Atrial Natriuretic Peptide and Ciliary Transport

Atrial natriuretic peptide (ANP) is a human hormone which blocks movement of salts and water by several tissues of the body. The peptide hormone is formed and stored within the atria of the heart, and released in response to excessive retention of fluid within the cardiovascular system. Most attention has been focused on the action of ANP to block salt and water reabsorption by the tubules of the kidney, leading to an outpouring (diuresis) of salt and water in the urine. In addition, ANP affects other human tissues, including the eye. Its action to lower the intraocular pressure raises the possibility that the hormone may provide a new and effective approach for the medical treatment of glaucoma. ANP has been recognized for only 8 years, and its structure has been known for only half that time. Although the hormonal effects on the kidneys have been the subject of hundreds of scientific studies, very little information is available concerning its mechanism of action on the ciliary epithelium, the tissue which secretes aqueous humor into the eye. Among the questions which are as yet unanswered are: (1) Does ANP act directly on the ciliary epithelium, or indirectly by an action on the blood vessels of the ciliary body? In the kidney, ANP is now known to exert both effects. (2) What are the biochemical mediators of the hormone’s actions? Peptide hormones are known to act largely by stimulating production of “second messengers” within the cell, which in turn trigger cascades of biochemical events leading to the final effects on transport of salts and water. The information available suggests that ANP’s effects could be mediated either by increase in one second messenger (cGMP) and/or by decrease in a second common second messenger (cAMP). (3) Can knowledge of the basic cellular mechanism of action of ANP permit us to design novel pharmacologic approaches to lower intraocular pressure more effectively in experimental animals and perhaps in man?

Hypothesis

The aqueous humor is normally formed largely by a primary transport of salts from the blood to the aqueous. The salt transfer establishes an osmotic gradient driving water across the ciliary epithelium into the aqueous. We suggest that ANP acts directly on the ciliary epithelium by blocking entry of sodium from the blood into the transporting cells. We further suggest that this blocking of sodium movement can be enhanced by the simultaneous administration of drugs which reduce the permeability (ease of entry) of sodium and which reduce the electrical force driving sodium into the epithelium.

Specific Aims

Until very recently, it was unclear whether or not ANP had any direct effect on the tubules of the kidney to block transport. This question could not be answered satisfactorily by studying ANP in whole animals and intact organs and tissues. In those preparations, the issue could not be resolved because of the many uncontrolled variables (such as hormonal levels, release of intracellular second messengers, and redistribution of regional blood flow) which could not be control led. The issue has, however, been unequivocally answered by the application of patch clamp techniques. This approach has revolutionized biomedical science, permitting an electrophysiologic approach to membrane events in essentially all areas of physiology. We aim to use a form of patch clamping, which permits us to control the composition of the fluids bathing the inside and outside of the ciliary epithelial cell. We are currently using this approach (“whole-cell patch clamping”) in my laboratory to study ciliary epithelial cells in culture. With this technique, we will be able to address the first two questions posed above. Therapeutic strategies based on the results of our cellular studies (the third issue) will be tested in the second year of support by extending the work to include study of the intraocular fluid dynamics and intraocular pressure in intact animals.

Long-Term Goals

I am a transport physiologist, board-certified in internal medicine. My major interest has been the physiologic regulation of salt and water transport across epithelia. Recently, I have become very interested in transport across the ocular epithelia, especially the ciliary epithelium. Currently, we are studying this problem by intracellular electrophysiologic recording in the rabbit iris-ciliary body, and by patch clamping and electronic cell sizing of ciliary epithelial cells in culture. My long-term goal is to develop a coherent, inclusive model of the basis and regulation of aqueous humor formation, and to formulate specific drug strategies for reducing intraocular pressure in humans. Dr. Richard Stone of the Dept. of Ophthalmology is acting as a consultant and will be invaluable in advising us in our efforts to: examine future biochemical aspects of the problem, formulate drug programs (and methods of drug delivery) and test our hypothesis in intact animals.