Jorge J. Gumucio

Physiologic significance and regulation of hepatocellular heterogeneity

Since the concept of hepatocellular heterogeneity was proposed a large body of evidence has accumulated that supports the notion that hepatocytes differ according to their location in the hepatic acinus. Several reviews (l-4) have summarized the numerous studies that have provided evidence that there is morphologic, biochemical, and functional (transport and metabolic) heterogeneity of hepatocytes. What is lacking, however, is a broad understanding of the physiologic meaning of this heterogeneity and the mechanisms by which it is established and maintained. Therefore, the objective of this review is twofold. First, several areas of liver function in which zonal differences in hepatocytes have been established will be analyzed to determine how the observed heterogeneity of hepatocytes may be important for the normal physiologic function of the liver. The areas to be discussed include the metabolism of glucose and ammonia, uptake of substances from sinusoidal blood, and bile secretion. Second, the mechanisms that may be responsible for the regulation of hepatocyte heterogeneity will be discussed. This analysis will draw mainly on data derived from experiments investigating the molecular mechanisms of the heterogeneous expression of the cytochrome Ph5” enzymes within the hepatic acinus. We have attempted to provide not an exhaustive review of the abundant descriptive data on hepatocellular heterogeneity, but rather a biological framework for understanding the functional significance of liver cell heterogeneity, while acknowledging that aspects of this proposal are speculative.

Quantitative Morphology of the Sinusoids of the Hepatic Acinus: Quantimet Analysis of Rat Liver

The transport of solutes from sinusoidal blood into the hepatocyte depends on the interaction of solutes with membranes. Several factors determine the probability of such interaction, among them being the configuration of the solute carrying space, the liver sinusoids. Thus, a quantitative morphologic study of hepatic sinusoids was carried out to determine the structural characteristics of the sinusoids of the hepatic acinus, the microcirculatory unit of the liver. Rat livers were perfusion-fixed, paraffinembedded, and sectioned at 5 µm. The volume fraction and surface/volume ratio of sinusoids were determined in each acinar zone by means of an automatic image analyzing system, the Quantimet 720. In separate experiments, the effect of route and pressure of perfusion on these parameters was studied. The variability associated with instrument stability, thresholding and section thickness was also assessed, and the reproducibility of measurements was determined. It was found that the structure of the sinusoids of acinar zone 1 (periportal) was quantitatively different from that of zone 3 (perivenular). While the volume fraction of sinusoids was significantly greater in the perivenular area of zone 3, the surface/volume ratio of zone 1 sinusoids was significantly larger than that of zone 3. Both findings suggested a structural organization favoring a greater probability of solute-membrane interactions in zone 1, the acinar inlet.

Demonstration by in situ hybridization of the zonal modulation of rat liver cytochrome P-450b and P-450e gene expression after phenobarbital

The various physiological processes that constitute liver function are compartmentalized within the hepatic acinus. The molecular mechanisms modulating the development and maintenance of this hepatocyte heterogeneity have not been defined. The objective of this study was to determine whether transcriptional or posttranscriptional zonal modulation of cytochromes P-450b,e gene expression was responsible for the heterogeneous induction of the P-450 proteins, which is observed after phenobarbital (PB) administration. The exact localization in liver tissue of hepatocytes responding to PB with induction of either P-450b,e mRNA or proteins was established by in situ hybridization and by immunofluorescence, respectively. As demonstrated by quantitative assessment of autoradiographs of approximately 20 hepatocytes located between a terminal portal venule and a hepatic venule, PB induced the P-450b,e mRNA up to sixfold in the 12-15 hepatocytes located closer to the hepatic venules (zones 2 and 3). In contrast, there was only a twofold induction in the 4-6 hepatocytes surrounding the terminal portal venules (zone 1). Quantitative immunofluorescence using an MAb showed that the acinar distribution of PB-induced P-450b,e proteins was similar to that of the mRNA. This combined approach indicated that, most likely, an increased rate of transcription of cytochromes P-450b,e genes in hepatocytes of zones 2 and 3 concomitantly, with a relative lack of activation, or repression, of these genes in hepatocytes of zone 1, were responsible for the heterogeneous phenotype observed after PB administration. Therefore, modulation of gene expression among hepatocytes of the liver acinus is one mechanism by which the functional heterogeneity of hepatocytes is attained. Experiments in which the induction of cytochromes P-450b,e genes was studied after administration of either PB or para-hydroxyphenobarbital, a main hepatic metabolite of PB, suggested that the species involved in the inductive process is the parent PB molecule rather than para-hydroxyphenobarbital.

Bile Salt Transport after Selective Damage to Acinar Zone 3 Hepatocytes by Bromobenzene in the Rat

To quantitate the contribution of acinar zone 3 hepatocytes (centrilobular cells) to the transport of physiological concentrations of taurocholate, selective acinar zone 3 cell damage was induced in female rats by the administration of bromobenzene. Forty-eight hours later, livers were perfused in situ. Oxygen consumption, gluconeogenesis, transport of [14C]taurocholate into bile, bile flow and capacity for removal of indocyanine green were assessed. The removal of single doses of [14C]taurocholate injected into the portal vein at loads of 0.025 and 0.11 μmol/kg was then measured. Bromobenzene-treated livers removed 80–85% of the injected dose while control livers removed about 98%. The percentage of the injected dose removed by each group at each load of taurocholate remained fairly constant. In separate experiments, [14C]taurocholate was administered in successive single injections at loads between 0.20 and 75 μmol/kg. The percentage of the injected dose removed remained constant and was similar to that observed at lower loads of taurocholate until a dose of 1.0 μmol/kg was used. At higher loads, the percentage of the injected dose removed decreased progressively, suggesting that the uptake process was reaching saturation. These results showed that acinar zone 3 hepatocytes contributed to the removal of 13–18% of a physiological load of taurocholate. Furthermore, a 50% decrease in bile flow was observed after bromobenzene administration. This suggested that damage to the centrilobular area is followed by alterations in bile flow and by the passage of at least 13–18% of the bile salts reaching the acinus into the general circulation. In addition, taurocholate removal after carbon tetrachloride-induced damage to the centrilobular area was studied. Twelve to eighteen percent of the injected dose of [14C]taurocholate was found in the hepatic vein effluent, values similar to the results obtained after bromobenzene-induced damage.

Bile Acid Metabolism

Transport and metabolism of bile acids by the liver have been the subject of several recent reviews.1–3 In this chapter, however, the problem is analyzed in a different context. An attempt is made to integrate the development of these functions in the fetal and newborn liver with the transport and metabolic regulation of bile acids observed in the adult liver. Moreover, these processes are analyzed in the context of the functional heterogeneity of the hepatic parenchyma observed in both the fetal and the adult liver. While significant progress has been made recently in this area, the available data are still scarce. Many more questions can be raised than answers given in dealing with the transport and metabolism of bile acids by the functional units of the fetal liver or by the zones of the adult hepatic acinus. We hope this review will stimulate research in these areas.

A Double Embedding Technique For The Electron Microscopic Analysis Of The Liver Acinus

A double embedding technique allowing accurate and systematic sampling of zones of the hepatic acinus by electron microscopy was developed. The method involved the primary embedding of large (10 mm × 30 mm × 0·5 mm) liver slices and the preparation of 10 μm thick tissue sections by means of a rotary microtome with a steel knife. Veins 20–40 μm in diameter, the landmarks of the hepatic acinus, were localized by light microscopy, dissected from surrounding tissue and re‐embedded for ultramicrotomy. The method facilitated the systematic evaluation of hepatocyte ultrastructure in each zone of the microcirculatory unit of the liver, the hepatic acinus.

Effect of Hypokalemia on the Reductive Amination of α-Ketoglutarate by Mitochondria from Normal and Cirrhotic Rat Liver

The reductive amination of α-ketoglutarate by glutamic dehydrogenase with malate as the hydrogen donor has been studied under anaerobic conditions, with added adenosine triphosphate, in normal and cirrhotic rat liver mitochondria isolated from normokalemic and hypokalemic animals. The formation of glutamate and aspartate, the products of the reductive amination of α-ketoglutarate, was measured as an index of the amount of ammonia incorporated. There was no difference in the amount of ammonia incorporated by normal and cirrhotic rat liver mitochondria. Hypokalemia lowered the ability of liver mitochondria to incorporate ammonia by the reductive amination of α-ketoglutarate in normal animals, and more strikingly in cirrhotic animals, although the concentration of potassium in the ,.mitochondria was unchanged. Incubation of mitochondria from hypokalemic animals with potassium partially restored the ability to incorporate ammonia, but without a significant change in the total mitochondrial potassium concentration.

The steady-state relaxation time and the product at equilibrium

1. 1. According to Bucher and his co-workers, the steady-state relaxation time (i.e. the reciprocal of the first-order process by which an enzyme reaction reaches equilibrium) determines, in part, the apparent metabolic flow rate through a given enzymatic step in vivo. 2. 2. A new method for the determination of the kinetic constants required for the calculation of the apparent metabolic flow rates according to Bucher, is developed. The method is based on the measured concentrations of substrate or product in the course of incubation, and has been adapted for use by a digital computer. 3. 3. The relevance of the data thus obtained to the calculated metabolic flow rates are briefly discussed.

Possible nizatidine-induced subfulminant hepatic failure

Subfulminant hepatic failure has not been reported to occur with the histamine-2 receptor antagonists. We report a possible case of nizatidine-induced subfulminant hepatic failure leading to the eventual development of cirrhosis.

Pulmonary Hypertension and Liver Disease

Excerpt To the Editor:We read with interest the article by Goldsmith and coworkers (1) on primary pulmonary hypertension in five patients with classic hemophilia. Pulmonary hypertension is a recogn...