Synthesis of Ethanolamine Phosphoglycerides in Rat Cerebral Cortex Subjected In Vitro to Experimental Hypoxia with and without Hypocapnia

Slices and homogenates from rat cerebral cortex were used to study the effect of hypoxia, with or without hypocapnia, on phosphatidylethanolamine synthesis. The incorporation of [1-³H]ethanolamine into the corresponding phospholipid was greatest in slices treated with pure nitrogen, intermediate when the nitrogen contained 5% CO₂, and least in slices treated with 95% O₂-5% CO₂. The role of hypocapnia in reinforcing the effect due to hypoxia did not require the integrity of the cell because similar results were obtained by treating homogenates with pure nitrogen or nitrogen plus 5% CO₂. In both cases the synthesis of phosphatidylethanolamine was abolished by the addition of EGTA and the degradation of newly synthesized phospholipid by phospholipases was similar to that obtained in controls. When the homogenate was not buffered, changes in the pH due to experimental treatment influenced the response to Ca²⁺ and to hypoxia plus hypocapnia. Intracellular calcium ions are thought to play a role in the response of cerebrocortical slices to N₂-treatment. In fact, although the incorporation was greater in complete medium that contains 2 mM Ca²⁺ than in the same medium prepared without the addition of this ion, the relative increase of incorporation due to N₂-treatment was greater in the medium lacking added Ca²⁺.     

Acidosis and Ca2+ distribution in myocardial tissue of flounder and rat

Summary The effect of acidosis on the myocardial Ca²⁺ distribution was examined at 15°C in ventricular strips of the flounder (Platichthys flesus) and at 30°C in atrial strips of the rat (Rattus norvegicus).Lowering the Ringer pH from 7.6 to 6.9 by increasing its CO₂ (flounder 2% to 12%, rat 4% to 14%), resulted in an elevated Ca²⁺ efflux in resting strips as well as in strips stimulated (12/min) to contraction. A decrease in pH of the Ringer used for the flounder myocardium by a lowering of bicarbonate (30 mM to 5 mM) also resulted in an elevation of the Ca²⁺ efflux, but the effect was smaller than that produced by an increased CO₂.With 11 mM Ca²⁺ and 10 mM EGTA added to the Ringer to reduce the amount of⁴⁵Ca²⁺ bound to extracellular sites, an increased CO₂ with a concomitant drop in Ringer pH resulted in an increased Ca²⁺ efflux in both myocardia. The Ca²⁺ efflux was only marginally elevated in the flounder myocardium and unchanged in that of rat when the same drop in Ringer pH was produced with a lowering in bicarbonate.In a nominally Ca²⁺-free Ringer with 0.1 mM EGTA the⁴⁵Ca²⁺ efflux was stimulated for both myocardia by an increase in CO₂.The flounder myocardium was exposed to high CO₂ in a nominally Na⁺, Ca²⁺-free Ringer and again the⁴⁵Ca²⁺ efflux increased. After a return to Na, Ca and low CO₂ in the Ringer, a higher efflux persisted in the strips being subjected to a high CO₂ than in the controls.The Ca²⁺ uptake rate was about the same at high and low CO₂ for both myocardia.Based on these results the measured increase in Ca efflux following an increase in CO₂ or a decrease in bicarbonate probably results from an elevated cytoplasmatic Ca²⁺ activity. It seems unlikely that an increased uptake rate of Ca²⁺ or a direct stimulation of Ca²⁺ transporting mechanisms in the cell membrane are responsible for the change.     

Stimulation of rat colonic mucosal prostaglandin synthesis by calcium and carbamylcholine: Relationship to alterations in cyclic nucleotide metabolism

The present study examined the relationships between prostaglandin (PG) synthesis and cyclic nucleotide metabolism in rat colonic mucosal slices. Ca²⁺, Ca²⁺ plus A23187 and carbamylcholine all increased [¹⁴C]-arachidonate release from prelabeled slices and stimulated production of PGE. Actions of A23187 and carbamylcholine required Ca²⁺ and were suppressed by tetracaine or mepacrine, whose known actions include inhibition of acyl hydrolase activity. Exogenous arachidonate or linoleate stimulated PGE synthesis in the absence of Ca²⁺ or in the presence of the inhibitors, suggesting a role for Ca²⁺ dependent acyl hydrolase activity in the mediation of the actions of Ca²⁺, A23187 and carbamylcholine on PGE synthesis. Accumulation of both cAMP and cGMP in colonic mucosal slices was enhanced by carbamylcholine, Ca²⁺, Ca²⁺ plus A23187, arachidonate or linoleate. Stimulatory actions of each of these agents on PGE production and cyclic nucleotide accumulation were inhibited by O₂ exclusion or indomethacin (100 μg/ml). The results support a role for local PG production in the mediation of carbamylcholine and Ca²⁺ actions on cyclic nucleotides. Endogenous ionic, neurohumoral and dietary factors may modulate colonic mucosal PG synthesis and cyclic nucleotide content, and thereby influence the physiologic expression of the actions of these putative local cellular regulators.     

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which may result from activation of Ca²⁺ permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca²⁺]_i from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca²⁺ channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30–48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37°C) significantly increased glycation of membrane proteins, hemoglobin (HbA_1c), TRPC3/6/7, and L-type Ca²⁺ channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca²⁺]_i, and phosphatidylserine exposure, and led to significant cell shrinkage. Ca²⁺ removal and addition of Ca²⁺ chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca²⁺ entry from extracellular space.     

The Uptake of Free CO2 and HCO3- during Photosynthesis of Plankton Algae with Special Reference to the Coccolithophorid Coccolithus huxleyi

From a re-evaluation of experiments with the coccolithophorid Coccolithus hurleyi made by Paasche (1964), curves are presented showing the rate of photosynthesis as a function of the concentration of both free CO₂ and bicarbonate CO₂. It is shown that photosynthesis in a naked clone is due only to the uptake of free CO₂. The problem concerning the high concentration of free CO₂ necessary for photosynthesis in Coccolithus huxleyi is discussed. It is shown that it is not due to lack of the enzyme carbonic anhydrase. Hence it is probable that the formation of coccoliths is the mechanism by which, in Coccolithus, the utilization of HCO₃^- ions in photosynthesis becomes possible. The OH^- ions produced during photosynthesis (₊Ca²⁺ taken up together with the HCO₃^- ions) are thus neutralized. This situation is different from other aquatics utilizing HCO₃^- in photosynthesis. Here the OH^- ions are neutralized via an ion exchange of Ca²⁺ with H⁺ in the surrounding medium.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

A comparison of the effects of Ca2+ and gibberellic acid on enzyme synthesis and secretion in barley aleurone

The effects of the addition and withdrawal of gibberellic acid (GA₃) and Ca²⁺ on enzyme synthesis and secretion by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. Incubation of layers in GA₃ plus Ca²⁺ affects the total amount of secreted α-amylase (EC 3.2.1.1) and acid phosphatase (EC 3.1.3.2) by promoting the appearance of different isoenzymic forms of these enzymes. The release of α-amylase isoenzymes 1–4 in response to GA₃ plus Ca²⁺ has a lag of 6 h. When layers are incubated in GA₃ alone for 6 h prior to the addition of Ca²⁺, isoenzymes 1–4 appear in the medium after only 30 min. When the addition of Ca²⁺ to layers pretreated in GA₃ is delayed beyond 12 h, its effectiveness in stimulating the synthesis and release of isoenzymes 3 and 4 is diminished. After 35 h of preincubation in GA₃, addition of Ca²⁺ will not stimulate synthesis of α-amylase isoenzymes 3 and 4. Aleurone layers preincubated for 6 h in GA₃ will respond to Ca²⁺ when the GA₃ is withdrawn from the incubation medium by producing α-amylase isoenzymes 1–4. The converse is not the case, however, since layers preincubated in Ca²⁺ for 6 h will not produce all isoenzymes of α-amylase when subsequently incubated in GA₃. The Ca²⁺-stimulated release of α-amylase from GA₃ pre-treated layers is dependent on the time of incubation in Ca²⁺ and the concentration of the ion. The response to Ca²⁺ is temperature-dependent, and other divalent cations such as Mg²⁺ cannot substitute for Ca²⁺. We conclude that Ca²⁺ influences α-amylase release by influencing events at the biochemical level.     

Role of calcium and calmodulin antagonist in photosynthesis and salinity tolerance inChlorella vulgaris

To cast light upon the role of Ca¹⁺ and calmodulin on photosynthetic rate (P_n), dark respiration (R_D) and amino acid and protein contents in salinity stressed and non-stressedChlorella cultures, the Ca²⁺ chelator EGTA [ethylene glycol-bis-(2-aminoethyl ether)-N,N- tetraacetate] and the calmodulin antagonist TFP (trifluperazine) were used. TFP markedly inhibited P_N while EGTA exerted a slight, if any, effect on P_N. NaCl tolerance, on the other side, was markedly abolished by TFP that inhibited P_N and lowered rate of proline accumulation. Calmodulin might be involved in osmoregulation and salt tolerance ofChlorella. R_D, however, was markedly enhanced by EGTA and Ca²⁺-free medium and hence the Ca²⁺ deprivation increased stress severity exerted by NaCl. Combinations of Na⁺ and Ca²⁺ enhanced P_N, decreased R_D and proline content in comparison with an osmotically equivalent reference culture containing only NaCl. Addition of Ca²⁺ to TFP treated cultures failed to reactivate calmodulin for proline synthesis. However, when Ca²⁺ was added to EGTA-treated cultures, only relatively reduced proline contents were recorded.     

Effect of 2-deoxy-d-Glucose on Aminoacids Metabolism in Rats’ Cerebral Cortex Slices

We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-¹⁴C]leucine, l-[1-¹⁴C]leucine and [1-¹⁴C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-¹⁴C]leucine and from [1-¹⁴C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO₂ production and lipid synthesis from l-[U-¹⁴C] leucine. This compound did not inhibit either CO₂ production, or lipid synthesis from [1-¹⁴C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-¹⁴C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO₂ production, or on lipid synthesis from l-[U-¹⁴C]lactate 10 mM and glucose 5.0 mM.     

Operation of the glycolate pathway in isolated bundle sheath strands of maize and Panicum maximum

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C₄ species was demonstrated from ¹⁴C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing ¹⁴CO₂ under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total ¹⁴C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO₂ fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since ¹⁴C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α-hydroxypyridine-methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [¹⁴C]-glycine was 90%. These results suggest that the pathway glycolate → glycine → serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells. Operation of ribulose-1,5-bisphosphate oxygenase activity in competition with ribulose-1,5-bisphosphate carboxylase is demonstrated by the lowering of total ¹⁴CO₂ fixation when O₂ is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose-1,5-bisphosphate labelling when the O₂ level was decreased. This was accompanied by an increase in CO₂ fixation. This could indicate an increased rate in synthesis of ribulose-1,5-bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.     

The role of cytosolic free calcium in the regulation of pyruvate dehydrogenase in synaptosomes

Calcium homeostasis and mitochondrial oxidative metabolism interact closely in brain and both processes are impaired during hypoxia. Since the regulation of the pyruvate dehydrogenase complex (PDHC) may link these two processes, the relation of cytosolic free calcium ([Ca²⁺]_i) to the activation state of PDHC (PDH_a) was assessed in isolated nerve terminals (i.e. synaptosomes) under conditions that alter [Ca²⁺]_i. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a and both responses required external calcium. Treatment with KCN, an in vitro model of hypoxia decreased ATP and elevated [Ca²⁺]_i and PDH_a. Furthermore, in the presence of KCN, PDH_a became more sensitive to K⁺ depolarization as indicated by larger changes in PDH_a than in [Ca²⁺]_i. The calcium ionophore Br-A23187 elevated [Ca²⁺]_i, but did not affect PDH_a. K⁺ depolarization elevated [Ca²⁺]_i and PDH_a even if [Ca²⁺]_i was elevated by prior addition of ionophore or KCN. Previous in vivo studies by others show that PDH_a is altered during and after ischemia. The current in vitro results suggest that hypoxia, only one component of ischemia, is sufficient to increase PDH_a. These data also further support the notion that PDH_a is regulated by [Ca²⁺]_i as well as by other factors such as ATP. Our results are consistent with the concept that PDH_a in nerve endings may be affected by [Ca²⁺]_i and that these two processes are clearly linked.Abbreviations (PDH_a) Activation state of the pyruvate dehydrogenase complex - ([Ca²⁺]_i) cytosolic free calcium concentrations - (MOPS) 3(N-morpholino)propanesulfonic acid - (fura-2AM) fura-2 acetoxymethyl ester - (AABS) p-(p-aminophenylazo)benzene sulfonic acid - (PDHC) pyruvate dehydrogenase complex - (TES) N-tris{[hydroxymethyl]methyl}-2-amino-ethanesulfonic acid - (SNK-test) Student-Newman-Keuls test     

Influence of calcium and magnesium on ethylene production by apple tissue slices

The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca²⁺, Mg²⁺, and Ca²⁺ plus Mg²⁺. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca²⁺ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.     

The Physiological Behaviour of IMR-32 Neuroblastoma Cells is Affected by a 12-h Hypoxia/24-h Reoxygenation Period

Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N₂: 2% O₂: 5% CO₂, v:v) by evaluating cell viability, modifications of NO, intracellular Ca²⁺ concentration [Ca²⁺]_i and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca²⁺]_i immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O₂ hypoxia induces variations of NO and [Ca²⁺]_i with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

Abstract: CO₂ fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with ¹⁴CO₂ and the incorporation of ¹⁴C into medium or cell extract products was determined. After chromatographic separation of ¹⁴C-labelled products, the fractions of ¹⁴C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the ¹⁴C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO₂ fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [¹⁴C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net ¹⁴CO₂ fixation, but did alter the distribution of ¹⁴C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or ¹⁴C incorporation). Low rates of conversion of α-[¹⁴C]ketoglutarate to [¹⁴C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.     

Analysis of the sensing and transducing processes implicated in the stomatal responses to carbon dioxide in Commelina communis L.

It has been previously debated whether CO₂ would depolarize the guard cell plasma membrane through malate‐mediated activation of the anion channel. Moreover, it has been assessed that the CO₂ signal would be transduced via cytosolic free Ca^{2 +} . In the present study, the CO₂ sensing and transducing processes were reinvestigated in Commelina communis (L.) mainly by studying how L ‐(–)‐malic acid and Ca^{2 +} flux modulators affected different CO₂ stomatal responses. L ‐(–)‐malic acid (1 m M ) inhibited by about 50% both CO₂‐induced stomatal closing and CO₂‐triggered inhibition of the stomatal opening response to the auxin indolyl‐3‐butyric acid. Stomatal closing in response to atmospheric CO₂ was strongly inhibited by the 1,4 dihydropyridines SDZ‐202 791 R(–) (SDZ (–)) and nifedipine, and this inhibition was attenuated by the 1,4 dihydropyridines SDZ‐202 791 S( + ) and S‐(–)‐BAY K8644. Suboptimal concentrations of the slow anion channel blockers 5‐nitro‐2,3‐phenylpropyllamine benzoic acid and anthracene‐9‐carboxylic acid increased the 50% inhibition of the CO₂ closing response by the Ca^{2 +} flux modulators SDZ (–) and 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N ′ N ′ ‐tetraacetic acid in a stronger manner than an additive one. Together, these results support the view that CO₂ is sensed through reducing proton extrusion. Moreover, they might suggest that the CO₂ signal is transduced through Ca^{2 +} signalling arising from depolarization‐mediated activation of a putative plasma membrane voltage‐gated L‐type Ca^{2 +} channel and for which the plasma membrane slow anion channel is a potential target.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.     

Role of anion exchange and thiol groups in the regulation of potassium efflux by lead in human erythrocytes

Pb²⁺ is thought to enter erythrocytes through anion exchange (AE) and to remain in the cell by binding to thiol groups. To define the role of AE mechanism and thiol groups in Pb²⁺ toxicity, we studied the effects of drugs and conditions that modify AE and that modify thiol groups on the ability of Pb²⁺ to stimulate potassium efflux as measured with ⁸⁶Rb. The most potent stimulation of ⁸⁶Rb efflux by Pb²⁺ occurred when conditions were optimal for the AE mechanism—that is, when bicarbonate was included in the buffer or a buffer made with Nal or NaCl rather than NaClO₄ or NaNO₃ was used. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid, potent inhibitors of the AE mechanism, completely inhibited stimulation of the ⁸⁶Rb efflux by Pb²⁺. These conditions or inhibitors did not affect stimulation of the ⁸⁶Rb efflux by ionomycin plus Ca²⁺. A role for Ca²⁺ channels was dismissed because the inorganic Ca²⁺ channel blockers, Cd²⁺ or Mn²⁺, did not prevent stimulation of ⁸⁶Rb efflux by Pb²⁺ but did inhibit stimulation by ionomycin plus Ca²⁺. ⁸⁶Rb efflux was more sensitive to Pb²⁺ if erythrocytes were treated for 15 min with thiol-modifying reagents that enter cells, such as iodoacetamide, N-ethylmaleimide, or dithiothreitol, than to reduced glutathione, a thiol-modifying reagent that is not permeable to the cell. Thus, in erythrocytes the AE mechanism and internal thiol groups are critical factors that affect the stimulation of a Ca²⁺-dependent process by Pb²⁺. © 1996 Wiley-Liss, Inc.     

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca²⁻]_i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca²⁻]_i and intracellular pH (pH_i) in Na⁺-containing and Na⁺-deficient bicarbonate/CO₂-buffered media to investigate the role of Na⁺ in these P-initiated changes. We found that P failed to initiate the AR in Na⁺-deficient medium, and that the initial rise in [Ca²⁺]_i following P (1 μg/ml) stimulation was similar for both media; however, the [Ca²⁺]_i in the Na⁺-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca²⁺]_i. Moreover, the differences in plateau [Ca²⁺]_i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca²⁺]_i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca²⁺]_i and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na⁺-dependent mechanism is important in the P-initiated human sperm AR. Such a Na⁺ requirement may reflect the involvement of this ion in pH_i regulation, as capacitated sperm that were incubated in a Na⁺-deficient medium for ≥ 30 min displayed a significantly lower pH_i. © 1996 Wiley-Liss, Inc.