Our bodies maintain a state of homeostasis through a complex network of cell-cell communications by numerous circulating and local mediator molecules, or hormones. Among various classes of intercellular communication molecules, our laboratory focuses on small peptide hormones, or so-called neuropeptides. These tiny proteins, usually consisting of a chain of less than 40 amino acids, are nevertheless versatile and important cellular languages. We search for novel neuropeptides and their receptors with various biochemical techniques and define their physiological functions at the whole-animal level through the powerful genetic techniques available in mice. We are particularly interested in neuropeptides involved in the physiological regulation of essential bodily functions in mammals, such as circulation, appetite, and sleep.
Fishing for Ligands of Orphan G Protein–Coupled Receptors
Most neuropeptides work though a class of cell-surface receptors called G protein–coupled (or seven-transmembrane) receptors. There are numerous "orphan" G protein–coupled receptor genes in the human genome; the cognate ligands or hormones for these receptor molecules have not been identified. We have been using a new approach–reverse pharmacology–that aims to identify neuropeptide ligands for orphan G protein–coupled receptors. We express orphan receptor genes in transfected cells and use them as a reporter system to detect ligand substances in tissue extracts that can activate signal transduction pathways in receptor-expressing cell lines.
We have discovered a novel pair of neuropeptides, termed orexin-A and -B (from the Greek orexis, meaning appetite), as the ligands for two orphan G protein–coupled receptors. Neurons producing orexins are located exclusively in the lateral hypothalamus, the classical feeding center of the brain. Furthermore, we found that orexin mRNA is up-regulated during fasting. These findings suggested that orexins may be involved in the regulation of feeding behavior. Indeed, animals eat significantly more when injected with orexin cerebroventricularly. However, soon after our discovery of orexin and orexin receptors, we and other researchers found that orexins also elicit various other pharmacological actions, such as augmentation of sympathetic outflow and increase in arousal and waking.
Orexins: A Link Between Sleep and Feeding?
Although we spend one-third of our life asleep, very little is known about the molecular mechanisms of sleep. Our recent gene-targeting experiments in mice have unexpectedly uncovered an essential role of orexins in sleep-wake regulation. We found that mice deficient for orexins suffer from narcolepsy, a disorder characterized in humans by excessive daytime sleepiness (for mice, nighttime sleepiness) and pathological intrusions of various REM-sleep phenomena into wakefulness, such as cataplexy (a sudden bilateral loss of posture muscle tone).
Using nighttime infrared video surveillance, we discovered that orexin-knockout mice experience numerous cataplectic and sleep attacks, and electroencephalographic and electromyographic analyses confirmed the diagnosis of a sleep disorder strikingly similar to human narcolepsy. A Stanford University group discovered that canine narcolepsy, previously the only known genetic model of this disease, is caused by mutations in an orexin receptor gene product, called the OX2 receptor. Moreover, recent reports by other researchers suggest that the orexin system is dysfunctional in a majority of human narcolepsy patients: orexin peptides cannot be detected in their cerebrospinal fluid (although there is no evidence of mutation in their orexin genes).
These findings firmly link orexin neuropeptides and their receptors to narcolepsy in both animal models and humans. The orexin system may be a promising lead in uncovering the mysterious brain mechanisms controlling the sleep-wake cycle in general. We also speculate that orexins may be a link between two apparently separate functions of the brain, feeding and sleep. It is well known that people are sleepier after a meal and more awake when hungry. Further investigation into the orexin system may explain these adaptive phenomena.
Endothelins: Regulators of Cardiovascular Function
Disorders of blood vessels, including all forms of heart attack and stroke, are the leading cause of disease and death in developed countries. The inner surface of blood vessel walls is lined with an extremely thin single layer of cells, called the vascular endothelium. In 1988, our laboratory discovered endothelins, a family of 21–amino acid peptides that are potent vasopressor molecules in the vascular endothelium. Three closely related members of the endothelin family, ET-1, ET-2, and ET-3, are now known to be expressed not only in blood vessels but also in many nonvascular cells of the heart, kidney, brain, and other organs. These peptides work locally within the tissue, acting on two types of G protein–coupled endothelin receptors, termed ETA and ETB receptors.
Our initial gene-targeting experiments in these molecular components of the endothelin pathway unexpectedly revealed the peptides' essential roles in normal development of certain neural crest– derived tissues, such as pharyngeal arch–derived structures of the jaw, throat, and heart; the nervous system of the gut; and skin pigment cells. Thus the knockout mice deficient for one of the endothelins or endothelin receptors suffer from birth defects ranging from craniofacial and cardiac malformations to aganglionic megacolon (Hirschsprung disease) and piebaldism. We and others also found that some human patients with Hirschsprung disease harbor mutations in the ET-3 or ETB receptor genes.
While providing novel insights into the fundamental embryology of neural crest development, these birth defects also killed the mice shortly after birth, prohibiting physiological assessments in adult animals. Recently, we have used several genetic tricks to circumvent the juvenile lethality of these mutant mice, either by supplying a wild-type transgene in a tissue-specific manner to the cells that need it during embryonic development (e.g., enteric neurons), or by knocking out the genetic components in a tissue-specific manner using the cre-loxP recombinase technology. These techniques have so far revealed several distinct roles of the endothelin system in the physiological regulation of blood pressure.
Antagonists for endothelin receptors are currently well into phase II and III clinical trials for various cardiovascular diseases. Our findings will help define how these peptides contribute to cardiovascular regulation in health and disease.
The studies in mice were supported in part by the Perot Family Foundation and the W. M. Keck Foundation.
As of April 29, 2003