Benjamin F. Trump

Cell death and the disease process. The role of calcium

The cellular processes involved in the death of the cell are of vital importance to biology and medicine: in biology because they occur as a normal part of the economy of every organism even during development and, of course, in its ultimate demise, and in medicine because cell death forms an important part of virtually every disease. We have been studying the events associated with cellular death for over 20 years and, in this chapter, will briefly review not only the knowledge that we have gained but also correlate it with data obtained from other laboratories. In addition, we will review our current working hypothesis concerning ion redistribution, particularly calcium, and their effects which lead to cell death.

HgCl2-induced changes in cytosolic Ca2+ of cultured rabbit renal tubular cells

Fura 2 was used to measure changes in cytosolic [Ca2+] ([Ca2+]i) in cultured rabbit kidney proximal tubule cells exposed to HgCl2. Treatment with 2.5-10 microM HgCl2 resulted in an extracellular [Ca2+] ([Ca2+]e)-independent 2- to 12-fold increase in [Ca2+]i above resting levels of about 100 nM. Treatment with 25-100 microM HgCl2 caused a rapid [Ca2+]e-independent 10- to 12-fold increase in [Ca2+]i within 1 min followed by a recovery to about 2-fold steady state by 3 min. With 25-100 microM HgCl2, both magnitude and rate of Ca2+ increase were similar, but recovery was greater with increasing doses. A slower, secondary increase in [Ca2+]i followed which varied with HgCl2 concentration and required [Ca2+]e. The first increase in [Ca2+]i represents release from intracellular pools. Calcium channel blockers, calmodulin inhibitors, and mitochondrial inhibitors do not alter the patterns of [Ca2+]i changes due to HgCl2. The recovery response with higher HgCl2 concentrations appears to be triggered by Hg2+ and not by the increased [Ca2+]i. Sulfhydryl modifiers N-ethylmaleimide, PCMB and PCMBS produced [Ca2+]e-independent [Ca2+]i increases similar to those induced by low HgCl2 concentrations. Cell killing with HgCl2 was about 50% greater with normal [Ca2+]e than with low [Ca2+]e, suggesting that [Ca2+]e influx is important in accelerating injury leading to cell death.

Studies of Ultrastructure, Cytochemistry, and Organ Culture of Human Bronchial Epithelium

In these studies human bronchi removed at time of surgery of “immediate autopsy” from both cancer and non-cancer patients are studied by light and electron microscopy and cytochemistry and cultured in vitro with serial studies of morphology and metabolism. Segments removed from humans are cultured in CMRL 1066 containing insulin and hydrocortisone with or without 5% inactivated fetal calf serum and with or without addition of vitamin A. Segments have been stored at 0–4°C in L-15 medium for up to 2 days prior to successful culture. Cultures are carried out in plastic Petri dishes on rocking platforms in chambers gassed with 45% oxygen, 50% nitrogen, and 5% CO2. The epithelial preservation can be seen by both light and electron microscopy and cultured tissues show normal areas of metaplasia and dysplasia. Specimens have so far been maintained for as long as 4 months with good maintenance of ultrastructure.

Chapter 1 Methods of Organ Culture for Human Bronchus

Publisher Summary This chapter discusses the methods of organ culture for human bronchus. One goal of organ culture is the maintenance of in vivo characteristics and specific cellular relationships. Both the human and bovine trachea and bronchus were cultured as 1-cm-square pieces in 60-mm Falcon plastic petri dishes with the epithelial surface uppermost. The culture medium was added to just reach the epithelial surface of the explant. Normal human bronchial epithelium is composed of a single layer of pseudostratified cells. The tall, columnar ciliated cells are the most numerous. Tall, columnar mucous cells are of two types: (1) goblet cells whose apices are dis tended with large mucous granules and (2) small mucous granule cells that have tiny mucous granules in the apex and are invisible by light microscopy unless stains for mucosubstances are used. The xeno-transplant studies show that the culture-induced metaplastic changes are reversible, because when such CMRL-cultured explants are transplanted into nude mice, the epithelium reverts to normal and columnar differentiation takes place and is maintained for over 1 year.

The Network of Intracellular Membranes

If the external membrane organizes the cell as a unit, the internal membranes make possible the differentiation of activities within the cell, greatly increasing its working surface. An overview of the membranes that define the organelles within the cell sap notes they are structurally of two types, relates structure to function, and describes the processes by which the cytoplasmic bodies communicate with each other and the “outside.”

The Measurement of Free Calcium to Assess Cellular Function and Injury Using Digital Imaging Fluorescence Microscopy

The purpose of this chapter is to introduce the technology involved in digital imaging fluorescence microscopy (DIFM) and its application to the study of toxic cell injury. A compelling issue in toxicologic pathology is the characterization of cellular and molecular events as they occur in the living cell following injury. The development of phase and Nomarski techniques for light microscopy has greatly improved the solution to this problem. Electron microscopy and analytical electron microscopy has enabled visualization of cell membranes and organelles at fixed points in time following an injury and has permitted correlation with observations of living cells in real time using phase or Nomarski microscopy often coupled with time-lapse cinematography or video microscopy. An important limitation of microscopy of living cells, however, has been that low levels of illumination and contrast problems have been difficult to solve and that information on chemical changes in the cell during phase microscopy has not been possible. Even the introduction of fluorescent probes has alone not solved these problems because of the phenomenon of photobleaching at normal levels of illumination. Moreover, the problems of contrast in examining the faint images of organelles were difficult to overcome even with advances in the sensitivity of photographic techniques. The development of DIFM has provided new opportunities for studying dynamic cellular events, particularly when coupled with traditional methods of fluorescence microscopy (for review see Taylor and Salmon, 1989).

Ion Deregulation, Cell Injury and Tumor Promotion

The purpose of this paper is to critically review and analyze the literature concerning the role of cellular ion deregulation in growth and differentiation specifically as it relates to tumor promotion. It has become evident in recent years that ion deregulation plays a significant role in cell division and cell death; concurrently, studies have been performed suggesting significant differences in neoplastic versus normal cells and in the effects of tumor promoters such as phorbol esters. Although many regulatory processes including ions are involved, the focus of this review will be limited to the roles of sodium, calcium and proton deregulation. We will also present our hypothesis correlating such ionic changes with a variety of cell effects and responses related to tumorigenesis in which we have developed a construct of the interaction between ion regulation, cell division, and cell differentiation. Implicit in this hypothesis is the intimate relationship between acute toxic cell injury, normal regeneration, and neoplasia; therefore, it has important regulations for nongenotoxic mechanisms of neoplasia including tumor promotion and enhancement.