Studies on the transport of carnitine in the brain using synaptosomes isolated from guinea-pig cerebral cortex.

Synaptosomes isolated from guinea pig cerebral cortex accumulate L-carnitine from the medium in an active process, dependent on the sodium gradient across the plasma membrane and on (Na+ + K+)-ATPase activity. L-Carnitine uptake is inhibited by oxidative phosphorylation uncouplers and by ouabain, a known inhibitor of (Na+ + K+)-ATPase. In addition, the omission of Na+ or its replacement by Li+ inhibited the transport, which was also competitively inhibited by gamma-aminobutyrate. The kinetics of carnitine uptake show that the overall process would consist of two components: a passive diffusion and a carrier-mediated transport which is saturated at 1-2 mM carnitine concentration.

Hypotonic shock activated Cl- and K+ pathways in human fibroblasts.

The exposure of human fibroblasts to hypotonic medium (200 mosmolal) evoked the activation of both 36Cl- influx and efflux, which were insensitive to inhibitors of the anion exchanger and of the anion/cation cotransport, and conversely were inhibited by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 36Cl- efflux was linked to a parallel efflux of 86Rb+; thus conductive K+ and Cl- pathways are activated during volume regulation in human fibroblasts. This conclusion is supported by evidence that, in hypotonic medium, 36Cl- influx and 86Rb+ efflux were both enhanced by depolarization of the plasma membrane. Depletion of the intracellular K+ content, obtained by preincubation with the ionophore gramicidin in Na(+)-free medium, had no effect on Cl- efflux in hypotonic medium. This result has been interpreted as evidence for independent activation of K+ and Cl- pathways. It is also concluded that the anion permeability is the rate-limiting factor in the response of human fibroblasts to hypotonic stress.

Pertussis toxin- and PMA-insensitive calcium mobilization by sphingosine in CFPAC-1 cells: evidence for a phosphatidic acid-dependent mechanism.

In a pancreatic duct adenocarcinoma cell line (CFPAC-1) sphingosine (10 microM) induced both mobilization of intracellular Ca2+ and stimulation of inositol phosphates accumulation. Whereas this latter effect was significantly inhibited by treatment with pertussis toxin or by short-term incubation with phorbol 12-myristate 13-acetate, Ca2+ mobilization was completely insensitive to both treatments. Experiments with permeabilized cells showed that sphingosine or the sphingosine metabolites sphingosine-1-phosphate and sphingosylphosphorylcholine were unable to directly release Ca2+ from internal stores, whereas phosphatidic acid, but not arachidonic acid, was effective. Phosphatidic acid formation was markedly enhanced (2.9-fold over control) by sphingosine, this effect being significantly reduced by preincubation with the diacylglycerol kinase inhibitor R59022. Ca2+ mobilization by sphingosine was also cut down by preincubation with R59022. In conclusion, the results suggest that sphingosine activates phospholipase C through a mechanism functionally coupled through a G protein and under control of PKC. Mobilization of [Ca2+]i by sphingosine is independent of phospholipase C stimulation and likely due to elevation of phosphatidic acid generated by stimulation of diacylglycerol kinase activity.

Characterization of chloride transport pathways in cultured human keratinocytes.

In human keratinocytes, mediated transport of Cl- was found to occur mainly by two mechanisms: an anion exchange and an electrically conductive pathway. The contribution of the anion exchange, which accounted for about 50% of overall Cl- efflux, was assessed either by its sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and by means of Cl- substitution experiments. The anion exchange exhibited a saturation behaviour over the range 10-135 mM Cl-; Cl- was more efficient than HCO3-, Br- and NO3- in increasing Cl- efflux rate, whereas SO4(2-) and I- inhibited Cl- efflux. The electrically conductive Cl- pathway, which accounted for about 40% of total Cl- efflux, was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and was at least partially sensitive to variation of the plasma membrane potential. The Cl- channel was insensitive to elevation in the intracellular concentration of either cyclic AMP and calcium ions. Indomethacin, an inhibitor of the cyclooxygenase, failed to reduce Cl- efflux, whereas nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase, induced 50% inhibition of Cl- efflux. These results support the conclusion that endogenous production of lipoxygenase-derived arachidonic acid metabolite(s) might be responsible for high basal Cl- permeability in human keratinocytes.

The phorbol ester PMA and cyclic AMP activate different Cl(-) and HCO3(-) fluxes in C127 cells expressing CFTR.

In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase C with the phorbol 12-myristate 13-acetate (PMA). Conversely, bicarbonate influx, determined by intracellular alkalinization of cells incubated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF-AM), was stimulated by cyclic AMP elevation, but not by PMA. Patch clamp analysis revealed that PMA activated a Cl(-) current with the typical biophysical characteristics of swelling-activated current and not of CFTR.

A volume-sensitive chloride conductance revealed in cultured human keratinocytes by 36Cl- efflux and whole-cell patch clamp recording.

The Cl- transport mechanism responsible for the stimulation of 36Cl- efflux after exposure to hypotonic medium (210 mosmol/kg) was investigated in human keratinocytes. The involvement of the anion exchanger and of the Cl-/cation cotransporters was ruled out by the finding that replacement of extracellular Cl- by the poorly permeant anion gluconate, and the addition of bumetanide and furosemide, inhibitors of the Na+/K+/Cl- and K+/Cl- cotransporters, respectively, failed to significantly reduce the activation of Cl- efflux by hypotonic medium. 'Whole cell' configuration of the patch clamp technique directly revealed the presence of a macroscopic Cl- current, which was evoked by incubation with hypotonic medium and was reversed by elevation of the extracellular osmolality. Volume-sensitive current showed outward rectification of the current-voltage relationship and time-dependent inactivation at depolarizing voltages. This current was Cl- selective, because the zero-current reversal potential approached the Cl- equilibrium potential, when extracellular Cl- was replaced by gluconate. 0.1 mM 1,9-dideoxyforskolin significantly reduced either 36Cl- efflux and the Cl- current, suggesting that the Cl- efflux and the macroscopic current activated after exposure to hypotonic medium are mediated by the same pathway. Electronic cell sizing showed that in keratinocytes hypotonic swelling was not followed by a significant regulatory volume decrease response.

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance.

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.

Sphingosine-induced inhibition of capacitative calcium influx in CFPAC-1 cells.

Sphingosine (10 microM) induced mobilization of intracellular Ca2+ stores in the pancreatic duct adenocarcinoma cell line CFPAC-1. The effect was specific for sphingosine, since the sphingosine analog C2-ceramide had no effect. Sphingosine did not cause Ca2+ entry from extracellular medium, as also shown by following Mn2+ quenching of Fura-2 fluorescence. Furthermore, sphingosine, similarly to the mitochondrial inhibitors rotenone and oligomycin, strongly inhibited the rate of Mn2+ entry triggered by both thapsigargin- and agonist-induced depletion of intracellular stores. The uptake of rhodamine 123, a lipophilic cation which estimates mitochondrial energy level, was reduced by sphingosine to an extent similar to that observed in the presence of mitochondrial inhibitors. It is suggested that impairment of mitochondrial function might be responsible for inhibition of capacitative Ca2+ entry caused by sphingosine.

Sphingosylphosphorylcholine and sphingosine-1-phosphate mobilize cytosolic calcium through different mechanisms in human airway epithelial cells.

The sphingosine derivatives sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) caused a similar elevation of the intracellular Ca2+ concentration ([Ca2+]i) in an immortalized airway epithelial cell line (CFNP9o-) incubated in Ca(2+)-free medium. The maximal effect was obtained with 2 microM SPC and 0.1 microM S1P and was sensitive to pre-incubation with pertussis toxin, indicating the involvement of a Gi/G(o) type of G protein. In Ca2+ containing medium, [Ca2+]i elevation by SPC was significantly higher than that by S1P, due to the fact that SPC was able to stimulate Mn2+ entry, whereas S1P was ineffective. SPC, but not S1P, caused a dose-dependent production of total inositol phosphates. Conversely, S1P, but not SPC, increased the level of phosphatidic acid. These findings suggest the presence of two distinct receptors, specific for SPC and S1P, respectively. Depletion of intracellular Ca2+ stores by SPC makes cells unable to respond to a subsequent addition of S1P. Conversely, cells do respond to SPC after a challenge with S1P, suggesting that the two receptors likely share one or more intracellular signalling component(s).

Increased chloride efflux in fibroblasts from X-linked muscular dystrophies and clones from Duchenne carriers.

Previous studies have suggested an increased chloride membrane permeability in Duchenne muscular dystrophy (DMD) fibroblasts. We report that an increased chloride efflux with respect to controls is present not only in fibroblasts from DMD, but also from two other X-linked muscular dystrophies, Becker and Emery-Dreifuss, as well as in clones from DMD carrier females. The latter observation suggests that, at least in DMD, the increased chloride efflux phenotype might be subject to lyonization.

Inhibition of the mitochondrial bc1 complex by dibromothymoquinone.

We have studied the effects of dibromothymoquinone (DBMIB) in various redox activities of the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations higher than 50 mol/mol of cytochrome c1 the inhibitor produces a bypass of electron transfer on the substrate side of the bc1 complex, because of its autooxidation capability. This induces an artifactual overestimation of the real inhibition titer of the redox activity of this enzyme, which has been found to be 3-6 mol/mol of cytochrome c1 by following the ubiquinol-cytochrome c reductase activity. This action is reversed by addition of excess of sulphydryl compounds like cysteine.

Role of CFTR and anion exchanger in bicarbonate fluxes in C127 cell lines.

C127 cell lines transfected with wtCFTR, delta F508CFTR or vector were employed to determine HCO3- fluxes in the presence or absence of functional CFTR, using the pH-sensitive dye BCECF. Both cytosolic alkalinization and acidification were due to activity of anion exchanger and were similar in the three cell lines, indicating that expression of CFTR did not influence anion exchanger activity. In C127wt cells only, cAMP elevating agents significantly stimulated HCO3- fluxes, insensitive to the inhibitor of anion exchanger 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, suggesting that activated CFTR directly mediates both HCO3- influx and efflux and therefore can contribute to intracellular and extracellular pH regulation.

Kinetic alterations of cytochrome-c oxidase in cystic fibrosis.

We compared the kinetics of cytochrome-c oxidase (cytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) in fibroblasts derived from normal and cystic fibrosis individuals. The Km of the enzyme for reduced cytochrome c was significantly increased in CF cells; the change, however, was observed only at temperatures above 25 degrees C. The Vmax values were comparable in both types of individuals.

Extracellular 2-chloroadenosine and ATP stimulate volume-sensitive Cl- current and calcium mobilization in human tracheal 9HTEo- cells.

The perforated-patch whole-cell technique was used to record membrane currents in epithelial cells (9HTEo-) obtained from the human tracheal epithelium. Extracellular application of 2-chloroadenosine and ATP (0.01-100 microM) caused activation of Cl- currents similar to those regulated by cell volume in airway and intestinal cells. This response was inhibited by increasing extracellular osmolality, by omission of extracellular Ca2+, or by the addition of the A2 adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Fluorimetric measurements with fura-2 reveal that 2-chloroadenosine and ATP elicited both a Ca2+ influx through the plasma membrane and a release from intracellular stores.

Staurosporine induces apoptotic volume decrease (AVD) in ECV304 cells.

Incubation of ECV304 cells with 1 micro M staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl(-) (and possibly K(+)) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.

The response of chloride transport to cyclic AMP, calcium and hypotonic shock in normal and cystic fibrosis fibroblasts.

It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C.

The essentiality of coenzyme Q for bioenergetics and clinical medicine.

Coenzyme Q is an essential component of the respiratory chain, where it represents a mobile pool between dehydrogenases and cytochromes. The fact that Q is a free component, and its concentration is not in great excess over the Km of the respiratory complexes, renders this compound potentially rate-limiting in the respiratory chain. On the other hand, the rate of lateral diffusion of Q in the mitochondrial membrane is not a limiting step under physiological conditions. Quinoid compounds, which act as inhibitors of the respiratory chain at the level of Q, besides being useful tools for investigations of electron transfer, could be important in pathology as inhibitors of respiration.

Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1ß inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.