Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis

Cytosolic ferritin sequesters and stores iron and, consequently, protects cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses mouse MtFt under the control of a tetracycline-responsive promoter. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) along with an increase in transferrin receptor levels and decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase in cellular iron uptake from transferrin, most of which was incorporated into MtFt. The induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. In addition, iron inserted into MtFt was less available for chelation than that in cytosolic ferritin and the expression of MtFt was associated with decreased mitochondrial and cytosolic aconitase activities, the latter being consistent with the increase in IRP-binding activity. In conclusion, our results indicate that overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that shunting iron to MtFt likely limits its availability for active iron proteins.     

Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis

Amyloid β peptide (Aβ) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Aβ induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Aβ and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Aβ decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Aβ may be relevant to the chronic neurodegeneration observed in AD.     

Lactate alters plasma membrane potential, increases the concentration of cytosolic Ca2+ and stimulates the secretion of insulin by the hamster beta-cell line HIT-T15

We have studied the effect of lactate on a number of intracellular events which may be important in controlling the secretion of insulin by the hamster beta-cell line HIT-T15. Using the fluorescent dye Oxonol V, as well as intracellular recording techniques to measure changes in membrane potential, we found that lactate, glucose, K+ and tolbutamide caused depolarization of HIT cells, while valinomycin resulted in hyperpolarization. Consistent with these findings was the observation that 10 mM lactate caused an increase of 69.0 +/- 18.4% (S.E.M., n = 6) in the level of free cytosolic Ca2+ within HIT cells (assessed by fluorescence of quin 2). This was probably due to influx of Ca2+ through voltage-sensitive calcium channels, since it was dependent upon the concentration of extracellular Ca2+ and inhibited by verapamil. Lactate also caused cytosolic acidification in HIT cells and increased the secretion of insulin. These findings are consistent with the view that the electrogenic efflux of lactate could be a determinant in the activation of HIT cells by lactate and possibly by glucose.     

pH homeostasis in pituitary GH4C1 cells: basal intracellular pH is regulated by cytosolic free Ca2+ concentration.

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pHi). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pHi from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P less than 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pHi by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pHi. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pHi to 6.75 +/- 0.03 (P less than 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pHi; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pHi markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pHi through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pHi in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.     

Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering

Residues important in the interaction between the 23-residue transmembrane (TM) domains of the integrin alpha(IIb)- and beta(3)-subunits were identified by mutating each non-Leu residue to Leu. Leu substitutions of alpha(IIb) at G972, G976, and T981, and of beta(3) at I693 and G708, increased ligand binding. Substitutions with other amino acids at alpha(IIb)G972 and beta(3)G708 could also increase ligand binding. The results are consistent with and extend the helical interface between the integrin alpha- and beta-subunit TM domains previously defined by cysteine scanning and disulfide bond formation. We differentiated between affinity- and valency-based modes of activation by TM domain mutations. The mutant alpha(IIb) W967C forms disulfide-linked alpha(IIb)-subunits within an (alpha(IIb)beta(3))(2) tetramer. This tetramer behaved as an ideal model for the valency mode of regulation, because it exhibited significantly increased binding to multivalent but not monovalent ligands and basally retained the bent conformation. By contrast, the activating Leu mutants showed increased binding to the monovalent, ligand-mimetic PAC-1 Fab and increased exposure of ligand-induced binding site (LIBS) epitopes, suggesting that they partially adopt an extended conformation. Furthermore, the previously described beta(3)G708N mutation in Chinese hamster ovary cells enhanced ligand binding affinity, not valency, and did not alter cell-surface clustering as defined by confocal microscopy. Our studies provide evidence that disrupting the integrin heterodimeric TM helix-helix interface activates ligand binding mainly by increasing the monomeric affinity for ligand, but not the receptor valency, i.e., clustering.     

Intracellular pattern of cytosolic Ca2+ changes during adhesion and multiple phagocytosis in human neutrophils. Dynamics of intracellular Ca2+ stores

The subcellular pattern of cytosolic free Ca2+ ([Ca2+]i) changes in human polymorphonuclear neutrophils (PMNs) was studied using imaging of fura-2 fluorescence (time resolution 12.5 ratios/s) to determine whether PMNs could obtain directional information from the [Ca2+]i signal. [Ca2+]i changes were observed during initial adherence, the subsequent chemotactic movement, and the phagocytosis of opsonized yeast particles. Initial adherence was followed by a rapid increase in [Ca2+]i (from 90 +/- 10 to 290 +/- 40 nmol/L in 6.5 +/- 2.5 seconds; +/- SEM, n = 10), apparently homogeneously distributed over the entire cytoplasm, which preceded the spreading of the PMNs. [Ca2+]i increases after the contact of the PMNs with yeast particles were of lower mean amplitude; [Ca2+]i increased simultaneously throughout the cytosol. In the absence of extracellular Ca2+, multiple phagocytotic events could proceed normally without a mandatory [Ca2+]i transient. In PMNs polarized on phagocytosis, gradients in [Ca2+]i could be observed. [Ca2+]i was more elevated in the periphagosomal area than in the remaining parts. Taken together, these data show that [Ca2+]i waves do not provide the neutrophil with directional information during chemotaxis and phagocytosis. Sustained small inhomogeneity of [Ca2+]i levels are consistent with a proposed redistribution of releasable Ca2+ stores on phagocytosis.     

Phot1 and phot2 mediate blue light-induced transient increases in cytosolic Ca2+ differently in Arabidopsis leaves

Phototropins (phot1 and phot2) are blue light (BL) receptors that mediate phototropism, chloroplast movements, and stomatal opening in Arabidopsis thaliana. Physiological studies have suggested that Ca2+ in the cytoplasm plays a pivotal role in these BL-induced responses. A phot1-mediated increase in cytosolic Ca2+ was reported in deetiolated seedlings of A. thaliana; however, the contribution of phot2 remains unknown. We examined a BL-induced transient increase in cytosolic free Ca2+ in leaves of transgenic A. thaliana of WT plants, phot1 and phot2 mutants, and phot1 phot2 double mutants expressing the Ca2+-sensitive luminescent protein aequorin. phot1 and phot2 had different photosensitivities: phot1 increased cytosolic Ca2+ at lower fluence rates (0.1-50 micromol x m-2 x s-1) and phot2 increased it at higher fluence rates (1-250 micromol x m-2 x s-1). By using Ca2+ channel blockers, Ca2+ chelating agents, and inhibitors of phospholipase C, we further demonstrated that both phot1 and phot2 could induce Ca2+ influx from the apoplast through the Ca2+ channel in the plasma membrane, whereas phot2 alone induced phospholipase C-mediated phosphoinositide signaling, which might result in Ca2+ release from internal Ca2+ stores. These results suggest that phot1 and phot2 mediate the BL-induced increase in cytosolic free Ca2+ differently.     

Low-calcium diet increases blood pressure and alters peripheral but not central angiotensin II binding sites in rats

The mechanism by which low-calcium (Ca) diet causes hypertension is unknown. We investigated angiotensin II (Ang II) receptor binding in brain, adrenals and urinary bladders in male Sprague-Dawley rats pair-fed a low-Ca (0.005% Ca; 0.5% P) and normal-Ca (1.4% Ca) diet for 8 weeks beginning at 4 weeks of age. The Ang II receptor sites in hypothalamus-thalamus-septum (HTS), adrenal glands and urinary bladder smooth muscle were measured by saturation isotherm binding using 125I-sarcosine1isoleucine8 Ang II (125I-SI Ang II). Systolic blood pressure was determined at 2-week intervals by tail-cuff method. Serum total Ca, Na+, K+ aldosterone and Ang II and bone density and mineral content were determined at the time of sacrifice. Chronic Ca deficiency in rats raised blood pressure and decreased Ang II receptor density in bladder smooth muscles and tended to increase adrenal Ang II receptors. Serum Ca. bone density and mineral content were significantly lower in the Ca-deficient rats, while serum Na+ was elevated in this group. Serum Ang II and aldosterone were unaltered after the 8-week dietary regimen. Possible mechanisms for the hypertensive actions of reduced dietary Ca intake involving the renin-angiotensin-aldosterone system are discussed.     

Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay.     

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.     

Acute hypoxia activates store-operated Ca2+ entry and increases intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle cells

Rationale

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side.

Objectives

To investigate if acute hypoxia caused the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and Ca²⁺ influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs).

Methods

Fluorescent microscopy and fura-2 were used to measure effects of 4% O₂ on [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) in isolated rat distal PVSMCs.

Measurements and main results

In PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca²⁺ stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca²⁺ entry through L-type voltage-depended Ca²⁺ channels (VDCC), hypoxia markedly enhanced both the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence. Moreover, the increased [Ca²⁺]_i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl₂ at concentrations that SOCE >85% was inhibited but [Ca²⁺]_i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca²⁺]_i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca²⁺]_i caused by hypoxia was completely abolished by perfusion with Ca²⁺-free KRBS.

Conclusions

These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca²⁺]_i in distal PVSMCs.KEYWORDS : Calcium signaling, pulmonary venous smooth muscle (PVSM), store-operated Ca²⁺ entry (SOCE), intracellular Ca²⁺ concentration ([Ca²⁺]_i)     

Altered ubiquitin causes perturbed calcium homeostasis,hyperactivation of calpain,dysregulated differentiation,and cataract

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca²⁺ elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca²⁺ elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.Many age-related diseases such as cataracts, macular degeneration, Alzheimer’s, Parkinson’s, Huntington’s, and several premature aging syndromes, appear to be causally associated with accumulation of abnormal proteins (1, 2). The accumulation of damaged proteins in many age-related diseases involves a vicious cycle of stress-induced postsynthetic modifications to bulk and catalytically critical molecules and limited capacity to remove the damaged proteins, thus accelerating accumulation of damaged proteins and protein precipitation (1–3). Clarity is essential for lens function. Age-related cataract is due to the aggregation and precipitation of proteins from the normally clear milieu and is the leading cause of adult blindness worldwide, affecting more than 18 million people (4). Congenital cataracts also involve protein precipitation (5).The lens is an excellent system to study specific relationships between proteolytic pathways, stress, and maintenance of protein quality because all of the cells are retained throughout life. The oldest lens tissue is found at the center or core of the lens. Crystallins, the major gene products of the lens, are very long-lived proteins, with half-lives of decades, and their aberrant synthesis or modification results in aggregation, insolubilization, and cataract (6). Common age-related stresses that confront proteins in the lens and other tissues during aging include oxidation, glycation, and methylation, as well as their sequels (3, 6–9). Effective stress-reducing systems including antioxidants, antioxidant and repair enzymes, and chaperone and proteolytic capacities help limit damage and maintain solubility and function in younger tissues (3).There are three general systems for intracellular proteolysis: lysosomal/autophagic mechanisms, calcium-activated proteases, or calpains, and the ubiquitin proteasome system (UPS). Because nuclei and lysosomes are removed from most of the lens cells in regions where most cataracts form, this leaves only cytoplasmic proteases, including the UPSs and calpains, to remove damaged proteins to retain lens function (3). There are two basic steps to the UPS: conjugation of substrates to multiple ubiquitins (Ubs) followed by degradation of the protein substrate. Ubiquitin is a highly conserved protein with seven lysines (10). The lysines are used, in the first step of the UPS, to form inter-Ub linkages that lead to Ub polymers that are conjugated to protein substrates. Commonly, proteins with K48-linked Ub oligomers attached are scheduled for degradation by the 26S proteasome. The UPS is also involved at multiple critical stages of proliferation, differentiation, and development in most tissues.It was recently noted that only a minute proportion of Ub conjugates use K6 on Ub (11). Thus, we were surprised to find that expression of higher levels of K6W-Ub in the lens produced cataracts (12). Other work showed that Ub mutations in which K6 is replaced by various amino acids, i.e., K6W-Ub, are conjugation competent but proteolytically incompetent. Thus, cells and tissues in which K6W-Ub is expressed accumulate Ub conjugates (13). Exchanging K for A or R has the same effects. Although structural information about Ub conjugates built using K6 is becoming available (14, 15), a complete understanding of features that render such conjugates biologically stable remain to be elucidated.Calpain is up-regulated by increased Ca²⁺. Gap junction proteins, or Connexins (Cxs), are required for maintaining Ca²⁺ homeostasis (16). UPS-dependent degradation of Cx has been observed in CHO and BWEM cells (17). The formation of cataracts in lenses that express K6W-Ub is compatible with novel functional connections between UPS activity, regulation of Cx and Ca²⁺, calpain activity, and lens clarity. To test the hypothesis, we targeted expression of K6W-Ub to the lens, and we monitored stability of multiple proteins, Cx function and ubiquitination, Ca²⁺, calpain activity, protein integrity, solubility, and localization, as well as lens clarity.     

Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP,contractility, and relaxation

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca²⁺-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca²⁺ fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, the extent of shortening was increased ~ 40% and maximal rate of shortening was increased ~ 80% in cardiomyocytes overexpressing Rrm1 + Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~ 150% with Rrm1 + Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1 + Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca²⁺], indicating increased contractility is primarily due to increased myofilament activity without altering Ca²⁺ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1 + Rrm2 overexpression as stimulation frequency was increased (1 Hz and 2 Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~ 1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca²⁺ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca²⁺. This article is part of a Special issue entitled "Possible Editorial".     

Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca²⁺-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca²⁺ fluorescence were monitored by video microscopy. At 0.5 Hz stimulation, the extent of shortening was increased ~ 40% and maximal rate of shortening was increased ~ 80% in cardiomyocytes overexpressing Rrm1 + Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~ 150% with Rrm1 + Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1 + Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca²⁺], indicating increased contractility is primarily due to increased myofilament activity without altering Ca²⁺ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1 + Rrm2 overexpression as stimulation frequency was increased (1 Hz and 2 Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~ 1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca²⁺ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca²⁺. This article is part of a Special issue entitled "Possible Editorial".     

Cellular responses to Ca2+ from extracellular and intracellular sources are different as shown by simultaneous measurements of cytosolic Ca2+ and secretion from bovine chromaffin cells.

Bovine adrenal medullary cells, cultured on quartz plates, were superfused with buffer to which pulses of stimulant were added. Cytosolic Ca2+ was measured by the fura-2 fluorescence method and the simultaneously released catecholamine was measured electrochemically. When stimulant concentrations were adjusted to given equivalent elevations of cytosolic Ca2+, secretion depended entirely on whether Ca2+ came from internal stores or from the extracellular medium. Calcium from internal stores did not support secretion under these conditions. This nonequivalence of the two sources of cytosolic Ca2+ points to important differences in the physiological roles of the two sources of calcium. Dimethylphenylpiperazinium (a cholinergic agonist) and elevated K+ increased cytosolic Ca2+ and caused secretion only in the presence of external Ca2+. Bradykinin, muscarine, and ATP elevated cytosolic Ca2+ in the presence and absence of extracellular Ca2+ but caused secretion only in the presence of extracellular Ca2+. UTP, which in the absence of extracellular Ca2+ elevated cytosolic Ca2+ as effectively as ATP, did not cause detectable secretion under any circumstance. Because of the high Ca2+-buffering capacity of the cytosol, we expected that Ca2+ gradients, perhaps quite steep, would be produced by a pulse of Ca2+ entering the cytosol. Fura-2 fluorescence measures only the average free cytosolic Ca2+. Our data show that Ca2+ entering across the plasma membrane was much more effective at triggering exocytosis than was Ca2+ released from internal stores, suggesting that the two sources of Ca2+ are effectively compartmentalized, probably by concentration gradients in the cytosol.     

Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells

The relations between membrane cholesterol content, basal (unstimulated) transmembrane 45Ca2+ movements, cytosolic calcium levels, and membrane fluidity were investigated in cultured rabbit aortic smooth muscle cells (SMCs) and isolated SMC plasma membrane microsomes. SMCs were enriched with unesterified (free) cholesterol (FC) for 18-24 hours with medium containing human low density lipoprotein and FC-rich phospholipid (PL) liposomes. This procedure increased cholesterol mass without affecting PL mass, resulting in an increase in the FC/PL molar ratio compared with controls in cells (67% FC increase, p less than 0.001; 43% FC/PL ratio increase, p less than 0.01) and in SMC microsomes (52% FC increase, p less than 0.05; 43% FC/PL ratio increase, p less than 0.05). Cholesterol enrichment also increased unstimulated 45Ca2+ influx (p less than 0.001) and efflux (p less than 0.05). Cellular cholesterol content correlated in a linear fashion with these changes (influx: r = 0.722, p less than 0.01; efflux: r = 0.951, p less than 0.05). In addition, cytosolic calcium levels increased approximately 34% (p less than 0.01) with cholesterol enrichment. The cholesterol-induced increase in 45Ca2+ influx was reversible with time and demonstrated sensitivity to the channel blockers. Fluorescence anisotropy measured from 5 degrees C to 40 degrees C using the fluorophore diphenylhexatriene showed decreased membrane fluidity in microsomal membranes obtained from cholesterol-enriched SMCs compared with controls (p less than 0.02). These results suggest that the SMC plasma membrane is very sensitive to cholesterol enrichment with liposomes or human low density lipoprotein and that increases in membrane cholesterol content increase cytosolic calcium levels in SMCs, are associated with a decrease in membrane fluidity, and unmask a new, or otherwise silent, dihydropyridine-sensitive calcium channel that may be involved in altered arterial wall properties with serum hypercholesterolemia.     

Changes in cytosolic 3,5,3'-tri-iodo-L-thyronine (T3) binding activity during administration of L-thyroxine to thyroidectomized rats: cytosolic T3-binding protein and its activator act as intracellular regulators for nuclear T3 binding

Changes in the amount of cytosolic 3,5,3'-tri-iodo-L-thyronine (T3)-binding protein (CTBP) and its activator during administration of L-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor. These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor.     

Polymorphonuclear integrins, membrane fluidity, and cytosolic Ca(2+) content after activation in essential hypertension

The purpose of this research was to obtain further information about the role of polymorphonuclear leukocytes in essential hypertension. These cells could be involved in the pathogenesis of organ injury. Thirty subjects (14 men and 16 women) with essential hypertension were enrolled. In these subjects we determined, at baseline and after in vitro activation with 4-phorbol 12-myristate 13-acetate and N:-formyl-methionyl-leucyl-phenylalanine, the polymorphonuclear leukocyte membrane fluidity, obtained by labeling the cells with 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3, 5-hexatriene, cytosolic Ca(2+) concentration, obtained by marking the cells with Fura 2-AM, and integrin pattern (CD11a, CD11b, CD11c, and CD18), by using the indirect immunofluorescence with a flow cytometer. At baseline there was no difference in membrane fluidity between normal subjects and hypertensives, whereas hypertensives showed an increase in cytosolic Ca(2+) content and an increase of the phenotypical expression of CD11a, CD11b, and CD18. In normal subjects and in hypertensives, after activation, no variation was found in membrane fluidity and cytosolic Ca(2+) content. In normal subjects, after activation, we observed a significant increase of the expression of all adhesion molecules, whereas in hypertensives we found an increase of the expression of CD11b, CD11c, and CD18 but also a decrease of CD11a. The behavior of the polymorphonuclear leukocyte integrin profile may have several explanations, and in particular, the trend of CD11a after chemotactic activation may be related to its cleavage or to an altered integrin phosphorylation/dephosphorylation balance hypothetically present in this clinical condition.     

Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1

Activated protein C (APC) exerts endothelial protein C receptor (EPCR)-dependent neuroprotective effects in a brain focal ischemia model and direct cellular effects on human umbilical vein endothelial cells (HUVECs) via protease-activated receptor-1 (PAR-1). Because PAR receptors are expressed in brain endothelium and mediate intracellular calcium concentration ([Ca2+]i) signaling, we hypothesized that APC may regulate intracellular [Ca2+] flux in human brain endothelial cells (BECs) via EPCR and PAR-1. Primary cortical BECs derived from human autopsies (early passage) and HUVECs were used for [Ca2+]i imaging fluorometry. Cells were exposed for 1 minute to APC, protein C zymogen, or mutant Ser360Ala-APC, and [Ca2+]i was monitored in the presence or absence of antibodies against PAR-1, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced dose-dependent [Ca2+]i release in human BECs (Delta[Ca2+]i max = 278.3 +/- 19.5 nM; EC50 for APC = 0.23 +/- 0.02 nM, n = 70 measurements). APC-induced [Ca2+]i signaling was abolished by a cleavage site blocking anti-PAR-1 antibody, whereas anti-PAR-2 and -PAR-3 antibodies were without effect. Antibody RCR252 that ablates APC binding to EPCR blocked APC-mediated [Ca2+]i signaling, whereas anti-EPCR antibody RCR92 that does not block APC binding did not abolish the APC-induced [Ca2+]i response. Experiments using HUVECs confirmed the findings for BECs. Thapsigargin inhibited the APC-induced [Ca2+]i signal, implicating the endoplasmic reticulum as a major source for the APC-induced [Ca2+]i release. These data suggest that APC regulates [Ca2+]i in human brain endothelium and in HUVECs by binding to EPCR and signaling via PAR-1.