Cl- absorption in European eel intestine and its regulation

The intestinal epithelium of the euryhaline teleost fish, Anguilla anguilla, absorbs Cl(-) transepithelially. This gives rise to a negative transepithelial potential at the basolateral side of the epithelium and to a measured short circuit current. Cl(-) absorption occurs via bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransport, localized on the luminal membrane. The cotransport operates in parallel with a luminal K(+) conductance that recycles the ion into the lumen. Cl(-) leaves the cell across the basolateral membrane by way of Cl(-) conductance and presumably via a KCl cotransport. The driving force for this process is provided by the electrochemical sodium gradient across the plasma membrane, generated and maintained by the basolateral Na(+)-K(+)-ATPase. The resulting NaCl absorption process is active and enables marine fish to take up water, thereby compensating for water that was lost passively from the body. Fresh water acclimatized eel also absorb Cl(-) actively, although in smaller quantities, utilizing the same ion transport mechanisms as marine eels. This mechanism is basically the same as the model proposed for the thick ascending limb (cTAL). Cl(-) absorption is regulated by a number of cellular factors, such as HCO(3) (-), pH, Ca(2+), cyclic nucleotides, and cytoskeletal elements. It is sensitive to osmotic stress, and therefore is a good physiological model to study ion transport mechanisms that are activated when osmotic stress induces cell volume regulation. The activation of these various ion transport pathways is dependent on cellular transduction mechanisms in which phosphorylation events (mainly by PKC and MLCK for the hypertonic response) and cytoskeletal elements, either microfilaments or microtubules, seem to play key roles.     

Metabolic suppression in anoxic frog muscle

Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates – most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K⁺ ions ([K⁺]) in interstitial dialysates remained stable (2–3 mM) throughout anoxia and recovery. Interstitial [lactate⁻] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K⁺] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristic of hypoxia-sensitive systems. Accepted: 15 January 1998     

Accelerated phospholipid degradation in anoxic rat hepatocytes

Accelerated degradation of membrane phospholipids characterizes the reaction of rat liver and myocardial cells to ischemia. A similar disturbance in phospholipid metabolism was sought in anoxic hepatocytes. Primary cultures of adult rat hepatocytes were made anoxic by evacuation of the CO₂O₂ atmosphere with N₂. The resulting loss of ATP was reversible upon reoxygenation after periods of anoxia up to 2 h. With 3–4 h of anoxia, the cells were incapable of regenerating ATP levels. Loss of viability was also indicated by the inability of over 90% of the cells after 3–4 h to exclude trypan blue. The baseline rate of turnover of [¹⁴C]-ethanolamine or glycerol prelabeled phospholipids was then established. A constant rate of turnover was found for, at least, the first 3 days the cells were in culture. No loss of total phospholipid occurred during this time. Anoxia induced very significant differences in the fate of prelabeled phospholipids. With [¹⁴C]-ethanolamine there was a 30% loss of total cellular radioactivity within 4 h. Total phospholipids determined as lipid phosphate decreased by 20%. This depletion of cellular phospholipids was paralleled by an accumulation of hydrophilic degradation products in the culture medium. Phosphorylethanolamine accounted for 50% of these, with equal amounts of glycerophosphorylethanolamine and ethanolamine the other 50%. A similar accumulation in the medium occurred with [¹⁴C]-glycerol- and [¹⁴C]choline-prelabeled phospholipids. The accelerated degradation of phospholipid was accompanied by evidence of membrane dysfunction as shown by the loss of 50% of the glucose 6-phosphatase activity in whole cell homogenates. The results of these studies establish that anoxia induces in cultured rat hepatocytes a similar disturbance to phospholipid metabolism as does ischemia of the same cells in the intact animal. This implies that the deprivation of oxygen per se determines the characteristic reaction of cells to ischemia. This conclusion allows further analysis of the effects of O₂ deprivation on cultured hepatocytes as a new experimental model with which to further explore the effects of ischemia on cells.     

pH regulation in anoxic plants

BACKGROUND: pH regulation is the result of a complex interaction of ion transport, H+ buffering, H+-consuming and H+-producing reactions. Cells under anoxia experience an energy crisis; an early response thereof (in most tissues) is a rapid cytoplasmic acidification of roughly half a pH unit. Depending on the degree of anoxia tolerance, this pH remains relatively stable for some time, but then drops further due to an energy shortage, which, in concert with a general breakdown of transmembrane gradients, finally leads to cell death unless the plant finds access to an energy source. SCOPE: In this review the much-debated origin of the initial pH change and its regulation under anoxia is discussed, as well as the problem of how tissues deal with the energy crisis and to what extent pH regulation and membrane transport from and into the vacuole and the apoplast is a part thereof. CONCLUSIONS: It is postulated that, because a foremost goal of cells under anoxia must be energy production (having an anaerobic machinery that produces insufficient amounts of ATP), a new pH is set to ensure a proper functioning of the involved enzymes. Thus, the anoxic pH is not experienced as an error signal and is therefore not reversed to the aerobic level. Although acclimated and anoxia-tolerant tissues may display higher cytoplasmic pH than non-acclimated or anoxia-intolerant tissues, evidence for an impeded pH-regulation is missing even in the anoxia-intolerant tissues. For sufficient energy production, residual H+ pumping is vital to cope with anoxia by importing energy-rich compounds; however it is not vital for pH-regulation. Whereas the initial acidification is not due to energy shortage, subsequent uncontrolled acidosis occurring in concert with a general gradient breakdown damages the cell but may not be the primary event.     

Propionate metabolism and its regulation by fatty acids in ovine hepatocytes

Propionate metabolism was studied in ovine hepatocytes. The main products of metabolism were CO2, glucose, L-lactate and pyruvate. The fatty acids, butyrate and palmitate inhibited propionate oxidation; butyrate inhibited but palmitate slightly stimulated gluconeogenesis from propionate. Butyrate and palmitate also inhibited lactate and pyruvate production from both endogenous substrates and from propionate.     

Characterization of lipoprotein secreted by cultured eel hepatocytes and its comparison with serum lipoproteins.

The lipoprotein secreted by cultured eel hepatocytes was fractionated by density gradient ultracentrifugation and compared with eel serum lipoproteins. Eel hepatocytes were cultured for 7 to 10 days as a monolayer in Williams' medium E containing 5% fetal bovine serum and 0.16 microM insulin on a dish precoated with fibronectin of horse serum. The only lipoprotein secreted by eel hepatocytes was a very-low-density lipoprotein like one which consisted of 69% triglyceride, 15% phospholipid, 4% cholesterol, and 12% protein. On the other hand, very-low-density lipoprotein and high density lipoprotein were found in eel serum, in which high density lipoprotein was a main lipoprotein. The secreted lipoprotein contained apo B and apo A as the main protein components. Furthermore, the lipoprotein contained proapo A-I in addition to apo A-I, which was proved by comparing the amino acid composition of both proteins. In our discussion, we noted that the lipoprotein secreted by eel hepatocytes was a good material for the study of high-density lipoprotein formation.     

Participation of high-density lipoprotein in vitellogenesis in Japanese eel hepatocytes

To investigate effect of estradiol-17β (E₂) treatment in vivo on binding of eel hepatocytes to HDL, we developed hepatocytes binding assay. When hepatocytes were incubated with 200 times excess of eel HDL, the binding of hepatocytes to HDL precoated on wells was inhibited competitively. This indicates that eel hepatocytes bound specifically to HDL. E₂ treatment in vivo induced vitellogenin (VTG) synthesis. Hepatocytes prepared from the same E₂ treatment eel showed 3-fold higher ability of binding to HDL compared to hepatocytes prepared from ells without E₂ treatment. We also examined effects of E₂ and HDL on VTG induction in cultured hepatocytes. VTG, determined by enzyme-linked immunosorbent assay (ELISA), induction was about two-times higher in the presence of both 10⁻⁵ M of E₂ and 400 μg of HDL than in the presence of 10⁻⁵ M E₂ alone. At concentrations below 10⁻⁶ M E₂, VTG was not induced in the presence or absence of HDL. By SDS–PAGE and immunoblotting, VTG was detected only in the presence of both 10⁻⁵ M of E₂ and HDL. Our findings strongly support the idea that HDL correlates with vitellogenesis in eel liver.     

AMPA receptors undergo channel arrest in the anoxic turtle cortex

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.     

Metabolic regulation of APOBEC-1 Complementation Factor trafficking in mouse models of obesity and its positive correlation with the expression of ApoB protein in hepatocytes

APOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals.     

Developmental regulation of anoxic stress tolerance in maize

Anoxia associated with flooding stress is detrimental to plant growth and productivity. When maize seedlings 2 to 7 d old were exposed to anoxic stress, 3-d-old seedlings were found to have much lower tolerance than 2-d-old seedlings. Ninety per cent of 2-d-old seedlings survived 72 h of anoxic stress compared with 0% of the 3-d-old seedlings. Since 2-d-old isolated root tips survived anoxic stress better than 3-d-old tips, the anoxic tolerance of 2-d-old seedlings was independent of the translocation of nutrient reserves from the endosperm to the root. The addition of glucose to the medium improved the anoxia tolerance of 2-d-old seedlings by 25% but had no effect on 3-d-old seedlings. Acclimation by pre-cxposure to 4% oxygen and pre-treatment with 100mmol m^?1 abscisic acid (ABA) improved the anoxia tolerance of 3-d-old seedlings by 2- and 4-fold, respectively. However, acclimation and ABA treatment had no effect on 2-d-old seedlings. The results indicate that anoxia tolerance in maize is develop-mentally regulated. The mechanism of anoxia tolerance innate to 2-d-old seedlings was inducible in 3-d-old seedlings by acclimation or treatment with ABA.     

Metabolic regulation of fatty acid esterification and effects of conjugated linoleic acid on glucose homeostasis in pig hepatocytes

Conjugated linoleic acids (CLAs) are geometric and positional isomers of linoleic acid (LA) that promote growth, alter glucose metabolism and decrease body fat in growing animals, although the mechanisms are poorly understood. A study was conducted to elucidate the effects of CLA on glucose metabolism, triglyceride (TG) synthesis and IGF-1 synthesis in primary culture of porcine hepatocytes. In addition, hormonal regulation of TG and IGF-1 synthesis was addressed. Hepatocytes were isolated from piglets (n = 5, 16.0 ± 1.98 kg average body weight) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Hepatocytes were cultured in William's E containing dexamethasone (10-8 and 10-7 M), insulin (10 and 100 ng/ml), glucagon (0 and 100 ng/ml) and CLA (1 : 1 mixture of cis-9, trans-11 and trans-10, cis-12 CLA, 0.05 and 0.10 mM) or LA (0.05 and 0.10 mM). Addition of CLA decreased gluconeogenesis (P < 0.05), whereas glycogen synthesis and degradation, TG synthesis and IGF-1 synthesis were not affected compared with LA. Increased concentration of fatty acids in the media decreased IGF-1 production (P < 0.001) and glycogen synthesis (P < 0.01), and increased gluconeogenesis (P < 0.001) and TG synthesis (P < 0.001). IGF-1 synthesis increased (P < 0.001) and TG synthesis decreased (P < 0.001) as dexamethasone concentration in the media rose. High insulin/glucagon increased TG synthesis. These results indicate that TG synthesis in porcine hepatocytes is hormonally regulated so that dexamethasone decreases and insulin/glucagon increases it. In addition, CLA decreases hepatic glucose production through decreased gluconeogenesis.     

Metabolic differences in hepatocytes of obese and lean pigs

There are important differences in terms of metabolic activity, energy utilization and capacity of protein and fat deposition when Iberian and modern pigs are compared. Primary culture of hepatocytes was used to evaluate hepatic function and sensitivity to hormones between breeds without the interference of circulating blood factors. Hepatocytes were isolated from pure Iberian (n=10) and Landrace (n=8) pigs of similar BW (24.5±12.1 and 32.9±6.1 kg BW, respectively), by collagenase perfusion. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to serum-free medium for the remainder of the culture period. Hepatocytes were maintained in William’s E supplemented with β-mercaptoethanol (0.1 mM), glutamine (2 mM), antibiotics (gentamicin, penicillin, streptomycin and amphotericin B), dimethyl sulfoxide (1 µg/ml), dexamethasone (10⁻⁸ M), insulin (0.173 and 17.3 nM) and glucagon (0.287, 2.87 and 28.7 nM) for 24 to 48 h. Gluconeogenesis (GNG), glycogen degradation, triglycerides (TG) content and esterification, β-hydroxybutyrate (BHB) synthesis, IGF-1 synthesis, albumin and urea synthesis were determined. Iberian pigs had greater capacity of GNG than Landrace (24%, P<0.05), although no difference in glycogen degradation was found (P>0.10). TG content and esterification tended to be lower in hepatocytes from Iberian compared with Landrace pigs (12% and 31%, respectively; 0.10<P<0.05). Furthermore, addition of free fatty acids (CLA or linoleic acid, 0.2 mM) increased TG content (64%, P<0.001) although no difference between fatty acids was found. When free fatty acids were compared, a trend toward increased esterification (41%, P=0.078) was found for CLA. Although glucagon stimulated and insulin inhibited BHB synthesis, no difference between breeds was found (P>0.10). IGF-1 synthesis was diminished in hepatocytes from Iberian compared with Landrace pigs (16%, P<0.05). On the contrary, rate of albumin synthesis was greater in Iberian compared with Landrace pigs (58%, P<0.05). Finally, the capacity of urea synthesis was lower in hepatocytes of Iberian compared with Landrace pigs (37%, P<0.05). When ammonia was added to the media, urea concentration increased (648%, 1108% and 2791% when 0 mM was compared with 2.5, 5 and 10 mM, respectively). Urea synthesis increased on increasing ammonia content (55% and 325% when 0 mM was compared with 5 and 10 mM, respectively; P<0.0001). In conclusion, the genetic background accounts for important differences in protein and energy metabolism pathways found in primary culture of hepatocytes from lean and obese pigs.     

Metabolic effects of D-glyceraldehyde in isolated hepatocytes.

The effects of D-glyceraldehyde on the hepatocyte contents of various metabolites were examined and compared with the effects of fructose, glycerol and dihydroxyacetone, which all enter the glycolytic/gluconeogenic pathways at the triose phosphate level. D-Glyceraldehyde (10 MM) caused a substantial depletion of hepatocyte ATP, as did equimolar concentrations of fructose and glycerol. D-Glyceraldehyde and fructose each caused a 2-fold increase in fructose 1,6-bisphosphate and the accumulation of millimolar quantities of fructose 1-phosphate in the cells. D-Glyceraldehyde caused an increase in the glycerol 3-phosphate content and a decrease in the dihydroxyacetone phosphate content, whereas dihydroxyacetone increased the content of both metabolites. The increase in the [glycerol 3-phosphate]/[dihydroxyacetone phosphate] ratio caused by D-glyceraldehyde was not accompanied by a change in the cytoplasmic [NAD+]/[NADH] ratio, as indicated by the unchanged [lactate]/[pyruvate] ratio. The accumulation of fructose 1-phosphate from D-glyceraldehyde and dihydroxyacetone phosphate in the hepatocyte can account for the depletion of the intracellular content of the latter. Presumably ATP is depleted as the result of the accumulation of millimolar amounts of a phosphorylated intermediate, as is the case with fructose and glycerol. It is suggested that the accumulation of fructose 1-phosphate during hepatic fructose metabolism is the result of a temporary increase in the D-glyceraldehyde concentration because of the high rate of fructose phosphorylation compared with triokinase activity. The equilibrium constant of aldolase favours the formation and thus the accumulation of fructose 1-phosphate.     

Metabolic activity in primary cultures of fish hepatocytes

In aquatic toxicology, isolated liver cells from fish can be used as a tool to generate initial information on the hepatic metabolism of xenobiotics, and on the mechanisms of xenobiotic activation or deactivation. This isolation of teleost liver cells is achieved by enzymic dissociation, and monolayer cultures of fish hepatocytes in serum-free medium maintain good viability for 3-8 days. During in vitro culture, fish liver cells express stable levels of phase I and phase II enzymes, such as cytochrome P4501A or glutathione S-transferase, and the cells show an induction of biotransformation enzymes after exposure to xenobiotics. The xenobiotic metabolite pattern produced by fish hepatocytes in vitro is generally similar to that observed in vivo. Limitations to more-intensive application of cultured fish hepatocytes as a screen in aquatic hazard assessment are partly due to the rather limited scope of existing studies, i.e. the focus on one particular species (rainbow trout), and on one particular biotransformation enzyme (cytochrome P4501A), as well as a lack of comparative in vitro/in vivo studies.     

Inotropic effects of adrenaline on the anoxic or hypercapnic myocardium of rainbow trout and eel

Summary The effects of adrenaline on the development of force under anoxia and hypercapnic acidosis (13% CO₂ in 30 mM HCO ₃ ^– ) were examined in isolated, electrically stimulated cardiac ventricle strips of rainbow trout and eel.During anoxia or acidosis applied 15 min in advance, the adrenaline concentration of the bathing solution was increased in 5 steps from 0 to 10^–4 M with 5 min at each step. Before levelling off the contractile tension increased by 145±42% (mean±SE) in the anoxic, 80±14% in the acidotic and 152±41% in the control trout cardiac strips. Except for the acidotic strips the corresponding values tended to be lower for the eel strips being 46±9%, 57±17% and 57±9%, respectively. The inotropic affinity for adrenaline was lower in the trout than in the eel myocardium. For the trout myocardium it remained unchanged, while it decreased somewhat for the eel myocardium under anoxia or acidosis.Adding to the muscle bath 10^–5 M adrenaline resulted in an increase in force development by about 90% for the trout myocardium and 50% for the eel myocardium. 5 min later anoxia or hypercapnic acidosis was applied for 30 min followed by 30 min at control conditions. Relative to the force values recorded just before anoxia or acidosis was applied, the changes in contractile force during these periods were the same with and without adrenaline. Thus adrenaline appears to have a persistent positive inotropic effect in the fish myocardium during and after oxygen lack or acidosis.