Robert A. Farley

Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib

A drawback of extensive coxib use for antitumor purposes is the risk of life-threatening side effects that are thought to be a class effect and probably due to the resulting imbalance of eicosanoid levels. 2,5-Dimethyl-celecoxib (DMC) is a close structural analogue of the selective cyclooxygenase-2 inhibitor celecoxib that lacks cyclooxygenase-2–inhibitory function but that nonetheless is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. To further establish the potential usefulness of DMC as an anticancer agent, we compared DMC and various coxibs and nonsteroidal anti-inflammatory drugs with regard to their ability to stimulate the endoplasmic reticulum (ER) stress response (ESR) and subsequent apoptotic cell death. We show that DMC increases intracellular free calcium levels and potently triggers the ESR in various tumor cell lines, as indicated by transient inhibition of protein synthesis, activation of ER stress–associated proteins GRP78/BiP, CHOP/GADD153, and caspase-4, and subsequent tumor cell death. Small interfering RNA–mediated knockdown of the protective chaperone GRP78 further sensitizes tumor cells to killing by DMC, whereas inhibition of caspase-4 prevents drug-induced apoptosis. In comparison, celecoxib less potently replicates these effects of DMC, whereas none of the other tested coxibs (rofecoxib and valdecoxib) or traditional nonsteroidal anti-inflammatory drugs (flurbiprofen, indomethacin, and sulindac) trigger the ESR or cause apoptosis at comparable concentrations. The effects of DMC are not restricted to in vitro conditions, as this drug also generates ER stress in xenografted tumor cells in vivo, concomitant with increased apoptosis and reduced tumor growth. We propose that it might be worthwhile to further evaluate the potential of DMC as a non-coxib alternative to celecoxib for anticancer purposes. [Mol Cancer Ther 2007;6(4):1262–75]

The cardiac sodium pump: structure and function

Abstract Cardiac sodium pumps (Na,K-ATPase) influence cell calcium and contractility by generating the Na+ gradient driving Ca++ extrusion via the Na+/Ca++ exchanger (NCX), and are the receptors for cardiac glycosides such as digitalis which increases cardiac contractility by decreasing the Na+ gradient driving Ca++ extrusion. There are multiple isoforms of the sodium pump expressed in the heart indicating the potential for isoform specific expression patterns, function and regulation.Regarding isoform expression patterns, human heart expresses α1, α2, α3, β1 and a small amount of β2. Within the human heart, α3, β1 and NCX levels are 30 – 50 % lower in atria than ventricles, associated with increased sensitivity to inotropic stimulation. Distribution at the cellular level has been studied in the rat heart where both a1 and a2 are detected in the T-tubules along with NCX.Regarding isoform function, we focussed on human sodium pumps as cardiac glycoside receptors. A study of human sodium pump expressed alone (α1) or in combination (α1 with α2, or α1 with α2 and α3) in their native membranes aimed to determine whether different isoforms had distinct affinities for the cardiac glycoside ouabain by evaluating whether the ouabain binding data were best fit with a single site or two site model. The results indicated that the affinity of these human a subunit isoforms for ouabain is indistinguishable, and that changes in sensitivity to cardiac glycosides during heart failure are likely due to a decrease in the total number of pumps rather than a shift in expression to a more sensitive isoform.Regarding isoform regulation, we hypothesized that a primary decrease in cardiac Na,K-ATPase expression would be associated with a secondary increase in cardiac Na+/Ca++ exchanger expression as a homeostatic mechanism to blunt an increase in cell Ca++ stores (and visa versa with an increase in Na,K-ATPase). Supporting the hypothesis: in a rat model of renovascular hypertension, or after treatment with amiodarone there are 50 % decreases in a2 levels with 35 – 40 % increases in NCX levels in left ventricle, while in the transition from hypo- to hyperthyroid, there are increases in both α1 (2-fold) and α2 (8-fold) with decreases in NCX (0.45-fold). In comparison, in transgenic mice overexpressing NCX, there was no secondary change in Na,K-ATPase α1 or α2 levels indicating that primary changes in NCX do not drive secondary changes in Na,K-ATPase in the heart.This information provides the basis for addressing the significant gaps in our understanding of the physiologic, structural and homeostatic coupling between sodium pump isoforms and Na+/Ca++ exchangers in the heart and how coupling is related to control of cardiac contractility in health and disease.

Significance of Sodium Pump Isoforms in Digitalis Therapy

Despite the long history of use of cardiac glycosides, questions persist relating to the very narrow range of therapeutic v toxic levels of the drug, and the factors, including hypokalemia, that predispose a patient to cardiac glycoside toxicity. The therapeutic receptor for cardiac glycosides is believed to be the alpha subunit of sodium pump, Na,K-ATPase. Three isoforms of this subunit are expressed in the heart, and the levels of cardiac sodium pump expression are depressed in heart failure. Which human sodium pump isoform(s) binds cardiac glycosides in the therapeutic range (1-2 nM for digoxin) in the failing heart has not been determined. Hypokalemia can potentially influence cardiac glycoside sensitivity at multiple levels: (1) it directly increases the affinity of cardiac glycosides for sodium pumps by decreasing competition with K+, (2) it decreases cardiac sodium pump expression which can augment or amplify the effects of decreased pump expression and activity due to heart failure itself and cardiac glycoside inhibition; (3) it decreases the expression of skeletal muscle sodium pumps which will influence the relative tissue and plasma distributions of cardiac glycosides. Establishing the therapeutic v toxic targets of cardiac glycosides will enable investigators to design isoform specific inhibitors that would potentially be specific for the therapeutic receptors and independent of plasma potassium levels.

Subunit requirements for expression of functional sodium pumps in yeast cells.

Na+/K(+)-ATPase from animal cell membranes is known to consist of an alpha-subunit and a beta-subunit. Amino acids within the alpha-subunit have been shown to participate in the catalytic functions of the enzyme and in the binding of cardioactive steroids. Although the function of the beta-subunit is not known, expression of both alpha- and beta-subunits is required for the functional enzyme. A putative third subunit, the gamma-subunit, has been suggested to be a part of the functional Na+/K(+)-ATPase complex, based on experiments showing that both the catalytic alpha-subunit and a small peptide of M(r) = 11,000 can be labeled by a photoreactive ouabain analog. Although the primary structure for the putative gamma-subunit from rat and sheep was recently deduced from cDNA clones, participation of this small protein in the catalytic activity of the Na+/K(+)-ATPase has not been demonstrated. In experiments described here, the heterologous expression of Na+/K(+)-ATPase in yeast cells was used to investigate whether the gamma-subunit is an essential component of the Na+/K(+)-ATPase. Yeast cells do not contain an endogenous Na+/K(+)-ATPase. The alpha- and beta-subunits or the alpha-, beta- and the putative gamma-subunits of Na+/K(+)-ATPase were expressed in the yeast Saccharomyces cerevisiae and ouabain-sensitive ATPase, p-nitrophenylphosphatase, and 86Rb uptake activities were measured either in membranes prepared from transformed yeast cells, or in intact yeast cells. Nontransformed yeast cells or yeast cells transformed with the gamma-subunit alone served as controls. Northern analysis and Western blots demonstrated that yeast cells do not contain an endogenous peptide with significant sequence homology to the putative gamma-subunit. Yeast samples containing only Na+/K(+)-ATPase alpha and beta subunits were capable of ouabain-inhibitable enzymatic activity and 86Rb transport. No gamma-subunit-dependent differences in the measured enzymatic activities or transport properties were detected in the different samples. These observations establish that the alpha beta-subunit complex is the minimum structural unit required for all the ouabain-sensitive reactions of Na+/K(+)-ATPase.

The influence of beta subunit structure on the interaction of Na+/K(+)-ATPase complexes with Na+. A chimeric beta subunit reduces the Na+ dependence of phosphoenzyme formation from ATP.

High-affinity ouabain binding to Na/K-ATPase (sodium- and potassium-transport adenosine triphosphatase (EC 3.6.1.37)) requires phosphorylation of the α subunit of the enzyme either by ATP or by inorganic phosphate. For the native enzyme (α/β1), the ATP-dependent reaction proceeds about 4-fold more slowly in the absence of Na than when saturating concentrations of Na are present. Hybrid pumps were formed from either the α1 or the α3 subunit isoforms of Na/K-ATPase and a chimeric β subunit containing the transmembrane segment of the Na/K-ATPase β1 isoform and the external domain of the gastric H/K-ATPase β subunit (α/NHβ1 complexes). In the absence of Na, these complexes show a rate of ATP-dependent ouabain binding from 75-100% of the rate seen in the presence of Na depending on buffer conditions. Nonhydrolyzable nucleotides or treatment of ATP with apyrase abolishes ouabain binding, demonstrating that ouabain binding to α/NHβ1 complexes requires phosphorylation of the protein. Buffer ions inhibit ouabain binding by α/NHβ1 in the absence of Na rather than promote ouabain binding, indicating that they are not substituting for sodium ions in the phosphorylation reaction. The pH dependence of ATP-dependent ouabain binding in the presence or absence of Na is similar, suggesting that protons are probably not substituting for Na. Hybrid α/NHβ1 pumps also show slightly higher apparent affinities (2-3-fold) for ATP, Na, and ouabain; however, these are not sufficient to account for the increase in ouabain binding in the absence of Na. In contrast to phosphoenzyme formation and ouabain binding by α/NHβ1 complexes in the absence of Na, ATPase activity, measured as release of phosphate from ATP, requires Na. These data suggest that the transition from E1P to E2P during the catalytic cycle does not occur when the sodium binding sites are not occupied. Thus, the chimeric β subunit reduces or eliminates the role of Na in phosphoenzyme formation from ATP, but Na binding or release by the enzyme is still required for ATP hydrolysis and release of phosphate.

Regulation of Na,K-ATPase activity.

The sodium pump Na,K-ATPase, a heterodimer of an alpha catalytic subunit and a beta glycoprotein subunit, is regulated by a wide array of hormonal, autocrine, and paracrine factors. Both short-term acute adjustments of activity and long-term adjustments of sodium pump pool size are important determinants of cellular Na,K-ATPase activity. Phosphorylation and dephosphorylation are implicated in the acute regulation of activity. Although there is not yet any direct demonstration of phosphorylation in vivo, in vitro studies on purified enzyme directly demonstrate that phosphorylation decreases Na,K-ATPase activity. In addition, it is likely that phosphorylation of other proteins regulates sodium pump activity and cellular distribution. In regard to long-term regulation, recent demonstration of differential translatability of alpha and beta mRNAs and differential stability of newly synthesized alpha and beta subunits suggests that beta subunit is synthesized in excess over alpha subunit and that the excess is rapidly degraded. The isoform composition of alpha beta heterodimers has been shown to affect enzymatic properties, and tissue-specific heterodimer patterns are emerging from regulation studies. In regard to Na,K-ATPase and hypertension, there is continued interest in the significance of the uncoupling of dopamine inhibition of proximal tubule Na,K-ATPase activity in hypertensive rat strains. The uncoupling has been shown to be specific to the proximal tubule, which has been shown to express DA1 dopamine receptors, and both receptor and postreceptor defects are implicated. Questions remaining include how activation of dopamine receptors is coupled to decreased sodium transporter expression in the proximal tubule (short- and long-term regulation) in normotensive rats, the precise nature of the defect in hypertension, and whether a similar defect is observed in human hypertensive patients.

Sodium pump isoform expression in heart failure: implication for treatment.

Abstract In the human heart several isoforms of the sodium pump (Na,K-ATPase, the cardiac glycoside receptor) are expressed (α1β1, α2β1, and α3β1). Their expression is regulated in a highly specific manner, so that there are region specific differences in the expression pattern. The isoform expression pattern is also known to be organ specific in many cases (e.g., kidney, skeletal muscle), suggesting isoform specific functions. In human heart, we have demonstrated that the isoform composition of the left ventricle is altered during heart failure in man and postulate a role of Na,K-ATPase isoforms in the compensatory mechanisms of this disease. When Na,K-ATPase isoforms were expressed separately in yeast cells, we found that the affinities of K and ouabain were lower for α2β1 than for α1β1 or α3β1. In addition, α3β1 had a lower turnover rate than α1β1. Similar results were found in a study, where Na,K-ATPase isoforms were expressed in Xenopus oocytes. Thus, there is evidence for specific biochemical properties of the Na,K-ATPase isoforms. In heterozygous knock-out mice, in which either α1 or α2 isoforms were selectively reduced, only the lower expression and activity of α2 led to a hypercontractile response as seen with cardiac glycosides. Therefore in mice, the effect of cardiac glycosides seems to be mediated specifically by α2.In summary, there is a tissue-specific regulation of Na,K-ATPase isoform expression in humans, as well as a highly specific regulation of the isoforms during disease, e.g., heart failure. There is also evidence for specific biochemical properties of different isoforms of the human Na,K-ATPase as well as for a specific functional impact on cardiac contractility in mice. Therefore, the isoforms of human Na,K-ATPase are not exchangeable and targeting specific isoforms by drugs or gene therapy may promise therapeutic benefit in diseases like heart failure or atrial fibrillation.

All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase β-subunit contain oligosaccharide

The beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.

Valine 904, Tyrosine 898, and Cysteine 908 in Na,K-ATPase α Subunits Are Important for Assembly with β Subunits

A 26-amino acid sequence in an extracellular loop of the Na,K-ATPase α subunit between membrane-spanning segments 7 and 8 has been shown to bind to the β subunit of Na,K-ATPase and to promote αβ assembly (Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255–8259) When this 26-amino acid sequence of the rat Na,K-ATPase α3 subunit was replaced by the corresponding sequence of the rat gastric H,K-ATPase α subunit, the chimeric α subunit assembled preferentially with the rat gastric H,K-ATPase β subunit (Wang, S.-G., Eakle, K. A., Levenson, R., and Farley, R. A. (1997)Am. J. Physiol. 272, C923–C930). In the present study, these 26 amino acids (Asn886–Ala911) of rat Na,K-ATPase α3 were replaced by the corresponding amino acids Asn908–Ala933 of rat distal colon H,K-ATPase. Site-directed mutagenesis of the chimeric α subunits and Na,K-ATPase α3 showed that Val904, Tyr898, and Cys908 in the Na,K-ATPase α3 subunit are key residues in αβ subunit interactions. The V904Q mutation in Na,K-ATPase α3 reduced the B max for ouabain binding and the ATPase activity of α3β1 complexes by ∼95%, and Y898R reduced theB max and ATPase activity by ∼60%. The complementary mutations Q904V and R898Y increased the amount of ouabain bound by yeast membranes expressing the chimera with the colon H,K-ATPase sequence. The amount of ouabain bound by complexes assembled between Na,K-ATPase α3 containing the Y898R,C908G mutations and gastric H,K-ATPase β was less than 10% of wild type Na,K-ATPase α3 expressed with the same β subunit. The R898Y,G908C mutations in the chimeric α subunits also increased ouabain binding.

Voltage-dependent transient currents of human and rat 5-HT transporters (SERT) are blocked by HEPES and ion channel ligands

The hyperpolarization‐activated transient current of mammalian 5‐hydroxytryptamine transporters (SERT) expressed in Xenopus oocytes was studied. Human (h) and rat (r) SERT transient currents are blocked by HEPES with changes in the waveform kinetics, and the blockade of hSERT has use‐dependent properties. HEPES also changes the time course of the prepriming step, especially for hSERT. Transient currents at hSERT and rSERT are also blocked by spermine and spermidine in the mM range, and by fluoxetine, cocaine, QX‐314, and QX‐222 in the μM range. These pharmacological and kinetic properties of transient current blockade emphasize the similarities between the transient current and phenomena at ion channels.