Calcium uptake by subcellular fractions of human umbilical artery.

Two different mechanisms for the active accumulation of Ca2+ by subcellular fractions of human umbilical artery are described. One, located in the mitochondrial fraction, was induced by exogenous ATP or respiratory substrates (ADP and succinate) and was inhibited by azide. The other, located in the microsomal fraction, was induced by ATP and potentiated by oxalate, but not inhibited by azide. Increasing ATP concentrations up to 4-5 mM increased microsomal Ca2+ accumulation, whereas increasing ATP concentration above 2-3 mM caused inhibition of mitochondrial Ca2+ uptake. Although changing pH from 7.4 to 7.2 had no effect on mitochondrial Ca2+ accumulation, it doubled microsomal uptake. Neither adenosine 3',5'-monophosphate nor guanosine 3',5'-monophosphate in the presence or absence of protein kinase and kinase modulator affected Ca2+ uptake by or phosphorylation of the subcellular fractions. Partially purified protein kinases from umbilical and beef skeletal muscle contained a component(s) distinguishable from the kinase on the basis of its heat stability that enhanced ATP-induced Ca2+ uptake by mitochondrial fractions from the umbilical artery. It is suggested that alterations in Ca2+ sequestration induced by changes in ATP concentration and intracellular pH in mitochondrial and microsomal fractions, respectively, could play a role in the control of arterial patency and closure with changes in PO2.     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

红细胞老化过程中囊泡化作用的探讨

红细胞存活120天,在体内老化的过程中,不断产生囊泡,膜脂及膜蛋白随囊泡丢失,红细胞逐渐形成球形,易破溶而消亡。本文用密度梯度方法将年轻及老化红细胞分离,加Ca~(2+)及钙离子载体(A_(23187))诱导红细胞囊泡化,比较年轻及老化红细胞囊泡化后及释放出的囊泡膜蛋白的变化,观察囊泡化后红细胞的变形性、溶血度及被吞噬细胞吞噬的能力。实验结果发现在Ca~(2+)诱导下年轻红细胞比老化者更易囊泡化;释放的囊泡中主要含区带3蛋白及少量区带4.5及4.9,另外,区带7及Hb明显增多,囊泡化后的红细胞膜有聚集。囊泡化后的红细胞变形性明显降低,易溶血及被吞噬细胞吞噬的量明显增多。     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules.

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoa). cGMP values ( approximately 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/ HCO3- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 microM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+/-]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity.     

Endothelin-1 improves the impaired filterability of red blood cells through the activation of protein kinase C

We previously showed that the deformability of human red blood cells (RBCs) is affected by intracellular signaling pathways by examining the effects of Ca2+ influx and the intracellular cAMP level on mechanically-impaired RBC filterability. In the present study, we investigated whether protein kinase C (PKC) participates in the regulation of RBC deformability by affecting membrane properties. The filterability of mechanically-stressed RBCs showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at 20-40 microM. The PKC activity, as measured in the membrane-rich fraction by an ELISA method using an antibody for the phosphorylated PKC substrate, maximally increased at the extracellular Ca2+ concentration where the filterability showed a marked improvement following the bottom of the V-shaped curve of the impaired filterability. At this Ca2+ concentration, the PKC activator endothelin-1 increased the PKC activity, and a PKC inhibitor (calphostin C) decreased it. Endothelin-1 improved and calphostin C worsened the impaired filterability. A specific type-B endothelin receptor agonist (IRL 1620) also improved the impaired filterability. A Western blot analysis revealed the presence of endothelin receptors in the RBC membrane. These results indicate that PKC improves the impaired filterability and that RBCs are the target of endothelin-1.     

Effects of subacute toluene exposure on protein phosphorylation levels in rat frontoparietal and striatal membranes

Effects of subacute toluene exposure (80 p.p.m. toluene in air, 5 + 4 days, 6 h day-1) were analysed on calcium (Ca2+)- and cyclic adenosine monophosphate (cAMP)-induced protein phosphorylation levels in membrane preparations from the frontoparietal cortex and the striatum of the adult male rat. After protein separation by gel electrophoresis, the amount of radioactive phosphate incorporated from adenosine 5'-[gamma-32P] triphosphate, tetra-(ethylammonium) salt ([32P]ATP) was measured indirectly by autoradiography. The 21 most phosphorylated protein bands were then analysed by computerized image analysis. In the frontoparietal cortex no protein bands were significantly affected after cAMP-induced back phosphorylation, while after Ca2+ stimulation there was a decreased incorporation of [32P]ATP in a 22,000 protein band. In the striatum there was a reduced incorporation of [32P]ATP in a 26,000 protein band after cAMP-induced back phosphorylation, and in four bands of 20,000, 21,000, 52,000 and 134,000, respectively, after Ca2+ stimulation. The reduced incorporation of [32P]ATP in these proteins indicated increased original phosphorylation levels after toluene exposure. A comparison between the frontoparietal cortex and the striatum showed a selective vulnerability of phosphorylation processes in striatal membrane protein bands. In conclusion, toluene exposure at low doses augments membrane protein phosphorylation levels in the rat forebrain and especially in the striatum, probably leading to changes in information handling and/or metabolic changes.     

Calcium channel activation facilitated by nitric oxide in retinal ganglion cells

We investigated the modulation of voltage-gated Ca channels by nitric oxide (NO) in isolated salamander retinal ganglion cells with the goals of determining the type of Ca channel affected and the signaling pathway by which modulation might occur. The NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 1 mM) and S-nitroso-cysteine (1 mM) induced modest increases in the amplitude of Ca channel currents recorded with ruptured- and permeabilized-patch techniques by causing a subpopulation of the Ca channels to activate at more negative potentials. The Ca channel antagonists omega-conotoxin GVIA and nisoldipine each reduced the Ca channel current partially, but only omega-conotoxin GVIA blocked the enhancement by SNAP. The SNAP-induced increase was blocked by oxadiazolo-quinoxaline (50 microM), suggesting that the NO generated by SNAP acts via a soluble guanylyl cyclase to raise levels of cGMP. The membrane-permeant cGMP analog 8-(4-chlorophenylthio) guanosine cyclic monophosphate also enhanced Ca channel currents and 8-bromo guanosine cyclic monophosphate (1 mM) occluded enhancement by SNAP. Consistent with these results, isobutyl-methyl-xanthine (IBMX, 10 microM), which can raise cGMP levels by inhibiting phosphodiesterase activity, increased Ca channel current by the same amount as SNAP and occluded subsequent enhancement by SNAP. Neither IBMX, the cGMP analogs, nor SNAP itself, led to activation of cGMP-gated channels. N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (2 microM), a broad spectrum inhibitor of protein kinase activity, KT5823 (1 microM), a specific protein kinase G (PKG) inhibitor, and a peptide inhibitor of PKG (200 microM) blocked SNAP enhancement, as did 5'-adenylylimidophosphate (1.5 mM), a nonhydrolyzable ATP analog that prevents protein phosphorylation. A peptide inhibitor of protein kinase A (10 nM) did not block the facilitory effects of SNAP. Okadaic acid (1 microM), a phosphatase inhibitor, had no effect by itself but increased the enhancement of Ca channel current by SNAP. These results suggest that NO modulates retinal ganglion cell N-type Ca channels by facilitating their voltage-dependent activation via a mechanism involving guanylyl cyclase/PKG-dependent phosphorylation. This effect could fine-tune neural integration in ganglion cells or play a role in ganglion cell disease by modulating intracellular calcium signaling.     

Effects of Ca2+on Erythrocyte Membrane Skeleton-Bound Phosphofructokinase,ATP Levels,and Hemolysis

Erythrocyte Ca²⁺overload is known to occur in several different disease states, and to affect the erythrocyte membrane deformability. We show here that an increase in intracellular Ca²⁺concentration in erythrocytes, induced by ionomycin, caused a reduction in ATP levels. Concomitant to the fall in ATP, a marked activation of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme in glycolysis, in the membrane skeleton fraction occurred. The increase in the membrane skeleton-bound PFK activity was most probably mediated by Ca²⁺, as direct addition of Ca²⁺to the membrane skeleton fraction from the erythrocyte induced an enhancement of the bound PFK activity. Time-response curves revealed that erythrocyte hemolysis did not occur during the first 30 min of incubation with ionomycin, when the membrane skeleton-bound PFK was activated. Longer incubation time resulted in solubilization of the membrane skeleton-bound PFK and a concomitant hemolysis of the erythrocytes. These results suggest that the Ca²⁺-induced activation of membrane skeleton-bound PFK, and thereby glycolysis, the sole source of energy in erythrocytes, may be a defense mechanism to surmount the damage induced by high Ca²⁺levels.     

Intracellular Ca2+ thresholds that determine survival or death of energy-deprived cells.

Increase of intracellular ionized or free Ca2+ is thought to play a central role in cell death due to ATP depletion. However, concurrently operative mechanisms of injury that do not require intracellular Ca2+ increases have made it difficult to test this hypothesis or to determine the concentrations at which intracellular Ca2+ becomes lethal. The predominant Ca2+-independent mechanism of injury during ATP depletion involves the loss of cellular glycine. This type of damage can be fully inhibited by adding the amino acid exogenously. Using glycine to suppress Ca2+-independent plasma membrane damage, we have examined the effect of intracellular Ca2+ elevations on cell viability during ATP depletion. Madin-Darby canine kidney (MDCK) cells were depleted of ATP by incubation with a mitochondrial uncoupler in glucose-free medium. Free Ca2+ concentration in the medium was varied between 26 nmol/L and 1.25 mmol/L in the presence of a Ca2+ ionophore. Measurements with the Ca2+ probes fura-2, furaptra, and fura-2FF showed that intracellular Ca2+ was clamped at extracellular levels under these conditions. Cell survival during ATP depletion was indicated by viable cells recovered 24 hours later. The results show that ATP-depleted cells can sustain high levels of intracellular Ca2+ (100 micromol/L) for prolonged periods and remain viable if plasma membrane damage is prevented by glycine. Cell death was observed only when intracellular free Ca2+ was allowed to increase beyond 100 micromol/L, and this was associated with dramatic nuclear alterations: chromatin condensation, loss of nuclear lamins, and breakdown of DNA into large 50- to 150-kb fragments. Our studies demonstrate unexpectedly high resistance of cells to calcium cytotoxicity if glycine that is lost during ATP depletion is restored. In addition, they provide insights into novel mechanisms of nuclear disintegration and DNA damage that are triggered when the high thresholds of intracellular Ca2+ required for cell death are exceeded.     

Endotoxicosis induced by Coxiella burnetii lipopolysaccharide stimulates a ribosomal protein S6 kinase: some properties of the partially purified enzyme.

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis.     

A high affinity Ca2+-dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense

Addition of Ca2+ (0.01-1 mM) to a standard Trypanosoma rhodesiense Mg2+-ATPase assay failed to elicit any increase in activity. However, in the absence of externally added Mg2+ and using calcium-EGTA or calcium-CDTA to precisely maintain free metal ion concentration, it was possible to measure a specific Ca2+-ATPase. Cell fractionation studies revealed this ATPase to be predominantly associated with subcellular particles having an equilibrium density of 1.22 g cm-3 and identified as surface membrane. Using a discontinuous sucrose gradient, a surface membrane enriched (SME) fraction, only slightly contaminated with mitochondria as judged by dichlorophenolindophenol-linked alpha-glycerophosphate dehydrogenase activity, was prepared. The SME fraction exhibited Ca2+-ATPase activity, using 200 nM free Ca2+, of 90 and 21 mU mg-1 protein, respectively, using CDTA and EGTA as buffering ligands. This latter result was most unexpected and indicated that the Ca2+-ATPase, in addition to having no Mg2+ requirement, was inhibited by submicromolar levels of Mg2+. The Ca2+-ATPase was found to have a K0.5 = 128 +/- 22 nM free Ca2+, the response to increasing Ca2+ concentration displaying an extremely high degree of co-operativity (Hill number (nH) = 4.9). The enzyme was found to be highly substrate-specific for ATP with K0.5 = 6.2 +/- 0.61 microM ATP. A Hill plot of the reaction velocity as a function of ATP concentration indicated two substrate binding sites (nH = 1.55). A range of potential modulators of ATPase activity were investigated, with only vanadate (V2O3-8) having any effect: 47% inhibition at 5.0 microM. The Ca2+-ATPase was unaffected by the calmodulin antagonists chlorpromazine (50 microM) and trifluoperazine (50 microM), whilst addition of calmodulin failed to produce any stimulation of activity. It is concluded that the kinetic properties of this ATPase are compatible with a potential role in the regulation of intracellular Ca2+ in bloodstream T. rhodesiense.     

Calcium oscillations in type-1 astrocytes, the effect of a presenilin 1 (PS1) mutation

Stimulation of type-1 astrocytes, by a number of agonists, has been shown to increase cytosolic Ca2+ concentrations in an oscillatory manner. However, it is unknown how these are driven or altered by aging, injury or disease. Therefore, we characterized the signaling properties of rat type-1 astrocytes in monolayer cultures. Ca2+ responses were recorded in astrocytes stimulated with ATP or glutamate. Oscillations were evident in cultures at 3 days in vitro (DIV 3) with a peak percentage (26%) of cells responding by DIV 7. The presence or absence of serum in the culture medium did not influence this percentage. Likewise, long-term culture (DIV 30+) did not increase the oscillating cell numbers. ATP was found to have a more potent effective dose (50 microM) than glutamate (10 mM). Membrane potential was recorded with fluorescent voltage-sensitive dye (membrane potential dye for FLIPR) and was similar regardless of the culture age and intracellular Ca2+ response. This suggests that mechanisms associated with the intracellular release of Ca2+ from endogenous Ca2+ stores, rather than ion fluxes across the plasma membrane, may contribute to the oscillations in the astrocytes. In order to identify a possible pathological significance of this response, we transfected astrocytes with wild-type presenilin (PS1) as well as PS1 harboring a mutation linked to familial Alzheimer's disease (FAD). PS mutation expressing astrocytes oscillated at lower ATP and glutamate concentrations when compared to wild-type PS1 expressing astrocytes. This indicates that PS1 mutation may introduce aberrations in the intracellular Ca2+ mobilization in astrocytes contributing to the accelerated pathogenesis in FAD.     

Nongenomic actions of thyroxine modulate intermediate filament phosphorylation in cerebral cortex of rats

The developmental effects of thyroid hormones (TH) in mammalian brain are mainly mediated by nuclear receptors regulating gene expression. However, there are increasing evidences of nongenomic mechanisms of these hormones associated with kinase- and calcium-activated signaling pathways. In this context, the aim of the present work was to investigate the signaling pathways involved in the mechanism of action of TH on cytoskeletal phosphorylation in cerebral cortex of 15-day-old male rats. Results showed that L-thyroxine (L-T4) increased the intermediate filament (IF) phosphorylation independently of protein synthesis, without altering the total immunocontent of these proteins. Otherwise, neither 3,5,3'-triiodo-L-thyronine (L-T3) nor neurotransmitters (GABA, ATP, L-glutamate or epinephrine) acted on the IF-associated phosphorylation level. We also demonstrated that the mechanisms underlying the L-T4 effect on the cytoskeleton involve membrane initiated actions through Gi protein-coupled receptor. This evidence was reinforced by the inhibition of cyclic adenosine 5'-monophosphate (cAMP) levels. Moreover, we showed the participation of phospholipase C, protein kinase C, mitogen-activated protein kinase, calcium/calmodulin-dependent protein kinase II, intra- and extracellular Ca2+ mediating the effects of L-T4 on the cytoskeleton. Stimulation of 45Ca2+ uptake by L-T4 was also demonstrated. These findings demonstrate that L-T4 has important physiological roles modulating the cytoskeleton of neural cells during development.     

Mitochondrial modulation of Ca2+ -induced Ca2+ -release in rat sensory neurons

Ca2+ -induced Ca2+ -release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.     

Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress

Fluid shear stress activates the endothelial nitric oxide (NO) synthase (eNOS) by a mechanism which does not require an increase in the intracellular concentration of free Ca2+ ([Ca2+]i), and is sensitive to several kinase inhibitors. Although phosphorylation of eNOS has been suggested to regulate enzyme activity, the mechanism of eNOS activation is still unclear. Here we demonstrate that fluid shear stress elicits the phosphorylation of eNOS on tyrosine and serine residues. Inhibition of phosphatidylinositol 3-kinase (PI3K), using wortmannin or a dominant negative mutant of its downstream target, Akt (protein kinase B), prevented the maintained serine phosphorylation and activation of eNOS. Enhancing eNOS phosphorylation by inhibiting serine/threonine phosphatases, increased eNOS activity by approximately twofold, as assessed by the accumulation of intracellular cyclic GMP, without increasing the intracellular concentration of free Ca2+. These data suggest that shear stress activates a pathway involving PI3K and the serine/threonine kinase Akt, which phosphorylates eNOS. This phosphorylation directly increases eNOS activity at resting [Ca2+]i, thus rendering the shear stress-induced activation of eNOS apparently Ca2+-independent.     

Total and free Mg2+ contents in erythrocytes: a simple but still undisclosed cell model.

The concentration of intracellular free Mg2+ ([Mg2+]i) in erythrocytes, measured by means of 31P NMR and using a dissociation constant for MgATP of 38-50 microM, amounted to 0.2 mM [Mg2+]i in the erythrocytes of various species, was not significantly different and was independent of their total Mg2+ content. The more probable value of [Mg2+]i using the more realistic KD of Mg ATP or the null-point method may amount to 0.4 mM [Mg2+]i in erythrocytes is lower than the [Mg2+]i in nucleated mammalian cell types. The lower [Mg2+]i may be caused by a different regulation of Mg2+ influx and Mg2+ efflux by intracellular Mg2+ in erythroblasts. Free and reversibly bound Mg2+ represent a Mg2+ buffer. The main Mg2+-binding substances are ATP and 2,3-bisphosphoglycerate (2,3-BPG). Total Mg2+ content in the erythrocytes of various species is correlated to the concentrations of ATP and 2,3-BPG. The changed Mg2+ level in erythrocytes during deoxygenation, maturation, cold storage, in Mg2+ deficiency and in sickle cell anemia was reviewed.     

Physiological roles of K+ channels in vascular smooth muscle cells

In this review, we present the basic properties, physiological functions, regulation, and pathological alterations of four major classes of K+ channels that have been detected in vascular smooth muscle cells. Voltage-dependent K+ (Kv) channels open upon depolarization of the plasma membrane in vascular smooth muscle cells. The subsequent efflux of K+ through the channels induces repolarization to the resting membrane potential. Changes in the intracellular Ca2+ concentration and membrane depolarization stimulate large-conductance Ca2+-activated K+ (BKCa) channels, which are thought to play an important role in maintaining the membrane potential. ATP-sensitive K+ (K(ATP)) channels underscore the functional bond between cellular metabolism and membrane excitability. The blockade of KATP channel function results in vasoconstriction and depolarization in various types of vascular smooth muscle. Inward rectifier K+ (Kir) channels, which are expressed in smooth muscle of the small-diameter arteries, contribute to the resting membrane potential and basal tone. Kir channel activation has been shown to raise the extracellular K+ concentration to 10-15 mM, resulting in vasodilation. Each of K+ channels listed above is responsive to a number of vasoconstrictors and vasodilators, which act through protein kinase C (PKC) and protein kinase A (PKA), respectively. Impaired Kv, KATP, and Kir channel functions has been linked to a number of pathological conditions, which may lead to vasoconstriction.     

老化红细胞变形能力与膜钙依赖中性蛋白酶活性的关系

研究表明,老化红细胞变形能力明显降低,且其降低与血红蛋白浓度增高及膜弹性降低有关［１］。红细胞膜钙依赖中性蛋白酶（Ｃａｌｐａｉｎ）和它的内源性抑制剂（Ｃａｌｐａｓｔａｔｉｎ）形成红细胞中一个蛋白水解系统,参与红细胞中的信号传导,调节细胞形状、体积和细胞膜通透性,与高血压、细胞老化等生理、病理现象密切相关。Ｃａｌｐａｉｎ可限制性水解红细胞膜骨架蛋白和其它膜内蛋白,导致红细胞损伤［２,３］。而老化红细胞变形能力降低与Ｃａｌｐａｉｎ的关系尚不清楚,为此我们检测了４２例健康人老化及年轻红细胞变形能力、Ｃ…     

Involvement of Calmodulin in Glucagon-Like Peptide 1(7-36) Amide-Induced Inhibition of the ATP-Sensitive K+ Channel in Mouse Pancreatic β-Cells

The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (K(ATP)) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic beta-cells. Membrane potential, single channel and whole-cell currents through the K(ATP) channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic beta-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the K(ATP) channels in mouse pancreatic beta-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane K(ATP) conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 microM) completely reversed the inhibitory actions of GLP-1 on the membrane K(ATP) conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the K(ATP) channel activity by 20 nM GLP-1 and its restoration by 20 microM W-7 or 10 microM calmidazolium at the single channel level. Bath application of 20 microM W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the K(ATP) channel activity accompanied by the initiation of electrical activity in mouse pancreatic beta-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system.