M.Eugenia Torres-Márquez

Amino Acid Osmolytes in Regulatory Volume Decrease and Isovolumetric Regulation in Brain Cells: Contribution and Mechanisms

Brain adaptation to hyposmolarity is accomplished by loss of both electrolytes and organic osmolytes, including amino acids, polyalcohols and methylamines. In brain in vivo, the organic osmolytes account for about 35% of the total solute loss. This review focus on the role of amino acids in cell volume regulation, in conditions of sudden hyposmosis, when cells respond by active regulatory volume decrease (RVD) or after gradual exposure to hyposmotic solutions, a condition where cell volume remains unchanged, named isovolumetric regulation (IVR). The amino acid efflux pathway during RVD is passive and is similar in many respects to the volume-activated anion pathway. The molecular identity of this pathway is still unknown, but the anion exchanger and the phospholemman are good candidates in certain cells. The activation trigger of the osmosensitive amino acid pathway is unclear, but intracellular ionic strength seems to be critically involved. Tyrosine protein kinases markedly influence amino acid efflux during RVD and may play an important role in the transduction signaling cascades for osmosensitive amino acid fluxes. During IVR, amino acids, particularly taurine are promptly released with an efflux threshold markedly lower than that of K+, emphasizing their contribution (possibly as well as of other organic osmolytes) vs inorganic ions, in the osmolarity range corresponding to physiopathological conditions. Amino acid efflux also occurs in response to isosmotic swelling as that associated with ischemia or trauma. Characterization of the pathway involved in this type of swelling is hampered by the fact that most osmolyte amino acids are also neuroactive amino acids and may be released in response to stimuli concurrent with swelling, such as depolarization or intracellular Ca++ elevation.

Oxidative phosphorylation is impaired by prolonged hypoxia in breast and possibly in cervix carcinoma

It has been assumed that oxidative phosphorylation (OxPhos) in solid tumors is severely reduced due to cytochrome c oxidase substrate restriction, although the measured extracellular oxygen concentration in hypoxic areas seems not limiting for this activity. To identify alternative hypoxia-induced OxPhos depressing mechanisms, an integral analysis of transcription, translation, enzyme activities and pathway fluxes was performed on glycolysis and OxPhos in HeLa and MCF-7 carcinomas. In both neoplasias exposed to hypoxia, an early transcriptional response was observed after 8h (two times increased glycolysis-related mRNA synthesis promoted by increased HIF-1alpha levels). However, major metabolic remodeling was observed only after 24h hypoxia: increased glycolytic protein content (1-5-times), enzyme activities (2-times) and fluxes (4-6-times). Interestingly, in MCF-7 cells, 24h hypoxia decreased OxPhos flux (4-6-fold), and 2-oxoglutarate dehydrogenase and glutaminase activities (3-fold), with no changes in respiratory complexes I and IV activities. In contrast, 24h hypoxia did not significantly affect HeLa OxPhos flux; neither mitochondria related mRNAs, protein contents or enzyme activities, although the enhanced glycolysis became the main ATP supplier. Thus, prolonged hypoxia (a) targeted some mitochondrial enzymes in MCF-7 but not in HeLa cells, and (b) induced a transition from mitochondrial towards a glycolytic-dependent energy metabolism in both MCF-7 and HeLa carcinomas.

Potentiation of the osmosensitive taurine release and cell volume regulation by cytosolic Ca2+ rise in cultured cerebellar astrocytes

Hyposmolarity (−30%) in cultured cerebellar astrocytes raised cytosolic Ca2+ concentration ([Ca2+]i) from 160 to 400 nM and activated the osmosensitive taurine release (OTR) pathway. Although OTR is essentially [Ca2+]i‐independent, further increase in [Ca2+]i by ionomycin strongly enhanced OTR, with a more robust effect at low and mild osmolarity reductions. Ionomycin did not affect isosmotic taurine efflux. OTR was decreased by tyrphostin A25 and increased by ortho‐vanadate, suggesting a modulation by tyrosine kinase or phosphorylation state. Inhibition of phosphatidylinositol‐3‐kinase activity by wortmannin markedly decreased OTR and the ionomycin increase. Conversely, OTR and the ionomycin effect were independent of ERK1/ERK2 activation. OTR and its potentiation by ionomycin differed in their sensitivity to CaM and CaMK blockers and in the requirement of an intact cytoskeleton for the ionomycin effect, but not for normal OTR. Changes in the actin cytoskeleton organization elicited by hyposmolarity were not observed in ionomycin‐treated cells, which may permit the operation of CaM/CaMK pathways involved in the OTR potentiation by [Ca2+]i rise. OTR potentiation by [Ca2+]i requires the previous or simultaneous activation/operation of the taurine release mechanism and is not modifying its set point, but rather increasing the effectiveness of the pathway, resulting in a more efficient volume regulation. This may have a beneficial effect in pathological situations with concurrent swelling and [Ca2+]i elevation in astrocytes.

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.

Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser748 autophosphorylation by Gαq

Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4⁻). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser⁷⁴⁴, a prominent and rapid PKC transphosphorylation site, and Ser⁷⁴⁸, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4⁻ potently induced PKD activation and Ser⁷⁴⁴ and Ser⁷⁴⁸ phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser⁷⁴⁸ phosphorylation induced by AlF4⁻, and largely abolished Ser⁷⁴⁴ phosphorylation. In contrast, Ser⁷⁴⁸ phosphorylation was almost completely intact, and Ser⁷⁴⁴ phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser⁷⁴⁸ phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser⁷⁴⁸.

Control of oxidative phosphorylation in AS-30D hepatoma mitochondria.

1. The distribution of control of the rate of state 3 respiration of AS-30D hepatoma mitochondria was determined. 2. The ATP/ADP carrier (flux control coefficient, Ci = 0.70) and the ATP synthase (Ci = 0.19-0.32) were the only steps that exerted significant control on the phosphorylating flux supported by either glutamate+malate, pyruvate+malate, or succinate+rotenone. This is in contrast to liver mitochondria where the control is distributed between several steps. 3. It is suggested that this pattern of control of phosphorylation in hepatoma mitochondria is a consequence of a lower content of adenine nucleotides or a higher content of Mg2+.

Dietary fatty acids effects on sucrose-induced cardiovascular syndrome in rats

Cardiovascular disease is one of the leading causes of morbidity and mortality in Mexico. We investigated the effects of omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids on the metabolic syndrome associated with cardiovascular disease in a high-sucrose-fed rat model. The metabolic syndrome-induced rats showed a significant increase in systolic blood pressure, serum insulin, nonfasting serum triglyceride and serum cholesterol levels. Experimental high-sucrose-fed animals received either a n-3 or n-6 enriched diet or a control diet during 6 weeks. Animals fed the n-3 enriched diet had a significant reduction in blood pressure and serum insulin and triglyceride levels. Serum triglyceride levels were also significantly reduced in the n-6-rich diet animals.

Cyanide-sensitive and cyanide-resistant respiration of dark-grown Euglena gracilis

Abstract The rate of endogenous respiration of Euglena gracilis Klebs grown in the dark in the presence of different carbon sources, both in late logarithmic and early stationary phases, was studied. Different sensitivity of endogenous respiration to cyanide was observed for each growth condition. However no significant difference in the ratio of cytochrome o to aa 3 was found. Inhibition constants for cyanide of endogenous and tetramethylphenylenediamine-stimulated respiration were also of a similar magnitude. The rate of respiration of isolated mitochondria was inhibited by cyanide to a different extent depending on the oxidizable substrate used. It is suggested that the sensitivity of endogenous respiration to cyanide in Euglena gracilis cells is related to the cytoplasmic availability of substrate generated by the particular growth conditions.

Identification of a functional Gs protein in Euglena gracilis

We found a Gs protein coupled to adenylyl cyclase in a free-living protist, Euglena gracilis. This Gs protein of approximately 42 kDa is substrate for cholera toxin and is recognized by an antibody against the C-terminal decapeptide of Gs. Furthermore, this protein is coupled to adenylyl cyclase, as shown by: (a) the activation of the enzyme by GTP-analogues and (b) the effect of cholera toxin on cAMP accumulation in intact cells and the continuous activation of adenylyl cyclase activity in membranes. These data indicate that the Gs-adenylyl cyclase-coupled system is already apparent in the protist kingdom.

Modulation of the ATP induced [Ca2+]c increase in as-30D hepatoma cells

1. The regulation of the increase in the cytosolic calcium concentration ([Ca2+]c) induced by extracellular ATP in AS-30D hepatoma cells was studied. 2. Homologous desensitization involving the refilling of intracellular calcium pools and the participation of protein kinase C was found. 3. Isoproterenol, forskolin and dibutyryl-cyclic AMP also induced an increase in [Ca2+]c. 4. Interestingly, synergism was found for isoproterenol or forskolin and ATP. 5. The results suggest that there are two pathways for mobilizing [Ca2+]c in AS-30D hepatoma cells; one is activated by ATP receptors and the other by cyclic AMP.