Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Effect of ouabain on growth regulation by serum components in Balb/c-3T3 cells: inhibition of entry into S phase by decreased protein synthesis

The effect of inhibition of the cell membrane Na+-K+ pump on the Balb/c-3T3 cell growth cycle was studied. Inhibition of the Na+-K+ pump resulted in a dose-dependent reduction of intracellular K+ concentration ((K+)i). However, inhibition of protein synthesis in Go/G1 and of subsequent entry into S phase occurred only after (K+)i fell below a critical threshold (50-60 mmoles/liter). Thus, when the (K+)i falls below a critical threshold, protein synthesis is inhibited, preventing cells from entering the S phase. The platelet-derived growth factor (PDGF) induces cells to become "competent" to traverse the cell cycle; the platelet-poor plasma component of serum allows competent cells to progress through G0/G1 and enter S phase. Inhibition of the Na+-K+ pump did not prevent the induction of competence by PDGF, but it did reversibly inhibit plasma-mediated events in early G0/G1. Similarly, cycloheximide inhibited plasma-mediated events but did not prevent PDGF-induced competence. Thus, protein synthesis may not be required for induction of competence; alternatively, the induction of the competent state may occur in these cells after removal of PDGF and protein synthesis inhibitor. Protein synthesis is required for subsequent plasma-mediated events in G0/G1.     

An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store.

There is considerable evidence, reviewed by Brostrom and Brostrom [1], that Ca2+ stores are involved in the regulation of protein synthesis. We provide evidence in HeLa cells that is consistent with their findings that depletion of Ca2+ stores and not changes in cytosolic free Ca2+ ([Ca2+]i) inhibit protein synthesis, but we also show that the mechanism leading to depletion is critical. Specifically, depletion of stores by the Ca(2+)-mobilizing hormone histamine does not inhibit protein synthesis. In assessing the role of Ca2+ stores in protein synthesis, experiments in certain cell types have been complicated by the use of Ca2+ ionophores, which simultaneously elevate [Ca2+]i and deplete Ca2+ stores. We have measured total cell Ca2+, [Ca2+]i and protein synthesis in HeLa cells under conditions that allowed evaluation of the separate contributions of stores and [Ca2+]i. Using 1,2-bis(2-aminophenoxyethane)-N,N,N'N'-tetraacetic acid (BAPTA) as an intracellular Ca2+, chelator and thapsigargin, which inhibits the membrane Ca(2+)-ATPase of storage vesicles, total cell Ca2+ can be depleted and this depletion is enhanced by extracellular EGTA which blocks Ca2+ influx; [Ca2+]i is actually lowered by BAPTA under these conditions. Protein synthesis is inhibited by BAPTA in the presence of EGTA and by thapsigargin with or without EGTA. However, histamine which with EGTA, affects an equal degree of Ca2+ depletion does not inhibit protein synthesis. Thus, it is suggested that Ca2+ stores are not homogeneous, and that the hormone-sensitive store specifically does not play a role in the regulation of protein synthesis. In this respect, the hormone-sensitive and insensitive stores do not functionally communicate and may be separately regulated.     

Relationship of the decreases in protein synthesis and intracellular Na+ during friend murine erythroleukemic cell differentiation

The earliest known ionic event during Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway is a 45% drop in intracellular sodium concentration ([Na+]i) due to a decrease in Na+ influx (Lannigan, D. A., and Knauf, P. A. (1985) J. Biol. Chem. 260, 7322-7324). We have analyzed the mechanism of the decreased Na+ influx. The Na+ influx in uninduced cells was insensitive to dimethylamiloride, bumetanide, and diisothiocyanostilbene disulfonate. The intracellular pH (pHi) did not change up to 15 h after dimethyl sulfoxide induction, at which time Na+ influx has decreased by approximately 40%; thus, the decrease in Na+ influx is not coupled to a change in pHi. A substantial amount of the decrease in Na+ influx seems to result from a drop in amino acid-dependent Na+ transport. This reduction in amino acid-dependent Na+ influx reflects a decrease in net Na+ influx rather than solely in Na+/Na+ exchange and can account for an appreciable portion of the reduction in [Na+]i seen during differentiation. The drop in amino acid-dependent Na+ influx could not be explained by membrane depolarization but was correlated with a decrease in protein synthesis. Inhibition of protein synthesis in uninduced cells by cycloheximide also caused a decrease in Na+ influx. We conclude that during differentiation the reduction in protein synthesis decreases amino acid-dependent Na+ influx which in turn causes a drop in [Na+]i leading to a reduction in the Na+/K+ pump rate.     

Association of increased intracellular free Ca2+ by platelet-derived growth factor with mitogenesis but not with proteoglycan synthesis in chondrocytes--effect of suramin

The effects of platelet-derived growth factor (PDGF) on the intracellular free Ca2+ concentration [( Ca2+]i) in chondrocytes were studied with a fluorescent Ca2+ indicator, fura 2, and compared with the effects of PDGF on mitogenesis and proteoglycan synthesis. PDGF evoked phasic and then tonic increase in [Ca2+]i dose-dependently in quiescent cultures of chondrocytes, and it also stimulated both DNA and proteoglycan syntheses dose-dependently similar to somatomedins. Suramin, which inhibits the interaction of PDGF with its receptors, caused dose-dependent inhibition of both the PDGF-evoked increase in [Ca2+]i and stimulation of DNA synthesis by PDGF. However, suramin rather enhanced the proteoglycan synthesis induced by PDGF without affecting the basal level of proteoglycan synthesis directly. These results suggest that [Ca2+]i may be an important signal for the action of PDGF on cell proliferation in chondrocytes, and that the initial signal for proteoglycan synthesis is different from that for DNA synthesis induced by PDGF after the activation of PDGF receptor.     

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.     

The sodium cycle. II. Na+-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na+ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na+-motive respiratory chain of V. alginolyticus. Na+ loaded cells incubated in a K+ or Li+ medium fail to synthesize ATP in response to lactate addition. The addition of Na+ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na+ level. Neither lactate oxidation nor delta psi generation coupled with this oxidation is increased by external Na+ in the Na+-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse delta pNa, i.e., [Na+]in greater than [Na+]out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K+-loaded cells incubated in a high Na+ medium, i.e., when delta pNa of the proper direction [( Na+]in less than [Na+]out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K+ medium (delta pNa is low) is decreased by CCCP even without monensin. Artificial formation of delta pNa by adding 0.25 M NaCl to the K+-loaded cells (Na+ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na+ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na+ instead of H+ as the coupling ion.     

Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.     

Inositol 1,3,4,5-tetrakisphosphate stimulates the initiation of DNA synthesis in Ca2+-deprived rat liver cells

The G1-S boundary of non-neoplastic cells requires extracellular Ca2+ for successful transition. Inositol 1,3,4,5-tetrakisphosphate but not inositol 1,4,5-trisphosphate can partially replace Ca2+ and stimulate the initiation of DNA synthesis of Ca2+-deprived T51B rat liver cells but only if sufficient extracellular Ca2+ (i.e., 0.075 mM) is present. The potent tumor promoter and protein kinase C activator 12-O-tetradecanoylphorbol acetate is also capable of replacing extracellular Ca2+ and partially stimulating the initiation of DNA synthesis. In addition, both inositol-1,3,4,5-tetrakisphosphate and 12-O-tetradecanoylphorbol acetate added together elicit a full DNA synthetic response.     

Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.     

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.     

The role of mitochondrial calcium transport during inflammation and the effect of anti-inflammatory drugs

The effects of inflammation induced by the inoculation of rats with Freund's adjuvant on calcium transport by isolated rat liver mitochondria and on mitochondrial in vivo protein synthesis were investigated. Mitochondria isolated from the liver of inflamed rats exhibited (i) a reduction in 45Ca2+ uptake and, (ii) a reduction in protein synthesis. Addition of ATP to the calcium uptake medium stimulate the uptake in inflamed rat liver mitochondria. After inflammation was controlled by treatment with a mixture of Clerodendron inerme flavonoidal glycosides and indomethacin, rat liver mitochondria showed (i) an increase in 45Ca2+ uptake and, (ii) an increase in mitochondrial in vivo protein synthesis. The mechanism of mitochondrial calcium transport and the mitochondrial protein metabolism during inflammation and after treatment with anti-inflammatory drugs were discussed.     

Synthesis and conformational studies of pseudopeptides containing an unsymmetrical triazine scaffold.

Solid-phase synthesis and conformational studies of two pseudopeptides constituted by a triazine scaffold bound to two peptidic arms are described. In this paper, a new scaffold based on unsymmetrical triamino 1,3,5-triazine bearing two alkyl chains has been designed, assisted by molecular modelling, as a mimic of the backbone of the i + 1 and i + 2 residues of a beta-turn. The results confirm the ability of the triazine scaffold to induce extended conformations of the peptidic strands and point out that this scaffold is a good candidate as a template to induce anti-parallel beta-sheet structure.     

Sodium-coupled ATP synthesis in the bacterium Vitreoscilla.

The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla.     

Lack of correlation between the activation of the Ca(2+)-phosphatidylinositol cascade and the regulation of DNA synthesis in the dog thymocyte.

Changes in the [Ca2+]i and/or activation of phospholipase C are thought to participate in the control by several growth factors of the mammalian cell proliferation. It has even been claimed that activation of the Ca(2+)-phosphatidylinositol cascade is sufficient to elicit cell proliferation [Jackson et al. (1988) Nature 335, 437-440; Julius et al. (1989) Science 244, 1057-1062]. In this work, we have evaluated the control of DNA synthesis by this cascade in a differentiated epithelial cell model: the dog thyrocyte in primary culture. We first observed that potent activators of the dog thyrocyte (2+)-phosphatidylinositol cascade such as carbachol or bradykinin failed to promote the onset of DNA synthesis in these cells. Moreover, carbachol inhibited the mitogenic effect of thyroid stimulating hormone (TSH) and of epidermal growth factor (EGF). The mitogenic effect of EGF was also reduced by bradykinin. Nevertheless, carbachol enhanced the expression of the protooncogenes c-fos and c-myc mRNAs. The time course of this enhancement was identical to the time course for the induction of c-fos and c-myc mRNAs by phorbol esters or EGF. On the other hand, in most experiments, TSH and EGF were able to trigger the onset of dog thyrocyte DNA synthesis without affecting their intracellular free Ca2+ concentration [Ca2+]i, 45Ca2+ efflux, or inositol phosphate generation. In several experiments, TSH increased the dog thyrocyte 45Ca2+ release and promoted a rise in the [Ca2+]i or the inositol phosphate accumulation but these effects were weak. In contrast to the effect of carbachol, the TSH effects on the [Ca2+]i and the 45Ca2+ efflux appeared slowly, were sustained, and were extremely sensitive to extracellular Ca2+ depletion. They were observed at hormone concentrations higher than the concentration achieving maximal stimulation of DNA synthesis. Similarly, in a few experiments, a slight increase in the [Ca2+]i or in the inositol trisphosphate generation were provoked by EGF. However, these modifications were not associated with an increased mitogenic potency of EGF. Finally, in all experiments, fetal calf serum slightly accelerated the dog thyrocyte 45Ca2+ efflux and increased their inositol phosphate generation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The sodium cycle. II. Na-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na⁺ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na⁺-motive respiratory chain of V. alginolyticus. Na⁺ loaded cells incubated in a K⁺ or Li⁺ medium fail to synthesize ATP in response to lactate addition. The addition of Na⁺ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na⁺ level. Neither lactate oxidation nor Δω generation coupled with this oxidation is increased by external Na⁺ in the Na⁺-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse ΔPNa, i.e., [Na⁺]_in > [Na⁺]_out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K⁺-loaded cells incubated in a high Na⁺ medium, i.e., when ΔpNa of the proper direction ([Na⁺]_in < [Na⁺]_out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K⁺ medium (ΔpNa is low) is decreased by CCCP even without monensin. Artificial formation of ΔpNa by adding 0.25 M NaCl to the K⁺-loaded cells (Na⁺ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na⁺ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na⁺ instead of H⁺ as the coupling ion.     

Regulation of steady state level of phosphoenzyme and ATP synthesis in sarcoplasmic reticulum vesicles during reversal of the Ca2+ pump.

The role of the Ca2+ concentration gradient in ATP synthesis and membrane phosphorylation by Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Pi concentration required to attain 50% of the maximal membrane phosphorylation varies significantly in the pH range of 5.5 to 4.5, the optimal being at pH 6.0. In the pH range of 6.0 to 7.0, this concentration of Pi was 4- to 10-fold higher in empty vesicles than in vesicles loaded with calcium phosphate, i.e. having transmembrane Ca2+ concentration gradient. ATP, ADP, and Ca2+ inhibit the membrane phosphorylation by Pi, the inhibition being greater at pH 7.0 than at pH 6.0. The pH profile for ATP synthesis shows a higher optimum than for membrane phosphorylation. The optimum pH for synthesis, but not for phosphorylation depends on whether the vesicles were previously loaded with calcium phosphate or with calcium oxalate. Addition of Ca2+ to the assay medium inhibits the extent of membrane phosphorylation and the rate of ATP synthesis to different extents. Evidence is presented that the rate of membrane phosphorylation by Pi is higher than the rate by which the phosphoprotein transfers its pohsphate to ADP for the ATP synthesis.     

Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line

Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.     

Stimulation and inhibition of the protein synthetic process by NAD+ in lysed rabbit reticulocytes.

NAD+ at 0.16 mM stimulates the initiation step of the protein synthetic process in lysed rabbit reticulocytes. This conclusion is based on the stimulation of (i) the transfer of formylmethionine from f[35S]Met-tRNAfMet into polypeptide, (ii) the accumulation of the initial dipeptide, methionylvaline, in the presence of pactamycin, and (iii) the formation of the 40 S initiation complex. The effect of NAD+ changes from a stimulatory role on protein synthesis to an inhibitory role at concentrations greater than 0.16 mM. At 4.0 mM NAD+, protein synthesis is inhibited. This has been demonstrated experimentally by using the same three assays described above. In addition, 4.0 mM NAD+ inhibits MettRNAfMet.initiation factor.GTP ternary complex formation. The elongation and termination steps of polypeptide synthesis are not affected by 0.16 to 4.0 mM NAD+. The data presented clearly show that the stimulatory activity of 0.16 mM NAD+ and the inhibitory activity of 4.0 mM NAD+ affects the initiation step of the protein synthetic process in lysed rabbit reticulocytes.