Peter Geck

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor α (TNFα) and Interleukin-1α (IL1α) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNFα and IL1α. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNFα (1, 10, 100, 1000 I.U./ml) and IL1α (0.1, 1, 10, 100 I.U./ ml).TNFα- and IL1α- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNFα and IL1α and a subsequent down regulation of PDH activity.

DRIVING FORCES OF AMINO ACID TRANSPORT IN ANIMAL CELLSfn1

Our observations on the response of TPP+ uptake into K+-depleted cells on the extracellular K+ concentration and on the addition of ouabain and amino acid appear to provide suggestive evidence of, or are at least consistent with, the operation of a powerful, K+-activated electrogenic pump. They are also consistent with the assumption that in these K+-depleted cells the active uptake of amino acid energized by the electric PD presumably generated by this electrogenic pump. It follows that an energy source other than the electrochemical potential gradient of Na+ ions need not be invoked to explain active amino acid transport with inverted Na+ and K+ distribution. In the presence of an electrogenic cation pump one should expect that owing to the activity of this pump the electrical PD is raised under these conditions to a value outweighing the opposed chemical potential gradient of Na+. The experimental evidence so far available is clearly consistent with such an assumption.

Na++ K++ 2 Cl− Cotransport in Animal Cells—Its Role in Volume Regulationa

Cell membranes of various vertebrate cells catalyze a Na+ + K+ + 2Cl- cotransport specifically inhibitable by furosemide and other high ceiling diuretics. The energetics of this process is not elucidated unequivocally. It was clearly shown that cotransport is no ATP-consuming process. We assume that transport is secondary active functionally coupled to the operation of the electrogenic Na+-K+ pump. The role of this transport system in transepithelial ion movement is that it serves as flux amplifier, doubling from 6 to 12 the number of osmotically active particles transported per ATP hydrolyzed. In concert with Na+-K+ pump, cotransport provokes net uptake of KCl into the cell and therefore cellular swelling. This process is regulated by a feedback control system for cell volume; if actual volume reaches reference value, cotransport is switched off to prevent further swelling. How cell volume is measured is not known, nor is the nature of the signal generated to switch cotransport from the operating to the nonoperating state or vice versa. cAMP-level or intracellular Ca2+ play no role as signals or as part of the volume-sensoring mechanism. Theophylline, other alkylxanthines, and some purine ribosides influence cotransport indirectly by reducing reference volume. The role of cytoskeleton in volume regulation is obscure. While high Concentrations of cytochalasin B and of colchicin do not influence cell volume, it is reduced by vinblastine and also by lectins, for example concanavalin A. Volume reduction is accompanied by reduction in cellular KCl content. The observation that during hypertonic incubation protein synthesis is inhibited can be traced back to a correlation between cell volume and protein synthesis and not to elevation of osmolarity per se. Reduction in cell volume under isotonic conditions by varying K+ and/or Cl- concentration or by furosemide inhibition of cotransport is strongly correlated to inhibition of protein synthesis. The reason for this correlation is not yet clarified. Not all cells showing furosemide-sensitive cotransport are able to regulate it, for example lymphocytes. For mammalian erythrocytes drastic species differences exist; while cells from man, rabbit, rat, and mouse all show cotransport, only cells from rat (and mouse?) are able to regulate cotransport.

Coupling in secondary active transport: Activation of transport by co-transport and/or counter-transport with the fluxes of other solutes

Abstract The transport of a solute may be activated by coupling to the fluxes of other solutes via co-transport and/or counter-transport. This coupling may stimulate the transport rate of the former and, under certain conditions, also contribute energy for the active accumulation. The coupling may occur in different ways, either by increasing the affinity of the carrier to the transported solute or by increasing the velocity of the carrier-solute complex, or by both effects at the same time. Each of these effects can be conceived as occurring either directly, in that the co- or counter-substrate alters the properties of the carrier to which it is bound, or indirectly, in a quasi-allosteric way by shifting the equilibrium between two conformations of the carrier in favor of that with the higher affinity or velocity, respectively. These possibilities have been treated kinetically in a more general way, taking into account all the mentioned possible effects. From the general formula two selected simplified model types are derived: the affinity type and the velocity type. Each of these types has been subdivided in a direct and a quasi-allosteric variant. It is found that both variants of the affinity type would predict that only the K m of the main transport is changed by the activation. Hence the affinity type in its pure form only applies to a limited number of experimentally investigated cases. Of the velocity-type models both variants would account for an increase in maximum velocity. But only the direct type would under rather stringent conditions explain an active accumulation, whereas the quasi-allosteric variant could not account for any accumulation at all. In that case the activation is merely catalytic, not energetic. In addition, a few combinations of velocity and affinity effects are considered and also a special model with three conformational modifications is presented. Each of these will account for a change in both K m and v max , as has usually been found in the systems so far studied. The differential effect of co- and counter-transport effects is evaluated for most of these.

Expression of novel genes linked to the androgen-induced, proliferative shutoff in prostate cancer cells

Androgens control cell numbers in the prostate through three separate pathways: (a) inhibition of cell death, (b) induction of cell proliferation (Step-1) and (c) inhibition of cell proliferation (Step-2, proliferative shutoff). The mechanisms underlying these phenomena are incompletely understood. The human prostate carcinoma LNCaP variants express these pathways as follows: LNCaP-FGC express both steps, LNCaP-LNO expresses Step-2, LNCaP-TAC expresses Step-1, and LNCaP-TJA cells express neither step. These cells facilitated the search for mediators of the androgen-induced proliferative shutoff pathway. Androgen exposure for 24 h or longer induced an irreversible proliferative shutoff in LNCaP-FGC cells. The Wang and Brown approach for identifying differentially expressed mRNAs was used to search for mediators of Step-2. Ten unique inserts were identified and from those ten, three genes were further studied. The basal expression of these genes in shutoff-negative variants was not affected by androgen exposure. They were induced by androgens in shutoff-positive LNCaP variants and the androgen receptor-transfected, shutoff-positive, MCF7-AR1 cells. These genes were induced only in the range of androgen concentrations that elicited the shutoff response. Time course analysis showed that their induction precedes the commitment point by 12-18 h. In addition, they were expressed in the normal prostate during proliferative shutoff. These features suggest that the candidate genes have a role in the regulation cascade for proliferative shutoff.

Concentrated, digestible DNA after hydroxylapatite chromatography with cetylpyridinium bromide precipitation

A method for the direct extraction of the DNA from the unfavorable phosphate eluant of hydroxylapatite chromatography is described. The DNA--reversibly precipitated with the cationic detergent cetylpyridinium bromide--can be subjected to further enzymatic manipulations within minutes. This method is applied to the rapid separation of pBR322 plasmid from the chromosomal DNA.

Early gene expression during androgen-induced inhibition of proliferation of prostate cancer cells: a new suppressor candidate on chromosome 13, in the BRCA2-Rb1 locus.

In the prostate gland cell numbers are regulated by androgens through three separate pathways: (a) inhibition of cell death (apoptosis), (b) induction of cell proliferation (step 1), and (c) inhibition of cell proliferation (step 2, proliferative shutoff). The precise regulation of these control pathways is still elusive. The human prostate carcinoma LNCaP cell line variants express a subset of proliferative pathways comparable to those present in normal prostate cells (LNCaP-FGC expresses both steps, LNCaP-LNO expresses step 2, LNCaP-TAC expresses step 1, LNCaP-TJA expresses neither). The purpose of the present work is to identify the genes involved in the androgen-induced proliferative arrest of these cells. Using a Wang-Brown subtracted library, a set of shutoff specific genes has been isolated. One of these new genes, AS3, shows high expression in the early regulatory phase of androgen-induced proliferative shutoff in the cell variants and in the prostates of castrated rats. The putative 1391-residue polypeptide has the molecular size of about 186 kDa. It has coiled-coil structures that usually participate in protein-protein interactions, a perfect leucine-zipper that suggests DNA binding, nuclear localization motifs, proline- and serinerich domains, unique C-terminal acidic-basic repeats, and ATP- and DNA-binding motifs. The transcript has 34 exons in a 200,000 bp region on chromosome 13q12-q13, downstream of the breast cancer susceptibility gene BRCA2, and centromeric to the retinoblastoma (Rb1) locus. This area is subject to frequent allelic losses in cancers, and is believed to carry a number of cryptic suppressor genes. The AS3 gene seems to be a novel candidate in the regulation of androgen-induced proliferative arrest of human prostate cells.

The efficiency of energetic coupling between Na+ flow and amino acid transport in ehrlich cells — A revised assessment

The overall efficiency of the coupling between transport of α-aminoisobutyrate and the entry of Na+ in Ehrlich cells has previously been determined to be 8–10%. It was concluded that the efficiency is grossly inadequate to account for the energization of amino acid transport by the electrochemical potential gradient of Na+, as postulated by the “gradient hypothesis”. This conclusion had, however, not taken into account that a major part of the Na+ entry is not coupled to a α-aminoisobutyrate transport. The “intrinsic efficiency”, which relates the amino acid transport to the coupled Na+ entry only, has now been evaluated from available experimental data and found to be approximately adequate to account for the highest accumulation ratios for this amino acid reported. It is concluded that the gradient hypothesis cannot be rejected on energetic grounds.

Identification of human estrogen-inducible transcripts that potentially mediate the apoptotic response in breast cancer

Hormone manipulation has been used for several decades with the purpose of inducing breast cancer regression. On the one hand, hormone ablation and antiestrogen administration were used on the rationale that estrogens induce proliferation of their target cells. Before the advent of the antiestrogen tamoxifen, on the other hand, the estrogen agonist DES was used to obtain clinical remissions. The rationale for the use of diethylstilbestrol (DES) was totally empirical. In fact, the efficacy of both treatments was comparable. A mechanistic explanation for estrogen-induced regression is urgently needed in order to provide a rationale for its use in therapeutic fields, and to develop markers to identify this phenotype in order to recognize responsive tumors. In this report, we use E8CASS cells (a MCF7 variant) as a model to study estrogen-mediated regression. The proliferation rate of E8CASS cells is decreased by estrogens. In order to isolate mRNA sequences induced by estradiol, a subtracted library was prepared from E8CASS cells grown in the presence and absence of estrogens. Twenty nine differentially expressed unique sequences were found. Seven of them were homologous to known genes, 12 of them were homologous to expressed sequence tags (EST), and 10 sequences had no homologues in the databases. The two sequences showing the highest induction by estradiol (E9 and E43) were chosen for further analysis. The sequence of the E43 coding region has 96% homology to the bovine actin2 gene and 100% identity to bovine actin2 protein, and it is homologous to the human actin-related protein 3 (Arp3). It has been suggested that Arp3 is involved in actin nucleation. The phenotype of E8CASS cells is clearly affected by estrogen treatment. It is likely that E43 may be involved in these morphological changes. The E9 cDNA is a putative zinc-finger protein of the PHD family of transcriptional transactivators. A member of this family, Requiem, is involved in apoptosis. The E9 mRNA is highly expressed in E8CASS cells treated with estrogens, a treatment which results in decreased proliferation rate and increased DNA degradation. This correlation suggests that E9 may be a mediator of estrogen-induced regression of breast cancer.

Regulation of the electrogenic (Na+ + K+)-pump of Ehrlich cells by intracellular cation levels

Abstract Ehrlich cells actively accumulate neutral amino acids even if both the Na + and K + gradients are inverted. The seeming contradiction of this observation to the gradient hypothesis is, however, explained by the presence of a powerful electrogenic Na + pump, which stongly raises the electrochemical potential gradient of Na + under these conditions. Since the evidence of this pump has so far been found only during abnormal concentrations of alkali ions (low K + , high Na + ) in these cells, the question arises whether the pump is equally powerful with completely normal cells, when the pump is not ‘needed’ for amino acid transport. Using the initial rate of uptake of the test amino acid (2-aminoisobutyrate) as a sensitive monitor of the electrical potential at constant cation distribution between cell and medium, a procedure has been devised to split the overall electrical potential into the diffusional and the pump component. With this procedure it could be shown that the electrogenic pump per se is most powerful in K + -depleted and Na + -rich cells but declines to a lower ‘resting’ value according as the electrolyte content of the cell approaches normality. A strong positive correlation between cellular Na + content and the electrogenic pumping activity suggests that the intracellular activity of this ion regulates the rate of the electrogenic pump. The low activity of the pump under normal conditions may explain why the existance of this pump has rarely come to attention previously.