AABRE: NETWORKING IN PUERTO RICO
PR-AABRE Researcher: Carmen Hernández, Ph.D., Department of Biology, UPR, Humacao Campus
Cluster: Neuroscience
Collaborators: Sandra Chinapen, Ph.D. (San Juan Bautista Medical School); James Porter, Ph.D. (Ponce School of Medicine); Nitza Lugo (Institute of Neurobiology, School of Medicine); Ralph Albrecht, Ph.D. (University of Wisconsin at Madison)
Mentor: Fernando González, Ph.D. (UPR, Rio Piedras Campus )
Project Title: Localization of Substance P and Acetylcholine in the Pathway Mediating Mucociliary Activity
WHEN A PERSON INHALES, bacteria and cellular debris are trapped in the mucus and transported by the cilia to the pharynx, where they are swallowed or ejected as phlegm from the throat or lungs. Cilia move fluid substances over their surface and are crucial to maintaining the respiratory system. But when a person suffers from asthma, chronic sinusitis, cystic fibrosis or bronchitis, mucociliary activity is impaired. Biologist Carmen Hernández and behavioral and neural scientist Sandra Chinapen believe that their studies of the mechanisms governing mucociliary activity at the cellular level could lead to development of pharmacological or physiotherapeutic remedies for quiescent cilia.
Scientists have found that mucociliary activity can be increased by electrical stimulation and by administration of the neurotransmitters acetylcholine (ACh) and substance P (SP) to the palate. Acetylcholine—originally called “vagusschtuff” because it was found to be the substance released by stimulation of the vagus nerve that altered heart muscle contractions, acetylcholine—is widely distributed in the peripheral and central nervous system. Substance P, also found in the peripheral and central nervous system, is involved in several other physiologic activities including pain mediation, vomit reflex, defensive behavior, change in cardio-vascular tone, stimulation of salivary secretion, smooth muscle contraction, and vasodilation.
Microtome cutting of rana pipiens palate.
To determine the neuronal pathways of SP and ACh, the researchers and the student researchers use the palate of the northern leopard frog, Rana pipiens, as an animal model. After they anesthetize the frogs, the palatine nerve is cut and exposed to the retrograde tracer Fluorogold. The anterograde tracer Biotinylated Dextran amine is injected in the trigeminal ganglion. After various time periods—24 hours, 48 hours, 72 hours, and 96 hours—the trigeminal ganglion and palate of the frogs are dissected, frozen, and cut with a cryostat, which slices it into micrometer sections. The researchers then analyze the sections of ganglion and palate under a fluorescent microscope to identify the cell of origin from the palatine nerve and nerve terminals of the palatine nerve. Immunocytochemistry and immunoelectronmicroscopy are used to determine if the cell of origin contains SP.
Photo: Louis M. Landy
In order to develop effective disease therapies, scientists need to discover the origin of the palatine nerve to understand the neuronal pathways that cause ciliary movement. The researchers hypothesize that the trigeminal ganglion, from which the palatine nerve emerges, contains the cells of origin of the palatine nerve. They are also trying to determine the neuronal pathway of ACh in mucociliary activity.
In–depth knowledge of the regulation of mucociliary activity may assist in the development of new treatments for diseases associated with quiescent cilia. “If SP is the neurotransmitter involved in mucociliary activity, we can isolate the gene for SP from the cell of the trigeminal ganglion and incorporate it in another cell using a plasmid in the place that previously incorporated the gene for SP. Once the plasmid with the gene of SP is taken into a bacteria cell, the cell will use that DNA to make RNA and proteins. In this way, the over–expression of SP can be accomplished and put into a drug that can manipulate mucociliary activity,” says Hernández.
c_hernandez@webmail.uprh.edu
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