IN FOCUS
Comparative models and biological stress

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

	ARTICLE

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COMPARATIVE STUDIES have been integral to biology and medicine from their earliest beginnings. However, the formal beginning of comparative physiology may well date to 1865 and Claude Bernard's often quoted admonition that "There are also experiments in which it is proper to choose certain animals which offer favorable anatomic arrangements or special susceptibility to certain influences. This is so important that the solution to a physiological or pathological problem often depends solely on the appropriate choice of the animal for the experiment so as to make the result clear and searching" [from Forster (8)]. The application of this basic premise has continued to provide important insights ever since. For example, picking only a few from the Nobel laureates list (http://www.nobel.se/), in 1908, Mechnikov was recognized for his work on cellular immunity, based on experiments conducted in starfish larvae and Daphnia; in 1920, Krogh received the prize for his insights into the mechanisms of capillary function, drawn heavily from comparative models; and in 1963, Hodgkin and Huxley were recognized for their studies of the ionic basis of the nerve impulse in the giant neurons of the squid. Little has changed today. The last two Nobel prizes were given for work grounded in comparative models: the first to Kandel, who shared the award in 2000 for his studies on the basis of learning, many of which used the sea slug, Aplysia, and in 2001, the award went to Hartwell, Nurse, and Hunt for their studies of the cell cycle and its control, once again conducted in alternative models, yeast and sea urchin eggs.

Their work highlights the traditional strengths of comparative physiology: 1) the investigation of shared problems in a variety of organisms to understand the general principles that govern physiological and evolutionary solutions and 2) the use of models, such as the giant squid axon or Aplysia, that make critical experiments possible. With the recent advances in molecular biology culminating in compilation of the genomes of humans and a number of "lower" animals comes a new appreciation for their similarity (28), reinforcing Bernard's argument and these traditional strengths as well as opening new avenues for comparative research. Because many of the genes and gene products are so similar, if an advantageous experimental system can be identified, be it insect, crustacean, teleost, or mammalian, the insights gained will often prove to be of general import. The American Journal of Physiology-Regulatory, Integrative and Comparative Physiology strives to provide a forum for both traditional and molecular work.

The recent review of Na-phosphate cotransport systems by Werner and Kinne (31) highlights one such example. Here a combination of functional and molecular studies identify "a clear vertical [phylogenetic] relationship" among the mammalian transporters of the NaPi-III family from the mammalian forms through homologs in Caenorhabditis elegans, yeast, and bacteria. Likewise, molecular evolution was shown to link the mammalian NaPi-II transporters to those of C. elegans and Vibrio cholerae. A second review by Briggs (2) on the zebrafish points to the power of models based in comparative physiology and linked to genetic tools. This model has a long history in developmental biology, but with the application of mutational screening, often combined with new imaging tools to take advantage of its transparency, it is emerging as an even more powerful model with broad application in biology.

In the sections to follow, a few recent examples of comparative studies examining responses to environmental stress are discussed. Stress has long been a major focus of comparative physiology, e.g., from environmental extremes of temperature or salinity or via limited availability of water or oxygen. In particular, the problem of an adequate oxygen supply has been widely studied (reviewed in Ref. 26). Anoxia is really two problems in one. The organism must initially adjust its metabolic requirements during the period of low oxygen (metabolic depression) and it must also protect itself against the oxidative stress during the reoxygenation period that follows. Because turtles and frogs routinely experience periods of low oxygen availability during diving or overwintering (hibernation), these organisms have provided important tools to assess the mechanisms that may be used to meet these challenging situations. A recent In Focus article by Scholz (23) summarized vascular responses to decreased tissue oxygenation. Here the primary focus is on metabolic depression and adaptations to the stresses of reoxygenation.

Three very different aspects of metabolic depression were explored recently: the signals initiating metabolic depression (12, 22), alterations in mitochondrial function (1, 7, 24), and regulation of glutamate in the brain, an adaptation that protects against anoxia-generated excitotoxicity (18). Metabolic depression protects by reducing energy demand and thus limits both utilization of substrate reserves and accumulation of toxic metabolites. During metabolic depression, energy-consuming processes are greatly reduced, e.g., Na-K- ATPase activity (3) and protein synthesis (13, 20), and mitochondrial respiration itself is damped (7, 24). In the turtle, there is significant right-to-left intracardiac shunting of blood during apnea, rapidly reducing arterial oxygen levels and triggering metabolic depression (12). Because these shunts are under vagal control, they may be readily induced experimentally, with consequent metabolic depression and decreased oxygen consumption. Using this preparation, Platzack and Hicks (22) present evidence suggesting that it is the decrease in systemic oxygen delivery, rather than arterial PO₂ per se, that triggers the hypometabolic state. At the subcellular level, it is clear that modulation of mitochondrial aerobic metabolism is an important contributor to acute metabolic depression in diving turtles (7, 24) and to the more gradual changes associated with estivation in snails (1). A very different set of responses underlies the resistance of the turtle brain to periods of oxygen deprivation. In mammals, hypoxia or ischemia leads to massive release of glutamate and other excitatory compounds, but in the turtle this does not occur (18). Recent data indicate that glutamine efflux is actually decreased, not increased as in mammals, apparently the result of activation of both adenosine receptors (by increased adenosine) and K_ATP channels (by decreased ATP) (18, 21).

The second problem posed by anoxia is reoxygenation and protection against associated production of reactive oxygen species (26). In anoxia-tolerant species, antioxidant defenses appear to play an important role (10, 11). In fact, Lushchak et al. (14) demonstrated that antioxidant defenses, e.g., liver catalase and brain glutathione peroxidase, were elevated during the oxygen-deprivation period, in effect physiologically "preconditioning" the animal to face the oxidative stress to come on reoxygenation. Interestingly, it was recently shown that preconditioning as seen in mammals (i.e., exposure to a brief anoxic period leads to decreased damage after a subsequent more-severe anoxic period) also occurs in fish, although it is as yet uncertain whether the same subcellular mechanisms are involved in the teleost as the mammal (9). A possible contributor to preconditioning may be induction of heat shock proteins (a family of molecular chaperones), inasmuch as their prior induction protects against a variety of physical and chemical stresses (e.g., Ref. 19). Interestingly, Chang et al. (6) showed differences in the induction and the pattern of heat shock protein expression in anoxia-tolerant vs. anoxia-sensitive species. However, it is as yet uncertain if differences in heat shock proteins actually contribute to anoxia tolerance.

A very different stress is imposed by exposure to toxic agents, both natural products and xenobiotics, but ability to detoxify or eliminate toxic chemicals is just as critical for survival. Recent work has shed new light on the protective mechanisms used by all organisms and introduced new models for future work. Miller (15) used confocal imaging of isolated teleost renal proximal tubules to show not only the presence of the ATP-driven drug pump p-glycoprotein (MDR) in the apical membranes, but also to demonstrate directly for the first time in the intact tubule of any species the participation of MDR in net transepithelial drug secretion (15). The skate liver has proven to be another productive model for understanding both function and evolution of drug transporters, including the ATP-driven bile salt export pump (4) and the organic anion transporting polypeptides (5). A novel drug and steroid transporter that is composed of two separate gene products was also discovered in this model (30). Others demonstrated the presence and functional importance of MDR in teleost liver (27). Interestingly, recent studies demonstrate that expression of the drug pumps is not limited to traditional excretory organs. Studies of the human placenta suggest the presence of several of multidrug resistance-associated protein (MRP) isomers that, by virtue of their localized expression in the apical membrane of the syncytiotrophoblast, appear to function in protecting the fetus from entry of organic anions and to promote excretion of conjugated metabolites into the maternal circulation (25). Perhaps more surprising was the recent demonstration that the shark rectal gland, in addition to its well studied role in fluid and electrolyte secretion, possesses drug excretory capacity (16, 17). The cationic drug pump, MDR, was not expressed in the rectal gland. Instead, rectal gland drug secretion was mediated by the anionic drug pump, MRP2. MRP2 activity could be modulated by the endothelin-1, apparently acting through protein kinase C. Finally, using a newly developed flat sheet preparation of choroid plexus from the dogfish shark mounted in Ussing flux chambers, Villalobos et al. (29) were able to demonstrate net absorptive (cerebrospinal fluid to blood) flux for organic anions with features similar to the secretory (blood to urine) flux of drugs and xenobiotics mediated by the cloned renal drug transporter OAT1. This is the first native plexus preparation permitting direct measurement of unidirectional fluxes and should prove extremely valuable for detailed analysis of the blood-cerebrospinal fluid barrier.

In summary, comparative models have played important roles in the development of our understanding of physiological processes. With new models, some of which are discussed above, and continuing developments in molecular biology and genomics, there is every reason to anticipate that comparative physiology will continue to provide critical insights for years to come.

	FOOTNOTES

Address for reprint requests and other correspondence: J. B. Pritchard, Laboratory of Pharmacology and Chemistry, 110 Alexander Drive, MD F1-03, Research Triangle Park, NC 27709 (E-mail: pritchard{at}niehs.nih.gov).

10.1152/ajpregu.00415.2002

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