The Fine Structure, Potassium Content, and Respiratory Activity of Isolated Rat Liver Parenchymal Cells Prepared by Improved Enzymatic Techniques

Abstract

Further modifications of the enzymatic technique for the preparation of isolated, intact, parenchymal cells from rat liver as previously described by this laboratory are presented together with a detailed account of several critical factors involved during the procedure . In addition, the fine structure of the cells as revealed by electron microscopy and the characteristics of their respiratory activity in different media and with several added substrates are described . It is shown that cells obtained by adding calcium during the preparative procedure retain approximately 34% more potassium than cells prepared solely in a calcium-free medium . The former cells also demonstrate a higher respiratory activity, which is not due to uncoupling of respiration . Electron microscopy reveals that the cells have an intact plasma membrane and well-preserved intracellular organelles . Glycogen particles are observed in all cells and are particularly abundant when either 20 mM pyruvate is added during the preparation or Eagle's Minimum Essential Medium is employed .

Oxygen Consumption and Oxidative Capacity of Hepatocytes from Young Male Obese and Nonobese Zucker Rats

“…Howard et al (19) report that this method yields almost entirely parenchymal cells with high glycogen and potassium contents, and high respiratory activity that is coupled. The values of hepatocyte oxygen consumption reported by us are consistent with reports of others for isolated hepatocytes (19,20,22), liver slices (23,24), and perfused livers (22) when data from various reports are recalculated to the identical mode of expression. The range of calculated values range from approximately 1 3 to 25 nmole/min/ 1 O6 cells.…”

“…A small piece of liver was saved for protein determination. Liver cells were isolated by a modification of the method of Howard et al (19) because it avoids the use of anesthesia that is required by the in vivo perfusion methods (20). The liver was perfused by syringe via the portal vein with 20 ml of ice-cold calcium-free Krebs-Ringer bicarbonate medium (KRB) (21), pH 7.4, which contained 0.05% collagenase, 0.1 % hyaluronidase, 5 m M glucose, 1 m M pyruvate, and 20 mlM 4( 2-hydroxyethy1)-1 -piperazineethanesulfonic acid (Hepes), and gassed with 95% 02-570 C 0 2 .…”

Isolated guinea pig adrenocortical cells in vitro: Morphology and steroidogenesis in control and ACTH‐treated cultures

“…Polar compounds were not separated on this system. Each value represents the average of determinations from duplicate disappointingly low at first glance, this level of recovery is similar on a per-gram basis to that obtained with large organs such as t4e liver (Howard et al, 1973;LaBrecque and Howard, 1976), and as noted above, is comparable with other protocols for the rat adrenal.…”

Metabolism of 2-Oxo-Acid Analogues of Leucine and Valine in Isolated Rat Hepatocytes

“…The animals were sacrificed under ether anesthesia, the livers quickly excised and chilled in ice-cold buffer. Liver cells were isolated according to Howard et al' 12 '. Viability of cells was more than 90% as judged from trypan blue exclusion.…”