Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Preparation of isolated rat liver cells.

1.

Ventricular myocytes were isolated by means of collagenase and hyaluronidase. Hearts of rats or guinea pigs were perfused in a retrograde manner; bovine ventricular tissue was incubated in from of tissue chunks.




2.

In “low Ca medium” (aCa=1 μM), the myocytes beat spontaneously. Resting potentials of −4 mV and input resistances of 1 MΩ, associated with a fast decay of aKi, indicated “hyperpermeability” of the cell membrane. Within 2 h, the cells aged: they granulated (swelling of mitochondria), contracted, rounded up and finally disaggregated. When the superfusate was changed to Tyrode solution (3.6 mM CaCl2), 90–98% of those cells responded with the “Ca paradox”. Initially, aCai increased from 0.2 μM to >10 μM, and the sarcomere length (SL) shortened to <1.5 μm. Within the following 3 min aCai renormalized but the contracture was sustained. After another 5 min, the SL shortened to <1.2 μm, but a delay of 1–2 min passed before aCai started to increase towards aCa0. The 2–10% Ca tolerant cells beat spontaneously. Their resting potentials were between −10 and −70 mV, their input resistances between 4 and 20 MΩ. Repolarization by anodal current flow could restore the sodium spike but not the plateau of the action potential.




3.

Freshly prepared myocytes were pre-incubated in a KB medium composed of 85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 5 mM Na2ATP, 5 mM pyruvate, 5 mM succinate, 5 mM β-OH-butyrate, 5 mM creatine, adjusted with KOH to pH 7.2 and with EGTA to pCa 7.5. The minimum time of preincubation was 1 h, the maximum 5 days when the cells were stored at 5°C. The conditioning with the KB medium improved both Ca tolerance and electrophysiology significantly. About 70% of the rod shaped myocytes were Ca tolerant. These cells had resting potentials between −75 and −90 mV, the membrane resistances were around 7 kΩ×cm2. The action potentials showed well pronounced plateaus and lasted for 125 ms (rat), 300 ms (guinea pig) or 400 ms (bovine). Their shape showed the typical species related pecularities.




4.

We discuss that both ageing and Ca intolerance of the un-conditioned cells result from Ca overload through the hyperpermeable membrane, and that Ca overload causes ATP depletion via mitochondrial disfunction. The constituents of the KB medium may act to prevent these processes.

Calcium tolerant ventricular myocytes prepared by preincubation in a "KB medium".

Rationale:Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of...

A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart.

The roles of changes in cellular redox, interorgan lactate flux and balance, and quantitative aspects of lactate metabolism in the pathogenesis of lactic acidosis are discussed. Altered metabolism of pyruvate is central to the development of lactic acidosis and hyperlactatemia. Lactic acidosis occurs as a result of a relative or absolute imbalance in lactate production and utilization. Lactate utilization for oxidative purposes and for the resynthesis of glucose is essential for the maintenance of acid-base balance. Because of its role in lactate homeostasis the liver may play a central role in acid-base balance. Impairment of hepatic utilization of lactate may produce lactic acidosis.

Lactate homeostasis and lactic acidosis.

Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells

Morphologically intact muscle cells were prepared by perfusing adult rat hearts with a balanced salt medium containing 0.1% collagenase and 0.2% hyaluronidase. Yields of intact cells representing up to 25% of the weight of the heart were obtained. The cells separated along the line of the intercalated discs, with cleavage of desmosomes but with tearing of the plasma membrane in the region of the gap junction, so that when two contiguous cells were parted, the gap junction was retained intact attached to one of the cells. The fine structure of undamaged cells was indistinguishable from that of normal myocardial cells in situ, whereas damaged cells characteristically revealed numerous cytoplasmic vacuoles, clumping of myofilaments, and blebbing of the cell membrane, but morphologically normal mitochondria. The earliest lesion detected was a dilatation of the T (transverse tubular) system. Respiration in the intact cells was linear for 30 to 60 minutes and approximately two to three times the rate observed w...

Morphology and metabolism of intact muscle cells isolated from adult rat heart.

Fluoromalate (an inhibitor of malate dehydrogenase and the malate carrier) or difluorooxaloacetate (an inhibitor of aspartate aminotransferase) inhibited gluconeogenesis from both pyruvate and lactate, but had no effect on glucose formation from fructose or endogenous substrates. When cells were incubated with both fluorocarboxylic acids simultaneously, an additive effect on the inhibition of glucose formation from lactate was observed, but the inhibition was not additive when pyruvate was the glucogenic precursor. Pyruvate removal was reduced 15% by difluorooxaloacetate and 44% by fluoromalate. Lactate production from pyruvate was inhibited 36% by fluoromalate but was almost unaffected by difluorooxaloacetate.



The results suggest that both malate-oxaloacetate and glutamate-aspartate shuttles participate, but to different extents, in glucose formation from either pyruvate or lactate. The malateoxaloacetate shuttle predominates during gluconeogenesis from pyurvate whereas the glutamateaspartate shuttle is of greater importance when lactate is the gluconeogenic precursor. However, the inhibition by fluoromalate of glucose formation from lactate indicates that not all the reducing equivalents arising during lactate oxidation are directly available for the reductive step of gluconeogenesis.



Neither of the two shuttles seems to compensate for inhibition of the alternative system. This lack of compensation implies that each shuttle is working close to or at its maximal capacity. Hence, transfer processes between mitochondria and cytoplasm must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. The failure of fluoromalate to inhibit ethanol oxidation suggests that alternatives to the malate-oxaloacetate shuttle exist for the transfer to the mitochondria of reducing equivalents generated in the cytoplasmic compartment.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1972.tb01850.x

Rate‐Limiting Steps of Gluconeogenesis in Liver Cells as Determined with the Aid of Fluoro‐Dicarboxylic Acids

LTHOUGH the association of hyperlactatemia with hyperventilation and A hypocapnia has been known for many years,l the cause of the lactate accumulation has not been established. A rising blood lactate concentration represents an imbalance between the rate of formation and the rate of disposal of lactic acid in various tissues. Available data indicate that erythrocytes and skeletal muscle are probably the chief sites of lactate production and that the liver, kidney, heart and brain are involved in its removal from the blood.” The liver has a large capacity for lactate uptake, and normal blood lactate levels can be found even in the presence of severe hepatic disease.“,4 Massive infusion of lactate fails to produce a persistent hyperlactatemia unless hepatic function is grossly impaired.“,” In the current study the possibility that lactate accumulation associated

Lactic Acid Metabolism in Hyperventilation, Metabolic Alkalosis and Shock

In order to identify rate-limiting processes of hepatic sorbitol and glycerol metabolism in the hyperthyroid state, suspensions of isolated liver cells were prepared from thyroxine-treated rats. Rates of utilization of sorbitol and glycerol by these cells were compared with the data obtained in previous studies with euthyroid animals. At saturating substrate concentrations hydrogen flux from sorbitol or glycerol to O2 was increased 50–60% over control values to 2.7–2.8 μmol × g−1× min−1 following thyroxine treatment. When cells were exposed to two sources of reducing-equivalents by incubation with sorbitol and glycerol in combination, the rate of hydrogen flux to O2 was increased about 160% above control levels to 4.7 μmol × g−1× min−1. However, this value was only 85% of the sum of the flux rates in cells incubated with sorbitol and glycerol separately, since each member of the substrate mixture partially inhibited the uptake of the other. Pyruvate, added as a cytoplasmic hydrogen acceptor, did not enhance sorbitol and glycerol uptake by liver cells from thyroxine-treated rats except when the cells were incubated with these substrates in combination. In this circumstance pyruvate addition overcame the inhibition of sorbitol uptake by glycerol. It is inferred from these findings that in the hyperthyroid state the transfer of reducing-equivalents from substrate to O2 is not rate-limiting for the hepatic metabolism of sorbitol or glycerol individually, as it is in control animals, but it remains the rate-limiting process when liver cells from thyroxine-treated rats are incubated with mixtures of these substrates.



Glucose was the major end-product of sorbitol and glycerol metabolism in cells from both euthyroid and hyperthyroid rats. As anticipated, therefore, rates of gluconeogenesis in cells from thyroxine-treated rats were increased in a manner corresponding to the enhanced capacity for hydrogen translocation. Maximal rates of glucose formation (3.39 μmol × g−1× min−1) were observed when cells were incubated with a mixture of sorbitol, glycerol and pyruvate.



It was found that up to 75% of the hydrogen flux to O2 during sorbitol and glycerol metabolism could utilize an antimycin-sensitive, rotenone-insensitive pathway, most likely involving mitochrondrial glycerolphosphate dehydrogenase. Less than 10% of the reducing-equivalent flux in these experiments was mediated by NAD-linked shuttles involving malate dehydrogenase.



Significant stimulation of hydrogen translocation was observed at 12 h after thyroxine administration and reached a plateau about 4 days after the initial thyroxine injection. This timecourse is consistent with the hypothesis that the effects of thyroxine are mediated through new protein synthesis and may reflect the time-course for the induction of mitochondrial glycerolphosphate dehydrogenase. It is suggested that induction of this enzyme may represent an example of a general action of thyroxine to increase the capacity of the carrier systems which mediate intercompartmental hydrogen and metabolite translocation and thereby stimulate hepatic intermediary metabolism.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03342.x

Stimulatory effects of thyroxine administration on reducing-equivalent transfer from substrate to oxygen during hepatic metabolism of sorbitol and glycerol.

Energy-dependent reduction of pyruvate to lactate by intact isolated parenchymal cells from rat liver

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