differentiation of VSMC. The data suggest that regulation of ion channel expression during differentiation may have physiological importance for normal smooth muscle function and may influence VSMC behavior under pathophysiological conditions.

Calcium-activated potassium channels and nitrate-induced vasodilation in human coronary arteries.

In some but not all arterial beds, smooth muscle cell calcium-activated K+ channels (KCa channels) play a central role in the mediation of the vasodilator response to nitric oxide (NO) and other nitrates. We investigated the effect of nitrates on KCa channels in the relaxation of human coronary arteries by means of isometric contraction experiments in arterial rings. We also measured whole-cell currents in freshly isolated human coronary artery vascular smooth muscle cells via the patch-clamp technique. Sodium nitroprusside, diethylamine-nitric oxide complex sodium salt and isosorbide mononitratre completely relaxed rings preconstricted with 5 microM serotonin and produced dose-dependent relaxations of 5 microM serotonin-preconstricted human rings. The relaxations were inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-oxyl 3-oxide (10 microM), which neutralizes nitric oxide. The KCa channel blockers iberiotoxin (100 nM) and tetraethylammonium ions (1 mM) significantly inhibited SNP-induced relaxations of human coronary arteries. Moreover, in the patch-clamp experiments, SNP (1 microM) stimulated KCa currents and spontaneous transient outward K+ currents carried by Ca spark activated KCa channels. The SNP-induced (1 microM) KCa current was strongly inhibited by iberiotoxin (100 nM). These data show that activation of KCa channels in smooth muscle cells contributes to the vasodilating actions of nitrates and nitric oxide in human coronary arteries. This finding may have unique clinical significance for the development of antianginal and antihypertensive drugs that selectively target K+ channels and Ca sparks.

Effects of pinacidil on K+ channels in human coronary artery vascular smooth muscle cells.

We investigated pinacidil-activated K+ currents in vascular smooth muscle cells (VSMC) from human coronary arteries with the patch-clamp method. In 19 of 54 VSMC, pinacidil (1 and 20 microM) induced a large, nonrectifying, outward current [IK(ATP)] and increased voltage-dependent outward K+ currents [IK(Ca)] positive to voltages of -25 mV. The pinacidil-induced (1 microM) IK(ATP) was blocked by glibenclamide (3 microM) but was not affected by iberiotoxin (100-300 nM). Pinacidil activated up to 150 functionally active ATP-dependent K+ channels (KATP channels) per cell with a single-channel conductance of approximately 17 pS at physiological membrane potentials (between -80 and -30 mV) and K+ gradients (6 mM/130 mM). In 26 of 54 VSMC, on the other hand, pinacidil (1-20 microM) failed to induce IK(ATP) but increased IK(Ca). This current was completely blocked by iberiotoxin (100-300 nM) and tetraethylammonium (1 mM) but not by glibenclamide (3 microM). The single-channel conductance of the channel underlying IK(Ca) was approximately 150 +/- 16 pS between -10 and +30 mV, consistent with large-conductance, maxi Ca(2+)-activated, K+ channels (BKCa channels). We conclude that pinacidil is a nonselective K+ channel opener targeting KATP and BKCa channels. Furthermore, the conductance of KATP channels in human coronary arteries is likely to be small under physiological conditions.

Regulation of spontaneous transient outward potassium currents in human coronary arteries.

BACKGROUND: Spontaneous transient outward potassium currents (STOCs) induce myogenic relaxation in small cerebral vessels. We found STOCs in human coronary artery vascular smooth muscle cells (VSMCs) and studied their regulation. METHODS AND RESULTS: K+ currents were recorded in human coronary VSMCs by current- and voltage-clamp techniques. STOCs were recorded in the presence of 200 mumol/L Cd2+ and 10 mumol/L verapamil, which block voltage-dependent Ca2+ channels. STOCs were inhibited by iberiotoxin (100 nmol/L), a selective blocker of Ca(2+)-activated potassium channels (BKCa), and disappeared in a Ca(2+)-free bath. Iberiotoxin depolarized the VSMCs within 20 minutes from -44 +/- 7 to -18 +/- 5 mV (n = 17). The Ca2+ ionophore A23187 increased intracellular Ca2+ and stimulated whole-cell BKCa current. Depletion of Ca2+ from the sarcoplasmic reticulum with caffeine (4 mmol/L) abolished STOCs for several minutes. Ryanodine (50 mumol/L) transiently stimulated STOCs but then completely inhibited STOCs within 10 minutes. The firing frequency of STOCs was directly correlated with intracellular Na+ concentrations from 0 to 24 mmol/L. Lowering intracellular Na+ to zero abolished STOCs. We next gave monensin (30 mumol/L) to increase intracellular Na+. This maneuver resulted in an increase in whole-cell current fluctuations and STOCs. Monensin-induced STOCs were abolished by either lowering extracellular Ca2+ to zero or chelating Ca2+ intracellularly with BAPTA-AM (30 mumol/L). CONCLUSIONS: STOCs resulted from BKCa activity and were dependent on extracellular Ca2+ but not significantly on voltage-dependent Ca2+ channels. STOCs were dependent on intracellular Na+ and intracellular calcium store refilling state. We suggest that Ca2+ entry into the cell through reverse-mode Na+/Ca2+ exchange determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.

Pituitary adenylate-cyclase-activating peptides relax human coronary arteries by activating K(ATP) and K(Ca) channels in smooth muscle cells.

Pituitary adenylate-cyclase-activating peptides (PACAPs) are potent dilators of arteries, including human coronary arteries. We tested the importance of specific K+ channel regulatory mechanisms in human arterial smooth muscle relaxation induced by PACAPs, using contraction and patch clamp measurements on human coronary artery vascular smooth muscle cells. PACAP27 and PACAP38 produced dose-dependent relaxations of 5 microM PGF2alpha-preconstricted rings, with half-maximal relaxations at 1.0 nM and 2.0 nM, respectively. Both peptides induced complete relaxation at 100 nM. Pretreatment of the vessels with the ATP-dependent K+ (K(ATP)) channel blocker glibenclamide (1 microM) or with the Ca2+-activated K+ (K(Ca)) channel blocker iberiotoxin (100 nM) inhibited PACAP27-induced relaxation in an additive manner. Moreover, in the patch clamp experiments on freshly isolated cells from human coronary arteries, PACAP27 (100 nM) induced a large, nonrectifying, outward (I(K)(ATP)) K+ current in a proportion of cells and a voltage-dependent outward (I(K)(Ca)) K+ current in other cells. The PACAP27-induced I(K)(ATP) was blocked by glibenclamide (3 microM), while the PACAP27-stimulated I(K)(Ca) was blocked by iberiotoxin (100 nM). These findings provide the first evidence that relaxation of arterial smooth muscle cells by PACAPs is mediated by opening of K(ATP) and K(Ca) channels. The data indicate that both K(ATP) and K(Ca) channels in vascular smooth muscle cells may serve as final common pathway to induce vasorelaxation by endogenous vasoactive signals in man.

High affinity endotoxin-binding and neutralizing peptides based on the crystal structure of recombinant Limulus anti-lipopolysaccharide factor.

Lipid A, the conserved portion of endotoxin or lipopolysaccharide, is the major mediator of septic shock, and therefore endotoxin-neutralizing molecules could have important clinical applications. The crystal structure of recombinant Limulus anti-lipopolysaccharide factor (rLALF) (Hoess, A., Watson, S., Siber, G. R., and Liddington, R. (1993) EMBO J. 12, 3351-3356), has been used to design synthetic peptides comprising different parts of the exposed amphipathic loop in the proposed endotoxin-binding domain of rLALF. We investigated the minimal requirements of rLALF for endotoxin and lipid A binding with linear 10-mer peptides. Only one linear peptide, corresponding to amino acids 36-45 of rLALF, was able to bind lipid A and endotoxin above background levels. Cyclic peptides, however, bind lipid A and endotoxin with high affinity, presumably by mimicking the three dimensional characteristics of the exposed hairpin loop. The cyclic peptide including amino acids 36-47, LALF-14, has a lipid A binding activity comparable to the high affinity endotoxin-binding peptide polymyxin B. LALF-14 has an improved serum half-life compared with its linear counterpart, and it is not toxic for cultured human monocytes or red blood cells. In mice, it blocks tumor necrosis factor-alpha induction after endotoxin challenge. The characterization of the minimal endotoxin-binding domain of rLALF and, importantly, its structure provided a basis for designing small molecules that could have prophylactic and/or therapeutic properties in humans for the management of septic shock.

High permeation of L-type Ca2+ channels at physiological [Ca2+]: homogeneity and dependence on the alpha 1-subunit.

Molecular cloning has identified multiple isoforms of dihydropyridine-sensitive C-class L-type Ca2+ channels. We tested the hypotheses that L-type (C-class) channels exhibit homogeneous high permeation properties at physiological Ca2+ concentrations and membrane potentials. We measured unitary currents through single dihydropyridine-sensitive omega-conotoxin-insensitive endocrine and smooth muscle L-type Ca2+ channels in rat pituitary GH3 and rat aortic A7r5 cell lines. We also measured unitary currents through smooth muscle (Cb) Ca2+ channel alpha 1-subunits in Chinese hamster ovary (CHO) cells. Our results show that single channel conductances of all three L-type (C-class) channels are uniform with high Ba2+ concentrations, e.g., approximately 23 pS with 110 mM Ba2+. The single channel conductances were reduced to similar values when the Ba2+ concentration was lowered to near-physiological values: 11.1, 9.3, and 8.4 pS in GH3, A7r5, and CHO cells at 2 mM Ba2+, respectively. The single channel conductances were not significantly different with near-physiological Ca2+ concentrations: 5.5, 5.9, and 4.9 pS in GH3, A7r5, and CHO cells at 2 mM Ca2+, respectively. The data suggest that L-type (C-class) channels are homogeneous in terms of Ca2+ permeation at physiological charge carrier concentrations and membrane potentials. Furthermore, the data indicate that the relatively high Ca2+ permeation under physiological conditions is determined by the intrinsic properties of the pore-forming Ca2+ channel alpha 1-subunit.

K+ currents in human coronary artery vascular smooth muscle cells.

K+ channels and their currents are important in vascular tone regulation and are potential therapeutic targets; however, K+ channels in human coronary artery vascular smooth muscle cells (VSMCs) have received little attention. We examined K+ currents in freshly isolated VSMCs from human coronary arteries (n=368 from 32 human hearts) with conventional patch-clamp or perforated-patch techniques with nystatin. We detected four different K+ currents: (1) the delayed rectifier K+ current, IK(dr); (2) the Ca2+-activated K+ current, IK(Ca); (3) the nonrectifying noninactivating outward ATP-dependent K+ current, IK(ATP); and (4) the spontaneous transient outward K+ current, IK(STOC). K+ channels underlying spontaneous transient outward currents probably represent a single clustered population of Ca2+-activated K+ channels functionally associated with Ca2+ release channels in the sarcoplasmic reticulum. Inwardly rectifying K+ currents were not observed. K+ currents were unevenly distributed in that they were not uniformly exhibited by all cells. The most prominent K+ currents were IK(Ca) (100%) and IK(dr) (46%). IK(STOC)s, which have not been previously described in humans, were present in 67% of VSMCs. IK(ATP) was small under physiological conditions; however, IK(ATP) increased markedly after cell stimulation with exogenous or endogenous coronary vasodilators. Thus, IK(ATP) may be particularly relevant in ischemia and could be of special importance as a therapeutic target. We conclude that human coronary VSMCs have unique K+ currents that differ sufficiently from those of other species, thus making the investigation of human material clinically relevant. The findings suggest potential avenues for further therapeutic research.

Radioprotective effects of a protein-free hemodialysate in human epidermis.

The accidental or therapeutic exposure of human skin to ionizing radiation is known to cause the radiation syndrome with its various manifestations. The aim of the study was to investigate the potential radioprotective effects of the protein-free hemodialysate Actovegin. After exposure to X-rays (single dose, 6 Gy), 70% of the cells died. In the presence of the hemodialysate, irradiation did not lead to cell death. Instead a slight increase in cell number was observed. A 5-fold increased cell number was found after 6 days when the cells were treated with the hemodialysate alone. To elucidate molecular mechanisms of the observed biological effects the correlation between the expression of the epidermal growth factor receptor (EGFR) and the demonstrated growth activation was investigated. Radiation alone resulted in a clear induction of EGFR, whereas the combination of irradiation and Actovegin treatment led to a strong downregulation after 2 days. Thus, the hemodialysate suppressed one of the radiation-induced effects. Further investigations have to elucidate the role of other proteins which are involved in the signal transduction cascade of tyrosine kinases (e.g. Ras, Raf, MAP kinases) leading to the transcription factor AP-1 in response to radiation under Actovegin treatment.

Pinacidil relaxes porcine and human coronary arteries by activating ATP-dependent potassium channels in smooth muscle cells.

We investigated the effect of the potassium channel opener pinacidil on ATP-dependent K+ channels (KATP) in the relaxation of porcine and human coronary arteries by means of isometric contraction experiments in arterial rings. We also measured whole cell currents in freshly isolated porcine and human coronary artery vascular smooth muscle cells with patch clamp. We first characterized serotonin-induced precontractions in our vessels and proved that the contractions were mediated by Ca2+ influx through voltage-dependent Ca2+ channels. Similarly, we observed that serotonin-induced contractions were strongly enhanced by small K(+)-induced depolarizations. Pinacidil completely relaxed rings preconstricted with 5 microM serotonin and produced dose-dependent relaxations of 5 microM serotonin-preconstricted rings, with an IC50 of 1.26 microM. Similar results were observed (IC50 = 1.15 microM) when the endothelium was removed. The KATP blocker glibenclamide (3 microM), inhibited pinacidil-induced relaxations (5-10 microM) by approximately 25% although the KATP blocker tetrapentylammonium (10 microM), inhibited pinacidil-induced (5-10 microM) relaxations completely. Pinacidil 10 microM had only a minimal effect on rings precontracted with a 50 mM external K+ concentration (IC50 = 60 microM). Porcine and human arterial rings did not differ qualitatively in their responses. Moreover, in the patch clamp experiments pinacidil (1 microM and 20 microM) induced a large, nonrectifying, outward current in both human and porcine cells. The reversal potential was close to the K+ equilibrium potential, suggesting an induction of pinacidil-activated K+ current. The pinacidil-induced (1 microM) current was strongly inhibited by glibenclamide (3 microM). These data show that the relaxation of porcine and human coronary arteries by pinacidil is primarily induced by an opening of KATP in smooth muscle cells. Furthermore, the vasorelaxant effect of pinacidil is not endothelium dependent.

Do band 3 protein conformational changes mediate shape changes of human erythrocytes?

The bilayer-couple model predicts a reversible membrane crenation for an increasing ratio of external to internal monolayer area. This was comprehensively proven. However, individual erythrocytes may undergo dramatic shape changes within seconds when the suspension medium is changed. In contrast, under physiological conditions with no addition of membrane active compounds, active phospholipid translocation and passive flip-flops are comparatively slow. We propose that conformational changes of the anion-exchange protein, band 3, may rapidly alter the monolayer area ratio. Band 3 occupies about 10% of the total membrane area of human erythrocytes. Under physiological conditions, its conformers are asymmetrically distributed with about 90% of the transport sites facing the cytoplasm. This distribution is altered when external conformations are recruited by changing the transmembranous Cl- gradient, the external pH, or by the application of inhibitors. In experiments, recruitment by low ionic strength caused a rapid, temporary formation of echinocytes. This suspension effect could also be found at high ionic concentrations, when Cl- was replaced by SO4(2-). Inhibitors known to recruit the external band 3 conformation, like DIDS, SITS and flufenamic acid, are echinocytogenic. For inhibitors not recruiting a certain conformation, e.g. phenylglyoxal and niflumic acid, no shape effect was found. Since band 3 ensures a fast equilibrium of internal and external anions these ions are usually distributed according to the transmembrane potential (TMP). In the literature, a correlation of TMP and band 3 conformation, as well as a correlation of TMP and red cell shape, is described. In the proposed model, low external Cl- concentrations, inhibitors, or a negative TMP may recruit the transport sit outwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel steroid derivative modulates gene expression of cytokines and growth regulators.

Topical steroid treatment is a common therapy for psoriasis. Steroids are known to bind to specific cytoplasmic receptors and to influence gene expression. We investigated the effects of the novel steroid derivative mometasone furoate on the expression of putative target genes in normal human epidermal cells (KC). Gene expression was measured by semiquantitative mRNA-PCR. In addition, cytokine receptor characteristics were assessed by ligand binding studies. We found a dose-dependent downregulation of proinflammatory mediators (IL-8, TNF alpha). Genes involved in growth regulation (HER-2, p53) were also modulated. IL-8 binding to KC was inhibited. We conclude that modulation of the expression of cytokine, cytokine receptor and growth factor genes may contribute to the antipsoriatic action of steroids.

Increased expression of the epidermal growth factor receptor in human epidermal keratinocytes after exposure to ionizing radiation.

The effect of exposure of human epidermal keratinocytes to ionizing radiation, both in vivo and in vitro, on the expression of the epidermal growth factor receptor (EGF-R) was studied on the protein, mRNA, and functional levels. Quantitative fluorometry of short-term organ cultures incubated with a monoclonal antibody against human EGF-R revealed a dose-dependent increase of EGF-R expression 24 h after irradiation with 4 and 6 Gy, with an additional increase after 48 h. In biopsy specimens from patients undergoing radiation therapy a markedly increased expression could be determined by quantitative fluorometry during radiation therapy which wa still considerably above the baseline level 4 weeks after termination of treatment. Radioligand binding assays demonstrated a 50% increase in 125I-EGF binding to primary keratinocytes and A431 cells, at doses of 1 Gy, with a further increase after 72 and 96 h. Northern blots were performed with total RNA from two human epidermal cell lines (SCLII and A431). In A431 cells, increased EGF-R transcript levels could be detected 48 h after irradiation. In cells of the SCLII cell line, EGF-R expression was not affected by irradiation. These results were confirmed by semiquantitative polymerase chain reaction of primary cultured keratinocytes, demonstrating an increase of transcripts of EGF-R 24 h after irradiation with single doses of 6 Gy. Thus exposure to ionizing radiation leads to an increased expression of functionally intact EGF-R in human keratinocytes, at the protein and mRNA levels, both in vitro and in vivo; we hypothesize that this effect is part of a stress program of epidermal cells in response to ionizing radiation, ensuring rapid repopulation of irradiated areas.

Interleukin-8 receptor-mediated chemotaxis of normal human epidermal cells.

Normal human keratinocytes show chemotactic behavior towards interleukin-8 (IL-8). Under physiological conditions this cytokine seems to be present in an equilibrium between monomeric and dimeric forms, as indicated by Western blotting data. Radioligand binding studies suggest that keratinocyte chemotaxis is mediated by receptors specific for IL-8 dimers. IL-8 receptor-specific mRNA can be detected in a keratinocyte cell line by polymerase chain reaction.