Does Nitric Oxide Regulate Pancreatic Secretion?

Project Overview

This project proposes to examine whether the recently discovered signaling molecule, nitric oxide (NO), contributes to the regulation of pancreatic secretion. The millions of cells that make up the pancreas must produce a coordinated response after stimulation by a diffuse hormonal signal from the small intestine. These hormones cause the pancreas to secrete digestive enzymes. Individual pancreatic cells vary widely in their response to intestinal hormones, yet the assembled tissue exhibits a very coordinated and uniform response to the hormones. The difference between the response of individual cells and the tissue suggests that the hormones may initiate the production of a secondary signal to coordinate the tissue into a uniform response. This type of tissue coordination has not been previously investigated. Nitric oxide has characteristics that make it an ideal candidate. Importantly, nitric oxide crosses cell membranes and can therefore be transferred between neighboring cells to foster coordination. Therefore, using techniques and procedures available in my faculty sponsor's lab, I will determine whether hormone stimulation of the pancreas results in NO production, whether direct application of NO is sufficient to initiate pancreatic secretion in the absence of hormone, and I will work to identify the biochemical target of NO. It is my desire to eventually gain a Ph.D. and to focus my career in the area of cancer research. Cancer is characterized by the disruption of cellular communication and its study utilizes many of the same skills and techniques. I hope that the experience I gain as an undergraduate researcher will therefore accelerate my scientific education and enhance my career potential.

Project Description

Introduction: Funds are requested to support laboratory research in cell biology. The proposed project will test the hypothesis that nitric oxide (NO) is produced by the pancreas in response to hormonal stimulation and that NO serves to promote and coordinate secretion throughout the pancreatic tissue. NO began to be recognized as an important regulator of blood pressure in the early 1990s. Since then, NO has been the subject of over 30,000 scientific papers. Therefore, a great deal is known about its mechanism of production (Stamler et al., 1992) and its distribution (Titheradge, 1998). The vast majority of the functional studies have focused on three areas; 1) regulation of blood pressure through NO influence on vascular smooth muscle; 2) immune cell function; and 3) apoptosis or programmed cell death important to development and control of cancer cells. However, the broad production of NO throughout the body, coupled to the local nature of its activity, suggests that this novel signaling molecule may have a fundamental influence on cell physiology and be a more general signaling molecule than initially assumed. Therefore, the more basic aim of this project will be to use the pancreas as a model system to demonstrate that NO influences virtually all cells through an interaction with a common component of cell physiology, and that the subsequent response is then tissue specific. This tissue specific premise has been demonstrated for other types of signaling systems. For example, calcium is a common signal in virtually all cells and changes in its concentration causes the type of tissue specific responses alluded to above. An increase in calcium in muscle causes contraction, in nerves it causes release of neurotransmitter, in the stomach it causes release of gastric acid, and in the kidney it causes concentration of urine. This project will approach the role of NO in an analogous fashion whereby NO may cause relaxation in vascular muscle, or antibody production in immune cells, or apoptosis in cancer cells, depending on the conditions, concentration, and type of cell. I will use the pancreas as the model to demonstrate a more general role for NO. The first step will be to demonstrate that NO is produced in the pancreas and that it can promote exocrine secretion. It should be noted that the first preliminary experiments that I have performed in the lab do suggest that NO promotes enzyme secretion by the pancreas and provided the stimulus for this proposal. The second step will be to identify the cellular element with which NO interacts. Much of the pancreatic signaling cascade that leads to the synthesis and secretion of digestive enzymes has previously been identified by other members of Dr. __________'s lab (Kein, 2000). Reports on other tissues suggest that NO can alter the activity of some aspects of these types of signaling molecules including guanylyl cyclase (Zhao et al., 1999; Malinski et al., 1999) and Ras (Lander et al., 1997). The pancreatic information will then serve as a starting point to identify the NO target in the pancreas by looking for specific increases in regulatory enzyme activity after treating the pancreas with NO or alternatively to look for decreases in activity in the presence of NO synthesis inhibitors. If the role of NO is to coordinate the response of pancreatic cells, then these signaling molecules should respond to the presence or absence of NO in the system.

Design Overview: Pancreatic cells will be maintained in a growth media and separated into three groups for experimental purposes. The untreated control group will provide a baseline response, a hormone treated group will provide the relevant physiological response, and the experimental group will provide the data on the various NO treatments. These experimental treatments will include NO alone, NO synthesis inhibitors in conjunction with hormone treatment, or NO scavengers that remove NO from the cells. The amount of secretion measured by the release of digestive enzymes will be monitored in the different groups of cells. This will be accomplished using colorimetric assays for the released digestive enzymes after harvesting the cell incubation solution. In other experiments the cells of the three groups will be lysed and the cellular proteins isolated and monitored for activity as a means to identify the NO target.

The identification of activated cellular proteins uses a procedure called immunoprecipitation. By this technique, antibodies to phosphotyrosine, a domain common to many activated proteins, is incubated with the cellular lysate. The antibody along with its attached cargo is then collected on protein A/G agarose beads. The collected beads are stripped and the liberated proteins are separated by electrophoresis. These proteins are then electrically transferred to reactive sheets of nitrocellulose. As the proteins are exposed on the surface of the nitrocellulose, they can be probed with protein specific chemiluminescent antibodies. Their presence will be monitored with blue light sensitive photographic film and then quantified by computer imagining (Adobe Photoshop and NIH Image). This procedure will allow me to identify the proteins that respond to NO, quantify the magnitude of the change, and correlate the change to corresponding changes in the secretion rate of the cells.

Finally, if changes in activity are identified, the results will be confirmed through the use of pharmacological manipulations. Previous work suggests that NO may influence either guanylyl cyclase, Ras, or calcium (Yolk et al., 1997) in cells. If NO acts through these agents then direct stimulation of these compounds, with commercially available drugs, should promote the same response as that observed for either the hormone or for NO.

Time Table: At the present time I am working in Dr. _________'s lab and am learning the techniques required to perform the experiments outlined above. I plan on completing all the secretion experiments this semester (Spring 2001) to conclusively demonstrate that the pancreas produces NO and that it promotes secretion by the pancreas. I also plan to gain experience in electrophoresis, immunoprecipitation, western blotting, and computer imagining. I will then use these skills to perform the experiments designed to identify the NO target in the Fall semester of 2001. The Spring semester of 2002 will be used to complete any experiments, write the final report, and hopefully contribute to the publication of the results in a scientific journal.

Experience: After graduating from NIU in the Spring of 2002, my goal is to continue my cell biology education in a graduate Ph.D. program and pursue a career in cancer research. Performing a long-term independent research project as an undergraduate will help to solidify those goals and provide me with incredibly valuable experience outside the common undergraduate program. The practical knowledge of cell biology along with laboratory and problem solving skills provided by this laboratory research project would be very advantageous in getting accepted into graduate schools and make a very productive start on my career goals.

Literature Cited

Lander, H., D. Hajjar, B. Hempstead, S. Campbell, and L. Quilliam. (1997) A molecular redox switch on p21 (ras). Structural basis for the nitric oxide-p21(ras) interaction. J. Bioi. Chem. 272:4323-4326.
Stamler, J., D. Singel, and J. Loscalzo. (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902.
Titheradge, M. (ed.) (1998) Nitric Oxide Protocols. Methods in Molecular Biology. Humana Press, Totowa NJ. pp. 324.
Volk, T., K. Mading, M. Hensel, and W. Kox (1997) Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP. J. Cell Physiol. 172: 296-305
Zhao, Y., P. Brandish, D. Ballou, and M. Marietta. (1999) A molecular basis for nitric oxide sensing by soluble guanylate cyclase. Biochemistry 96: 14753-14758.

Budget

Funding Period: March 2001 -December 2001

The following items represent specialized reagents and chemicals that are specific for the project described above. Most routine laboratory materials, including cells, common chemicals, electrophorsis reagents, and all the necessary equipment will be supplied by Dr. ________'s laboratory in the Department of __________________________..

Antibodies for protein detection anti-Shc $175
anti-phosphotyrosine $90
anti-PKC $175
anti-ERK $175
chemiluminescence probes $75
Immunoprecipiation Reagents Protein A/G Agarose $100
NO Donors NOC-9 $90
NO Synthesis Inhibitors L-NAME, N-propylarginine $85
Intestinal Hormone CCK $120
Protease inhibitors Complete Cocktail $250
Calcium -GC probes ionomycin, ODQ, thapsigargin $145
Disposable Commodities $350
TOTAL BUDGET REQUEST $1830

(The budget is based on current prices as listed in the catalogs of the relevant biochemical supply companies)