Cytological study of ischemic damage in rat kidney

Reparative regeneration in different periods following nephrectomy and the application of low-dose radiation has been studied by means of light and electron microscopy. Experimentally induced ischemia resulted in the destruction of renal corpuscles, the perishing of tubular epithelial cells, and the proliferation of connective tissue. Reparative regeneration is based on aseptic inflammation and its phase duration depends on the extent of organ injury. In nephrectomized rats, reparative regeneration is accompanied by necrosis and the deposition of calcium in the cortical substance. Calcium plays an important role in kidney metabolism and its increased content is characteristic of degenerative changes. The experiments showed that the use of low-dose radiation does not accelerate the process of reparative regeneration in rat kidney.     

Oxidative phosphorylation in nuclei isolated from rat thymus

1. Nuclei were isolated from rat thymus in 0.25 M sucrose-3 mM CaCl₂. Fractionation of thymus tissue showed that the nuclear fraction could be contaminated with 10% intact cells at the most.

2. The nuclear fraction contained about 50% of the cellular ATP. Under anaerobic conditions nuclear ATP is degraded. When, after an anaerobic incubation, the nuclei are incubated under aerobic conditions endogenous nuclear ATP is resynthesized. The amount of ATP found cannot be accounted for by mitochondria or intact cells. It could also be shown that the ATP synthesis is not due to such contamination.

3. Nuclear ATP synthesis and respiration were inhibited by amytal, rotenone, and carbon monoxide. The latter inhibition was reversed by light. 2,4-Dinitrophenol abolished ATP synthesis and accelerated respiration. Oligomycin inhibited respiration and phosphorylation. The inhibition of respiration by oligomycin could be released by dinitrophenol. ATP synthesis was also inhibited by lewisite. This inhibition could be reversed by BAL. No inhibition, however, was found with arsenite or arsenite + BAL.

4. A P:O ratio between 0.6 and 1.0 was obtained for nuclear phosphorylation. This must be considered as a minimal value.

5. It is concluded that thymus nuclei are capable of respiratory-chain-linked oxidative phosphorylation.     

Nucleotides and coenzymes in nuclei isolated from rat liver.

In rat-liver nuclei, isolated by the non-aqueous technique, the concentrations and labelling rates of the purine moiety of acid-soluble nucleotides were determined and compared with corresponding data for non-fractionated tissue and nuclei-free cytoplasm. Livers were used from untreated rats, from rats with a highly stimulated synthesis of NAD and from rats following a heavy metabolic load with adenosine. Under all circumstances, the nuclear and cytoplasmic concentrations of nucleotides (e.g. ATP and its dephosphorylated forms, pyridine nucleotides) and of free glucose were practically identical. Specific radioactivities after a pulse with formate also indicated a nucleo-cytoplasmic equilibrium for purine-containing nucleotides. It is concluded that precursor pools for nuclear biosyntheses as well as energy supply for other nuclear activities may be determined by an analysis of the non-fractionated tissue.     

Age-associated changes in initiation of ribonucleic acid synthesis in isolated rat liver nuclei.

Initiation of RNA synthesis was studied in an attempt to determine a possible molecular mechanism for age-related biochemical and physiological changes. Initiation of RNA synthesis was determined by incorporation of [gamma-32 P]ATP and of [gamma-32P]GTP into an acid-insoluble product by intact nuclei isolated from livers of Sprague-Dawley CD-strain rats of various ages. When the rats were grouped into young (0.75-9 months) and old (12-30 months) rats, a significant decrease (P less than or equal to 0.001) in incorporation of initiating nucleotides was observed. The rat population was divided into five age groups (0.75-3 months, 4-9 months, 12-18 months, 19-23 months and 30 months) for further analysis of the effect of age on the initiation of RNA synthesis. Analysis of data from these groups indicated a significant trend for an age-related decrease in RNA-synthesis initiation (correlation coefficient = 0.94). Long-term hypophysectomy coupled with minimal hormone-replacement therapy was shown to have a significant effect on the reversal of the age-related decrease in initiation of RNA synthesis. It was observed that initiation of RNA synthesis in nuclei from 19-month-old rats, hypophysectomized at 12 months of age, was closest to that in 3-month-old intact rats and was not significantly different from that in liver nuclei of 0.75-9-month-old intact rats.     

Polyanion induced physical changes in isolated nuclei

Small angle light scattering techniques were used to determine the effects of selected polyanions on isolated mouse spleen nuclei. The light scattering measurements made it possible to distinguish nuclear swelling from lysis and/or aggregation. Heparin and dextran sulfate were shown to induce nuclear swelling. Chondroitin sulfate, although structurally similar to heparin, had no observable effect on the nuclei. Polystyrene sulfonate and pyran copolymer caused nuclear aggregation and lysis, respectively.     

Properties of isolated frog liver and kidney nuclei

In order for a nuclear preparation to be used for analytical purposes, the method of isolation and composition of the suspension medium must be carefully examined. Accordingly, satisfactory techniques for the isolation of frog liver and kidney nuclei were developed. The medium for frog liver nuclei consisted of: 55% glycerol, 0.001 M magnesium chloride, 0.033 M sodium β-glycerophosphate and/or 0.002 M KH₂PO₄, K₂HPO₄ (pH 6.8), however, the addition of 0.15 M sucrose was essential for satisfactory isolation of kidney nuclei. Inclusion of sucrose (0.15 M ) in the isolation medium promoted nucleolar swelling and a decrease in nuclear volume in liver cell nuclei. Nucleolar migration and extrusion were noted in solutions with high cationic content. The morphological appearance of isolated nuclei was found to be extremely sensitive to the ionic strength of the isolation medium, as was the isolation procedure in toto. Effects were considered to be the result of precipitation and swelling of nucleoprotein. Dissociation of nucleoprotein was considered to be associated with temperature change. The uptake of supra-vital dyes aided in recognition of the morphological alterations and was also an indicator of nuclear viability. Trypsin readily altered the nuclear membrane and a rapid decrease in nuclear density occurred, but the nucleolus remained intact. The diverse response of liver and kidney nuclei as compared with the nucleated red blood cells (a contaminant) to treatment with trypsin was noted and its implications discussed.     

Volume-regulatory potassium release from isolated perfused rat kidney

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.     

DNA degradation in isolated rat liver nuclei

Purified rat liver nuclei were incubated at 4°C. After different intervals of incubation aliquots of the nuclear suspension were taken and DNA was extracted by a common SDS-phenol-chloroform procedure. The fractionation of DNA by agar gel electrophoresis revealed large DNA fragments. It was shown that the well-known DNA degradation to monomers and its multiples is preceeded by an earlier breakdown of DNA into characteristic large fragments.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca²⁺ _i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca²⁺ _i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca²⁺ _i levels of the heart. The fluorescence ratio technique was applied to estimate Ca²⁺ _i. The disappearance of mechanical activity of the heart preceded that of the Ca²⁺ _i transient in the first 2 min of ischemia. The slope of upstroke of the Ca²⁺ _i transient, reflecting Ca²⁺ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca²⁺ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca²⁺ _i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca²⁺ _i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca²⁺ _i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca²⁺ _i handling and sustained depression of mechanical performance.