The National Science Foundation's (NSF) Tokyo Office periodically receives and disseminates reports on research developments in Japan that are related to the Foundation's mission. NSF-sponsored researchers currently working in Japan prepare many of these reports. These reports present information for use by NSF program managers and policy makers; they are not statements of NSF policy.
Special Scientific Report #01-01 (July 13, 2001)
Collaborative Research on the Piscine Extracellular Calcium-Sensing Receptor
Christopher A. Loretz, a faculty member in the Department of Biological Sciences at the University at Buffalo, prepared the following report. Dr. Loretz was in Japan from March 4 to April1 on a Japan Society for the Promotion of Science (JSPS) Senior Invitation Research Fellowship. Professor Yoshio Takei in the Ocean Research Institute at the University of Tokyo served as his scientific host. Dr. Loretz can be reached by e-mail at: loretz@acsu.buffalo.edu.
Summary of Visit
The four-week visit that I conducted under the JSPS Short-Term Invitation Research Fellowship permitted the development of a new collaborative laboratory research project and afforded me the opportunity for extended scientific conversations with the staff of the Ocean Research Institute (ORI) at the University of Tokyo. I share with Professor Yoshio Takei at the ORI a common interest in the physiological adaptations of fishes to varying salinities and in the evolutionary history of vertebrate endocrine systems. The international collaborative relationship that developed from these shared interests brought me to the ORI for four months in 1995 during my sabbatical leave, and several times subsequently. These joint efforts utilized Drs. Takei’s and my expertise in biochemistry and electrophysiology, respectively. Dr. Takei has a long-standing interest in the evolutionary history and physiological actions of natriuretic peptides and of angiotensins in vertebrate animals. My research interests are complementary, and focus on the biophysics and cellular regulation of intestinal absorption of salts and nutrients. The fellowship opportunity from JSPS strengthened this collaborative relationship through both laboratory research and intellectual exchange. The results of the successful laboratory project are summarized in the following report.
In addition to the collection of important preliminary data on the existence of a calcium-sensing receptor in the Japanese eel (unagi), my extended discussions during the fellowship visit with Dr. Takei and his laboratory research staff focused on the development of future collaborative experiments that will utilize the skills represented in both of our laboratories and will involve an additional collaborator in the United States. Future work will confirm and extend the initial findings of this visit, viz., the existence of a calcium-sensing receptor in the intestine and in hormone-secreting endocrine cells of fishes, and the physiological involvement of that receptor in salinity adaptation. Plans for these collaborative efforts were continued and extended in May 2001 when Dr. Takei and I both attended the 14th International Congress on Comparative Endocrinology in Sorrento, Italy.
Also during this fellowship visit, I was very pleased to extend my acquaintance with two additional researchers in the Division of Physiology within the Department of Marine Bioscience at the ORI. Drs. Susumu Hyodo and Koji Inoue are experts in the field of molecular biology applied to fish systems, and their consultation and contributions of time and effort during my fellowship visit in March were critical in the achievement of such great success during this short-term visit. I look forward to continued interaction with these two talented researchers.
Collaborative Research Project
The roles for ionic calcium (Ca2+) in cellular and organismal function are many and varied, and essential for survival. Calcium-dependent cellular processes are numerous and include hormone secretion (stimulus-secretion coupling), cell motility (excitation-contraction coupling) and neurotransmission (Ca2+-based action potentials). This critical involvement of ionic calcium in numerous cellular functions is unique among ions. Calcium is simultaneously a controlled variable in physiological systems, a fluid-borne extracellular messenger in multicellular organisms, and an intracellular mediator in a variety of effector cells.
The collaborative experiments conducted with Drs. Takei and Hyodo at the Ocean Research Institute were designed to test the broad hypothesis that fish express calcium-sensing receptors (CaSR) that are homologous to recently identified mammalian and avian Ca2+-sensing receptors. Three approaches were used: (1) immunohistochemistry of CaSR in fish tissues, (2) western blot analysis of CaSR expression in fish tissues, and (3) reverse transcription-polymerase chain reaction (RT-PCR) analysis of CaSR expression in fish tissues. Since Dr. Takei and I have previously (and successfully) explored several aspects of the physiology of the Japanese eel (Anguilla japonica), this was an excellent system for us to use in these studies.
Our immunohistochemistry experiments were successful and provided initial evidence in support of the hypothesis; it appears that a number of piscine tissues, including the brain, gill, kidney and intestine, contain a CaSR-like protein. The western blot analyses were inconclusive; this may reflect the relatively low level of expression of CaSR in fish tissues, or perhaps limited cross-reactivity of the antibodies that we used. Using RT-PCR technique with oligonucleotide primers designed to recognize the highly conserved transmembrane domain of the CaSR protein, we were able to identify several messenger RNA (mRNA) species from the intestine of the eel that belong to the G-protein-coupled, seven-transmembrane-domain receptor family (the family of membrane receptor proteins that includes CaSR). On closer examination, we discovered that the mRNA species that we identified are more closely related to previously described fish pheromone receptors (also members of the same receptor family) than they are to CaSR. This is a somewhat surprising (but, nevertheless,very interesting) result, and it suggests that these so-called “pheromone receptors” may be multifunctional and serve in the intestine as receptors for substances such as amino acids (or even Ca2+). We are preparing to test a new series of oligonucleotide primers that are designed to recognize specifically the Ca2+-binding domain of the CaSR, and thereby exclude the related pheromone receptors. We expect to complete these experiments soon.
Site Visits
During my stay in Japan, I visited the National Cardiovascular Center Research Institute (Kokuritsu Junkankibyo Center) in Osaka. The Center is a comprehensive institution for treatment and research in cardiovascular disease. The research laboratories that I visited were thoroughly modern and remarkably well equipped by world standards. My host at the Research Institute was Dr. Hiroyuki Kaiya, a specialist in endocrinology.
Host Institution Profile
The Ocean Research Institute (ORI) of the University of Tokyo was founded in 1962 by an act of the Japanese Diet. Over the years preceding its foundation, marine scientists had emphasized the necessity of establishing a new oceanographic institution for the promotion of basic and comprehensive research on marine sciences. Since its establishment to address that need, the breadth of expertise in the Institute has steadily grown. As a result of its reorganization in 2000, the Institute now has 6 research departments (comprising 16 research groups) and three research centers. The Institute is equipped with sophisticated instruments and facilities on the Nakano campus and at a shore laboratory, the Otsuchi Marine Research Laboratory in Iwate Prefecture. The Institute operates two ocean-going research vessels, the Hakuho Maru (100 meters overall length) and the Tansei Maru (51 meters). The Institute’s facilities are available for use by oceanographers and marine scientists throughout Japan. Expertise in support of physical, chemical, and biological oceanography, meteorology, geology and geophysics, marine biology, and fisheries science is also provided for educating graduate students at the master's and doctoral levels. Many researchers, including scientists and students from foreign countries, are involved each year in various aspects of the research efforts of the Institute. The Institute participates in major international cooperative research projects and has hosted many international scientific congresses. The ORI is recognized for its interdisciplinary approach to marine research, and all major fields of study are addressed by the faculty.
Scientists in the Laboratory of Physiology (within the Department of Marine Bioscience), in which Prof. Takei and Drs. Hyodo and Inoue work, focus their attention on the resolution of adaptive mechanisms of many and diverse marine life forms to various environments. Current studies pay special attention to the relationships among fish migration and osmoregulation. Several scientists are working on the adaptation of marine plankton to environmental conditions of varying light intensity and nutrient availability. The physiological and biochemical adaptations of migratory or euryhaline fishes such as eel, salmon, trout, mullet and flounder to hypotonic fresh water or hypertonic seawater during seaward or upstream migration form a central research theme in this laboratory. Control mechanisms for sodium and calcium transport in gills, kidney and intestine are under investigation using physiological and electrophysiological techniques, such as measurement of ion movement or potential difference across membrane sheets isolated from osmoregulatory organs. Control of osmoregulatory processes by the endocrine system during seawater and freshwater adaptation in teleosts is being clarified through radioimmunoassay measurement of circulating concentrations of hormones such as prolactin, growth hormone, cortisol, thyroid hormone, angiotensins, natriuretic peptides and somatolactin which are known to be associated with hydromineral homeostasis. Identification of hormone receptors in osmoregulatory organs and changes in receptor characteristics during seawater and freshwater adaptation are also being studied by radioreceptor assay and biochemical techniques. Furthermore, molecular techniques such as in situ hybridization are developed and applied to learn fundamental mechanisms of migration and osmoregulation.
Research facilities available at the ORI include: laboratory for molecular cloning , DNA sequencing facility, computer facility, radioisotope laboratory, aquarium room complex, laboratory for cultivation of microalgae and bacteria, sample and specimen storage facility, low-temperature rooms, photographic darkroom, machine shop, glass-blowing shop, and instrument rooms. The Institute's library contains about 30,000 books and monographs and over 1,000 current scientific periodicals.
Impressions and Thoughts on the Present State of Science in Japan
I have been aware for quite some time of the great strength of the Japanese scientific community in the field of comparative endocrinology, and more recently in the rapidly expanding field of molecular biology. In a global community faced with limited potential to feed the world’s population, it is encouraging to witness the Japanese emphasis on food resources and the support that is provided to basic research with an eye to eventual practical application. Through collegial relationships at the ORI and attendance at various scientific conferences, I am familiar with advances in the fields of aquaculture and mariculture in Japan at the level of both production and maintenance of food resources, and the understanding of adaptations by natural populations to hostile environments. Greater understanding of these adaptations may benefit humankind in the future through exploitation of heretofore-unused habitats, sensitivity to the effects of endocrine disrupters, and in assessing the harmful effects of habitat destruction.
Collegial ties among Japanese researchers are remarkably broad and strong. Techniques and technologies are generously shared by my Japanese colleagues. There is an extraordinary level of cooperation in the research endeavor in Japan generally, and this is further promoted by governmental support in the form of grants and fellowships, and by industrial involvement (specifically, for example, by Japanese companies that maintain active research programs in physiology, endocrinology and pharmacology).
Comments Concerning the Fellowship Program
The JSPS Short-Term Invitation Fellowship Program presents a tremendous opportunity for foreign scientists to expand their professional and personal horizons through collaborative research projects with leading Japanese researchers. In a time when many scientific questions are complex and demand interdisciplinary approaches to their solution, international fellowship programs such as this provide a means to tap scientific resources that would otherwise be unavailable.
Personally, the JSPS Short-Term Fellowship furthered my collaborative association with Dr. Takei and his associates at the ORI (specifically, Drs. Hyodo and Inoue). The ongoing work of this group in the fields of fish osmoregulation and of cardiovascular and endocrine physiology points to continued powerful collaboration and, as mentioned above, planning efforts are underway for this next phase of research.
The greater cultural awareness that the Fellowship Program promotes is valuable and cannot be understated. The intellectual intensity, rigor, enthusiasm, and work ethic of Japanese scientists, generally, are extraordinary, and it is infectious to the visitor. The benefits to the foreign scholar of a personal visit greatly outweigh those that might be achieved using simply long-distance written, voice or electronic communication.
Other Comments
This photograph shows me at work in one of the ORI laboratories.
Christopher A. Loretz (JSPS Short-Term Invitation Fellow) at work in his host lab at the Ocean Research Institute, University of Tokyo