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At least 217 records · Page 12Linked to original sources
PubMed2003

Effects of bicarbonate buffered dialysate on human peritoneal mesothelial cell intracellular calcium homeostasis.

This study compares the biocompatibility of two bicarbonate-based peritoneal dialysis (PD) solutions using the measurement of intracellular free calcium (Ca(i)2+)) as a sensitive parameter of cell function in human peritoneal mesothelial cells (hPMC). Fura-2-loaded hPMC suspensions were exposed to bicarbonate (38 mmol/L) and bicarbonate (25 mmol/L), lactate-buffered PD (15 mmol/L) solutions at pH 7.4 and compared with Krebs-Ringer physiological saline (KRS; pH = 7.4). Resting Ca(i)2+ values and 4br-A23187 (1.0 micro mol/L) induced transients were compared in treatment and control groups. In separate studies, the effect that low saline pH had on Ca(i)(2+) homeostasis was examined. Suspended cells or cells attached to coverslips were bathed in citric acid-phosphate (McIllvaine's) buffered saline (MBS, pH = 7.4). Cells were acidified (pH = 5.3) with citric acid and then challenged with ionophore. Ionophore challenge produced a significantly reduced Ca(i)2+ transient response in cells exposed to the bicarbonate/lactate fluid compared with bicarbonate or KRS. Acidified cell suspensions produced a small monophasic Ca(i)2+ transient rise that was short lived. Gradual recovery of MBS to pH 7.4 produced no changes to Ca(i)2+ homeostasis of cell monolayers. Ionophore treatment produced a biphasic response identical to cells bathed in KRS. This study has demonstrated that short-term exposure to bicarbonate did not alter Ca(i)2+ homeostasis directly, or subsequent modulation of intracellular pH. The MBS system provided a reliable method of modifying the external pH during continuous Ca(i)2+ measurement.

Bicarbonates↗
PubMed2000

Altered intracellular calcium homeostasis in cerebellar granule cells of prion protein-deficient mice.

Previous studies have indicated that recombinant cellular prion protein (PrP(C)), as well as a synthetic peptide of PrP(C), affects intracellular calcium homeostasis. To analyze whether calcium homeostasis in neurons is also affected by a loss of PrP(C), we performed microfluorometric calcium measurements on cultured cerebellar granule cells derived from prion protein-deficient (Prnp(0/0)) mice. The resting concentration of intracellular free calcium [Ca(2+)](i) was found to be slightly, but significantly, reduced in Prnp(0/0) mouse granule cell neurites. Moreover, we observed a highly significant reduction in the [Ca(2+)](i) increase after high potassium depolarization. Pharmacological studies further revealed that the L-type specific blocker nifedipine, which reduces the depolarization-induced [Ca(2+)](i) increase by 66% in wild-type granule cell somas, has no effect on [Ca(2+)](i) in Prnp(0/0) mouse granule cells. Patch-clamp measurements, however, did not reveal a reduced calcium influx through voltage-gated calcium channels in Prnp(0/0) mice. These data clearly indicate that loss of PrP(C) alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage-gated calcium channels.

Animals↗
PubMed2003

Characterization of chronic low-level proteasome inhibition on neural homeostasis.

Increasing evidence suggests that proteasome inhibition plays a causal role in promoting the neurodegeneration and neuron death observed in multiple disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). The ability of severe and acute inhibition of proteasome function to induce neuron death and neuropathology similar to that observed in AD and PD is well documented. However, at present the effects of chronic low-level proteasome inhibition on neural homeostasis has not been elucidated. In order to determine the effects of chronic low-level proteasome inhibition on neural homeostasis, we conducted studies in individual colonies of neural SH-SY5Y cells that were isolated following continual exposure to low concentrations (100 nm) of the proteasome inhibitor MG115. Clonal cell lines appeared morphologically similar to control cultures but exhibited significantly different rates of both proliferation and differentiation. Elevated levels of protein oxidation and protein insolubility were observed in clonal cell lines, with all clonal cell lines being more resistant to neural death induced by serum withdrawal and oxidative stress. Interestingly, clonal cell lines demonstrated evidence for increased macroautophagy, suggesting that chronic low-level proteasome inhibition may cause an excessive activation of the lysosomal system. Taken together, these data indicate that chronic low-level proteasome inhibition has multiple effects on neural homeostasis, and suggests that studying the effects of chronic low-level proteasome inhibition may be useful in understanding the relationship between protein oxidation, protein insolubility, proteasome function, macroautophagy and neural viability in AD and PD.

Autophagy↗
PubMed2000

Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis.

Prior studies have established the requirement for enzymatic hydrolysis of glucosylceramides to ceramide for epidermal barrier homeostasis. In this study, we asked whether sphingomyelin-derived ceramide, resulting from acid-sphingomyelinase activity, is also required for normal barrier function. We showed first, that a subset of Niemann-Pick patients with severe acid-sphingomyelinase deficiency (i.e., <2% residual activity) demonstrate abnormal permeability barrier homeostasis, i.e., delayed recovery kinetics following acute barrier disruption by cellophane tape-stripping. To obtain further mechanistic insights into the potential requirement for sphingomyelin-to-ceramide processing for the barrier, we next studied the role of acid-sphingomyelinase in hairless mouse skin. Murine epidermis contains abundant acid-sphingomyelinase activity (optimal pH 5.1-5.6). Two hours following acute barrier disruption by tape-stripping, acid-sphingomyelinase activity increases 1. 44-fold (p<0.008 versus vehicle-treated controls), an increase that is blocked by a single topical application of the acid-sphingomyelinase inhibitor, palmitoyldihydrosphingosine. Furthermore, both palmitoyldihydrosphingosine and desipramine, a chemically and mechanically unrelated acid-sphingomyelinase inhibitor, significantly delay barrier recovery both 2 and 4 h after acute barrier abrogation. Inhibitor application also causes both an increase in sphingomyelin content, and a reduction of normal extracellular lamellar membrane structures, in the stratum corneum. Both of the inhibitor-induced delays in barrier recovery can be overridden by co-applications of topical ceramide, demonstrating that an alteration of the ceramide-sphingomyelin ratio, rather than sphingomyelin accumulation, is likely responsible for the barrier abnormalities that occur with acid-sphingomyelinase deficiency. These studies demonstrate an important role for enzymatic processing of sphingomyelin-to-ceramide by acid-sphingomyelinase as a mechanism for generating a portion of the stratum corneum ceramides for permeability barrier homeostasis in mammalian skin.

Adolescent↗
PubMed2003

Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities.

Prolonged exposure of human epidermis to excess endogenous or exogenous glucocorticoids can result in well-recognized cutaneous abnormalities. Here, we determined whether short-term glucocorticoid treatment would also display adverse effects, specifically on two key epidermal functions, permeability barrier homeostasis and stratum corneum integrity and cohesion, and the basis for such changes. In humans 3 d of treatment with a potent, commonly employed topical glucocorticoid (clobetasol), applied topically, produced a deterioration in barrier homeostasis, characterized by delayed barrier recovery and abnormal stratum corneum integrity (rate of barrier disruption with tape strippings) and stratum corneum cohesion (microg protein removed per stripping). Short-term systemic and topical glucocorticoid produced similar functional defects in mice, where the basis for these abnormalities was explored further. Both the production and secretion of lamellar bodies were profoundly decreased in topical glucocorticoid-treated mice resulting in decreased extracellular lamellar bilayers. These structural changes, in turn, were attributable to a profound global inhibition of lipid synthesis, demonstrated both in epidermis and in cultured human keratinocytes. The basis for the abnormality in stratum corneum integrity and cohesion was a diminution in the density of corneodesmosomes in the lower stratum corneum. We next performed topical replacement studies to determine whether lipid deficiency accounts for the glucocorticoid-induced functional abnormalities. The abnormalities in both permeability barrier homeostasis and stratum corneum integrity were corrected by topical applications of an equimolar distribution of free fatty acids, cholesterol, and ceramides, indicating that glucocorticoid-induced inhibition of epidermal lipid synthesis accounts for the derangements in both cutaneous barrier function and stratum corneum integrity/cohesion. These studies indicate that even short-term exposure to potent glucocorticosteroids can exert profound negative effects on cutaneous structure and function. Finally, topical replenishment with epidermal physiologic lipids could represent a potential method to reduce the adverse cutaneous effects of both topical glucocorticoid treatment and Cushing's syndrome.

Administration, Topical↗
PubMed2003

pH directly regulates epidermal permeability barrier homeostasis, and stratum corneum integrity/cohesion.

Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased beta-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities.

Animals↗
PubMed2001

Sulfate homeostasis, NaSi-1 cotransporter, and SAT-1 exchanger expression in chronic renal failure in rats.

BACKGROUND: It is known that hypersulfatemia, like hyperphosphatemia, occurs in chronic renal failure (CRF). The aim of this study was to assess the effects of CRF on sulfate homeostasis and on sodium sulfate cotransport (NaSi-1) and sulfate/oxalate-bicarbonate exchanger (Sat-1) expression in the kidney. In addition, sulfate homeostasis was compared with phosphate homeostasis. METHODS: Experimental studies were performed in adult male rats at three and six weeks after 80% subtotal nephrectomy (Nx) or sham-operation (S) (N = 9 per group). Transporter protein and mRNA expressions were measured by Western blot and RNase protection assay (RPA), respectively. Results were quantitated by densitometric scanning (Western) and electronic autoradiography (RPA), and were expressed in densitometric units (DUs; Western) and cpm (RPA). RESULTS: Creatinine clearance was lower in Nx-3 compared with S-3 rats (0.23 vs. 0.51 mL/min/100 g body weight, P < 0.001) and was further impaired in Nx-6 rats (0.15 vs. 0.48, P < 0.001). Sulfatemia was significantly higher in Nx-3 rats (1.08 vs. 0.84 mmol/L, P < 0.05) and further increased in Nx-6 rats (1.42 vs. 0.90 mmol/L, P < 0.01). Fractional sulfate excretion (FESO4) was increased by twofold in Nx-3 and Nx-6 rats compared with corresponding S rats. Phosphatemia did not differ between Nx-3 rats and controls, but was increased in Nx-6 rats (P < 0.01). Total amounts of both NaSi-1 and Sat-1 proteins were significantly decreased in both Nx-3 and Nx-6 rats when compared with controls. However, NaSi-1 protein and mRNA densities did not significantly change in Nx-3 rats, but were significantly increased in Nx-6 rats when compared with controls (4.8 vs. 3.7 DU/microg protein, P < 0.05, and 7.1 vs. 2.8 cpm/microg RNA, P < 0.01, respectively, for protein and mRNA). In contrast to NaSi-1, Sat-1 protein density was significantly decreased both in Nx-3 (2.9 vs. 3.6 DU/microg protein, P < 0.05) and Nx-6 rats (2.4 vs. 3.4 DU/microg protein, P < 0.05), and Sat-1 mRNA density significantly decreased in Nx-6 rats (10.7 vs. 14.7 cpm/microg RNA, P < 0.05). Na-PO4 cotransporter (NaPi-2) protein total abundance and density were decreased at three and six weeks in Nx rats. CONCLUSIONS: These results demonstrate that both NaSi-1 and Sat-1 total protein abundances are decreased in CRF, which may contribute to the increase in fractional sulfate excretion. Strikingly, NaSi-1 density was not decreased in CRF three weeks after Nx, and furthermore, increased six weeks after Nx, in contrast to NaPi-2 density, which was decreased at both times. The significance of this difference remains to be determined, but may explain why hypersulfatemia occurs earlier than hyperphosphatemia in CRF.

Animals↗
PubMed1998

Role of the liver in interorgan homeostasis of glutathione and cyst(e)ine.

The most widely recognized function of reduced glutathione (GSH) is its defense against toxic compounds, whether exogenous, such as electophilic xenobiotics, or endogenous, such as reactive oxygen species, generated during normal oxidative metabolism and/or stress. However another no less significant role of GSH-namely its function as a reservoir and vehicle for packaging and transport of cyst(e)ine-has been receiving increasing attention. Because GSH is relatively more auto-oxidation resistant and stable than cyst(e)ine (CYSH), it serves as the preferred form for storage and transport of the latter especially in the extracellular and relatively much less reduced (than intracellular) milieu, where CYSH oxidizes to cystine (CYSS) rapidly. Over the past two decades, significant work has been going on to delineate the intra- and extrahepatic (interorgan) turnover, transport, and disposal of GSH and define the quantitative role of these processes in interorgan homeostasis of GSH, CYSH, and CYSS. These studies have identified the liver as the central organ of interorgan GSH homeostasis, with sinusoidal GSH efflux as the major determinant of plasma GSH, CYSH, CYSS, and thiol-disulfide status of plasma. This article focuses on the principal components and determinants of interorgan homeostasis of GSH and its breakdown products. It also presents the current state of knowledge under both normal and diseased states.

Animals↗
PubMed2001

In vitro complement activation favoring soluble C5b-9 complex formation alters myocellular sodium homeostasis.

BACKGROUND: Deranged Na(+) homeostasis in skeletal muscle is closely associated with excessive complement activation that is encountered during sepsis. Recent evidence suggests that soluble C5b-9 complexes (SC5b-9), which are readily detected in plasma during sepsis and have long been considered irrelevant nonmembrane binding end products of complement activation, may have numerous biologic effects. The purpose of this study, therefore, was to determine the effects of SC5b-9 on myocellular ion homeostasis and its mechanism(s) of action. METHODS: Hindlimb fast-twitch extensor digitorum longus (EDL) was freshly isolated from rats weighing 50 to 70 g and then incubated at 30 degrees C for 60 minutes in normal Krebs-Henseleit buffer (KHB, pH 7.4) containing 10% zymosan-activated rat serum (10 mg/mL at 37 degrees C for 60 minutes) as a source of SC5b-9. Zymosan particles were removed by centrifugation after activation to exclude any noncomplement direct effects. Heat-inactivated rat serum (56 degrees C for 30 minutes) was used as control. EDL muscle was also incubated with pertussis toxin (1 microg/mL), in Ca(2+)-free KHB, with thapsigargin (0.3 or 3 micromol/L), or with ouabain (0.01, 0.1 or 1 micromol/L) before and/or during incubation with 10% zymosan-activated or heat-inactivated rat serum. Intracellular Na(+) and K(+) contents ([Na(+)](i) or [K(+)](i)) of EDL muscle were determined by using flame photometry after washing in ice-cold Na(+)-free Tris-sucrose buffer. SC5b-9 in zymosan-activated human serum was determined by SC5b-9 enzyme-linked immunoassay. RESULTS: SC5b-9 in zymosan-activated human serum significantly increased by 400% as compared with nonactivated, normal human serum. Zymosan-activated rat serum markedly increased [Na+]i without affecting [K(+)](i) in fast-twitch EDL muscle, which was completely inhibited by pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) with thapsigargin. The addition of ouabain (at micromolar concentrations) increased myocellular [Na(+)](i) and decreased myocellular [K(+)](i) in both the zymosan-activated and the heat-inactivated rat serum groups. The effects of ouabain on myocellular [Na(+)](i) and [K(+)](i) were equivalent in these 2 groups. Zymosan-activated and heat-inactivated rat serum had similar effects on myocellular [K(+)](i) in the presence or absence of pertussis toxin, removal of extracellular Ca(2+) or depletion of intracellular Ca(2+). CONCLUSIONS: Zymosan-activated rat serum (presumed SC5b-9 enriched) selectively alters Na(+) homeostasis in isolated fast-twitch skeletal muscle. The mechanisms for such effects may be linked to G-proteins, Ca(2+) flux and Na(+),K(+)-adenosine triphosphatase pump binding site blockade.

Animals↗
PubMed1983

Effects of hypolipidemic therapy on cholesterol homeostasis in freshly isolated mononuclear cells from patients with heterozygous familial hypercholesterolemia.

Freshly isolated mononuclear leukocytes have been reported to show changes in cholesterol synthesis and high-affinity degradation of low-density lipoproteins (LDL) that parallel those that occur in the liver. To examine whether hypolipidemic therapy in patients with heterozygous familial hypercholesterolemia influences cholesterol homeostasis in their mononuclear cells we assessed the effects of colestipol and nicotinic acid (alone and in combination) on the rates of high-affinity 125I-labeled LDL degradation and on the rates of cholesterol and phosphatidylcholine biosynthesis by freshly isolated cells. Rates of 125I-labeled LDL degradation were lower in mononuclear cells from patients with heterozygous familial hypercholesterolemia on no medication (3.1 ng per 4 X 10(6) cells per 5 hr) than in cells from normal control subjects (6.1 ng per 4 X 10(6) cells per 5 hr) and, in the former patients, the values were not significantly affected by therapy with nicotinic acid. In contrast, freshly isolated mononuclear cells from patients receiving colestipol degraded 125I-labeled LDL at near-normal rates (5.0 ng per 4 X 10(6) cells per 5 hr). The rates of cholesterol synthesis were also higher in mononuclear cells isolated from patients treated with colestipol than in cells from untreated patients or from those receiving nicotinic acid; in contrast the rate of synthesis of phosphatidylcholine did not show any consistent changes. Similar results were obtained in a smaller number of patients studied longitudinally, in which colestipol therapy significantly increased rates of cholesterol synthesis and high-affinity degradation of 125I-labeled LDL by freshly isolated mononuclear cells. We conclude that previously observed changes in cholesterol homeostasis in the liver of patients treated with bile acid sequestrants are paralleled by similar changes in freshly isolated mononuclear cells and that these cells offer an accessible model for further studies on how diet and pharmacologic agents influence cellular cholesterol homeostasis in humans.

Cholesterol↗
PubMed1990

Cytosolic calcium homeostasis in fungi: roles of plasma membrane transport and intracellular sequestration of calcium.

Cytosolic free calcium ([Ca2+]c) has been measured in the mycelial fungus Neurospora crassa with Ca2(+)-selective microelectrodes. The mean value of [Ca2+]c is 92 +/- 15 nM and it is insensitive to external pH values between 5.8 and 8.4. Simultaneous measurement of membrane potential enables the electrochemical potential difference for Ca2+ across the plasma membrane to be estimated as about -60 kJ.mol-1-a value that cannot be sustained either by a simple Ca2(+)-ATPase, or, in alkaline conditions, by straightforward H+/Ca2+ exchange with a stoichiometric ratio of less than 5 H+/Ca2+. We propose that the most likely alternative mechanism of Ca2+ efflux is ATP-driven H+/Ca2+. In accord with this proposal, depletion of the ATP level from 2.5 to 0.5 mM by CN- elicits an increase in [Ca2+]c, but only in alkaline conditions in which the putative H+/Ca2(+)-ATPase would be selectively stalled. The insensitivity of Ca2+ homeostasis to CN- in more acid conditions implies that the Km (ATP) of the transport system is 100 microM or less. The increase in [Ca2+]c in the presence of CN- at pH 8.4 (50 nM.min-1) is compared with 45Ca2+ influx (0.62 mM.min-1) under the same conditions. The proportion of entering Ca2+ remaining free in the cytosol is only 8 x 10(-5), and since the concentration of available chelation sites on Ca2(+)-binding proteins is unlikely to exceed 100 microM, a major role for the fungal vacuole in short-term Ca2+ homeostasis is indicated. This notion is supported by the observation that cytosolic Ca2+ homeostasis is disrupted by a protonophore, which rapidly abolishes the driving force (a transmembrane pH difference) for Ca2+ uptake into fungal vacuoles.

Adenosine Triphosphate↗
PubMed1999

Modulation of mitochondrial Ca(2+) homeostasis by Bcl-2.

We have investigated the role of mitochondrial Ca(2+) (Ca(m)) homeostasis in cell survival. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) preceded activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 led to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik led to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induced a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also led to apoptosis. We suggest that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, our results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival.

Calcium↗
PubMed1999

The Golgi apparatus plays a significant role in the maintenance of Ca2+ homeostasis in the vps33Delta vacuolar biogenesis mutant of Saccharomyces cerevisiae.

The vacuole is the major site of intracellular Ca2+ storage in yeast and functions to maintain cytosolic Ca2+ levels within a narrow physiological range. In this study, we examined how cellular Ca2+ homeostasis is maintained in a vps33Delta vacuolar biogenesis mutant. We found that growth of the vps33Delta strain was sensitive to high or low extracellular Ca2+. This strain could not properly regulate cytosolic Ca2+ levels and was able to retain only a small fraction of its total cellular Ca2+ in a nonexchangeable intracellular pool. Surprisingly, the vps33Delta strain contained more total cellular Ca2+ than the wild type strain. Because most cellular Ca2+ is normally found within the vacuole, this suggested that other intracellular compartments compensated for the reduced capacity to store Ca2+ within the vacuole of this strain. To test this hypothesis, we examined the contribution of the Golgi-localized Ca2+ ATPase Pmr1p in the maintenance of cellular Ca2+ homeostasis. We found that a vps33Delta/pmr1Delta strain was hypersensitive to high extracellular Ca2+. In addition, certain combinations of mutations effecting both vacuolar and Golgi Ca2+ transport resulted in synthetic lethality. These results indicate that the Golgi apparatus plays a significant role in maintaining Ca2+ homeostasis when vacuolar biogenesis is compromised.

Adenosine Triphosphatases↗
PubMed2000

Autocrine action and its underlying mechanism of nitric oxide on intracellular Ca2+ homeostasis in vascular endothelial cells.

The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.

Adenosine Triphosphate↗
PubMed2001

Cholesteryl ester transfer protein biosynthesis and cellular cholesterol homeostasis are tightly interconnected.

Cholesteryl ester transfer protein (CETP) mediates triglyceride and cholesteryl ester (CE) transfer between lipoproteins, and its activity is strongly modulated by dietary cholesterol. To better understand the regulation of CETP synthesis and the relationship between CETP levels and cellular lipid metabolism, we selected the SW872 adipocytic cell line as a model. These cells secrete CETP in a time-dependent manner at levels exceeding those observed for Caco-2 or HepG2 cells. The addition of LDL, 25OH-cholesterol, oleic acid, or acetylated LDL to SW872 cells increased CETP secretion (activity and mass) up to 6-fold. In contrast, CETP production was decreased by almost 60% after treatment with lipoprotein-deficient serum or beta-cyclodextrin. These effects, which were paralleled by changes in CETP mRNA, show that CETP biosynthesis in SW872 cells directly correlates with cellular lipid status. To investigate a possible, reciprocal relationship between CETP expression and cellular lipid homeostasis, CETP biosynthesis in SW872 cells was suppressed with CETP antisense oligonucleotides. Antisense oligonucleotides reduced CETP secretion (activity and mass) by 60% compared with sense-treated cells. When CETP synthesis was suppressed for 24 h, triglyceride synthesis was unchanged, but cholesterol biosynthesis was reduced by 20%, and acetate incorporation into CE increased 31%. After 3 days of suppressed CETP synthesis, acetate incorporation into the CE pool increased 3-fold over control. This mirrored a similar increase in CE mass. The efflux of free cholesterol to HDL was the same in sense and antisense-treated cells; however, HDL-induced CE hydrolysis in antisense-treated cells was diminished 2-fold even though neutral CE hydrolase activity was unchanged. Thus, CETP-compromised SW872 cells display a phenotype characterized by inefficient mobilization of CE stores leading to CE accumulation. These results strongly suggest that CETP expression levels contribute to normal cholesterol homeostasis in adipocytic cells. Overall, these studies demonstrate that lipid homeostasis and CETP expression are tightly coupled.

Biological Transport↗
PubMed2004

Proteasome inhibition alters neural mitochondrial homeostasis and mitochondria turnover.

Inhibition of proteasome activity occurs in normal aging and in a wide variety of neurodegenerative conditions including Alzheimer's disease and Parkinson's disease. Although each of these conditions is also associated with mitochondrial dysfunction potentially mediated by proteasome inhibition, the relationship between proteasome inhibition and the loss of mitochondrial homeostasis in each of these conditions has not been fully elucidated. In this study, we conducted experimentation in order to begin to develop a more complete understanding of the effects proteasome inhibition has on neural mitochondrial homeostasis. Mitochondria within neural SH-SY5Y cells exposed to low level proteasome inhibition possessed similar morphological features and similar rates of electron transport chain activity under basal conditions as compared with untreated neural cultures of equal passage number. Despite such similarities, maximal complex I and complex II activities were dramatically reduced in neural cells subject to proteasome inhibition. Proteasome inhibition also increased mitochondrial reactive oxygen species production, reduced intramitochondrial protein translation, and increased cellular dependence on glycolysis. Finally, whereas proteasome inhibition generated cells that consistently possessed mitochondria located in close proximity to lysosomes with mitochondria present in the cellular debris located within autophagosomes, increased levels of lipofuscin suggest that impairments in mitochondrial turnover may occur following proteasome inhibition. Taken together, these data demonstrate that proteasome inhibition dramatically alters specific aspects of neural mitochondrial homeostasis and alters lysosomal-mediated degradation of mitochondria with both of these alterations potentially contributing to aging and age-related disease in the nervous system.

Animals↗
PubMed2004

A mathematical model of the folate cycle: new insights into folate homeostasis.

A mathematical model is developed for the folate cycle based on standard biochemical kinetics. We use the model to provide new insights into several different mechanisms of folate homeostasis. The model reproduces the known pool sizes of folate substrates and the fluxes through each of the loops of the folate cycle and has the qualitative behavior observed in a variety of experimental studies. Vitamin B(12) deficiency, modeled as a reduction in the V(max) of the methionine synthase reaction, results in a secondary folate deficiency via the accumulation of folate as 5-methyltetrahydrofolate (the "methyl trap"). One form of homeostasis is revealed by the fact that a 100-fold up-regulation of thymidylate synthase and dihydrofolate reductase (known to occur at the G(1)/S transition) dramatically increases pyrimidine production without affecting the other reactions of the folate cycle. The model also predicts that an almost total inhibition of dihydrofolate reductase is required to significantly inhibit the thymidylate synthase reaction, consistent with experimental and clinical studies on the effects of methotrexate. Sensitivity to variation in enzymatic parameters tends to be local in the cycle and inversely proportional to the number of reactions that interconvert two folate substrates. Another form of homeostasis is a consequence of the nonenzymatic binding of folate substrates to folate enzymes. Without folate binding, the velocities of the reactions decrease approximately linearly as total folate is decreased. In the presence of folate binding and allosteric inhibition, the velocities show a remarkable constancy as total folate is decreased.

Allosteric Site↗
PubMed2005

A single PDZ domain protein interacts with the Menkes copper ATPase, ATP7A. A new protein implicated in copper homeostasis.

The homeostatic regulation of essential elements such as copper requires many proteins whose activities are often mediated and tightly coordinated through protein-protein interactions. This regulation ensures that cells receive enough copper without intracellular concentrations reaching toxic levels. To date, only a small number of proteins implicated in copper homeostasis have been identified, and little is known of the protein-protein interactions required for this process. To identify other proteins important for copper homeostasis, while also elucidating the protein-protein interactions that are integral to the process, we have utilized a known copper protein, the copper ATPase ATP7A, as a bait in a yeast two-hybrid screen of a human cDNA library to search for interacting partners. One of the ATP7A-interacting proteins identified is a novel protein with a single PDZ domain. This protein was recently identified to interact with the plasma membrane calcium ATPase b-splice variants. We propose a change in name for this protein from PISP (plasma membrane calcium ATPase-interacting single-PDZ protein) to AIPP1 (ATPase-interacting PDZ protein) and suggest that it represents the protein that interacts with the class I PDZ binding motif identified at the ATP7A C terminus. The interaction in mammalian cells was confirmed and an additional splice variant of AIPP1 was identified. This study represents an essential step forward in identifying the proteins and elucidating the network of protein-protein interactions involved in maintaining copper homeostasis and validates the use of the yeast two-hybrid approach for this purpose.

Adenosine Triphosphatases↗