Mortimer M. Civan - US grants

The basic aim of the research plan is to study the interrelationships between the composition of the intracellular fluids and sodium transport across epithelia. Although the program is primarily based upon intracellular recording with ion-selective microelectrodes and reference micropipets, complementary studies will be performed with 3 1p nuclear magnetic resonance, electron probe X-ray microanalysis and computer simulations. In order to facilitate integration of the information obtained, most of the work will be conducted with a single epithelial model, split and whole frog skin. The principal objective will be to characterize the mode of apical sodium entry from the outer medium into the cell, and in particular to examine the putative role of intracellular sodium and calcium in regulating this entry. A second objective will be to define the electrokinetic and thermodynamic properties of the Na,K-exchange pump a the basolateral membranes. A third concern will be to examine the functional integration of the entire epithelium under three experimental conditions.

Cyclic-3',5'-AMP (cAMP) plays a major role in regulating sodium transport across frog skin and other tight epithelia. This second messenger is thought to stimulate transepithelial Na+ movement by activating a cAMP-dependent kinase, which phosphorylates sites gating the apical Na+ channels. An increasing number of observations from this laboratory have suggested that the ubiquitous enzyme protein kinase C (PKC) may also be an important regulator of sodium transport across frog skin. The hypothesis has been developed that: (i) PKC phosphorylates sites identical with, or near, the regulatory sites phosphorylated by cAMP kinase, and (ii) that the natriferic action of insulin at the apical membrane is mediated by activation of PKC. The present proposal is focussed on this hypothesis, and aims at: (i) more rigorously examining whether PKC activation increases apical Na+ permeability (papNa), (ii) determining whether the apical natriferic action of insulin is mediated by PKC, (iii) examining whether translocation of PKC from cytosol to apical membrane is necessary for PKC activation to increase papNa, and (iv) determining whether the intracellular alkalinization (produced by both PKC activation and insulin in other cells) is important in the expression of this natriferic effect. Apical Na+ permeability, transepithelial Na+ transport and intracellular pH will be monitored with intracellular micropipets and pH-selective microelectrodes and transepithelial voltage clamping of intact isolated-epithelial and whole-thickness preparations of frog skin. Parallel studies of PKC activity and translocation will be conducted, using both enzymatic assays and monoclonal and polyclonal antibodies.

Cl- is the major anion in the aqueous humor. The final step in Cl- secretion is likely extrusion from the nonpigmented ciliary epithelial (NPE) cells through ion channels. These Cl- channels have been detected in the intact ciliary epithelium and in isolated NPE cells but only in low abundance, so that this site is likely a rate-limiting step to aqueous humor formation. Based on our volumetric, electrophysiologic and collaborative molecular biologic measurements conducted under hypotonic and isotonic conditions, we have formulated a hypothesis concerning ciliary epithelial Cl- secretion. We propose that the Cl channel CIC-3 and the Cl- channel regulator P1cln control the physiologic secretion of Cl-, that the same Cl- channels subserving aqueous humor formation are recruited during the response to hypotonic cell swelling, that these Cl- channels are regulated by protein kinase C, calcium/calmodulin and an epoxide, and that the regulation of these Cl- channels significantly modulates the rate of aqueous humor formation. We shall extend our studies of broken cell, isolated-cell and intact epithelial preparations, applying both electrophysiology and molecular biologic strategies. We shall study primary and/or continuous lines of NPB and pigmented ciliary epithelial (PE) cells (of human, rabbit, bovine, and cat origin) by patch clamping in the ruptured-membrane and perforated- membrane, whole cell modes and in the cell-attached and excised-patch modes. In addition, we shall monitor transport of the intact ciliary epithelium non-invasively with cell-attached patching. The objectives of the program are to test our hypothesis of ciliary epithelial Cl- secretion by addressing the following sets of issues (l) Do the NPB cells display only a single set of functional Cl-channels under hypotonic and isotonic conditions? (ii) Do the pigmented ciliary- epithelial (pE) cells display the same or different functional Cl- channels? Can transcripts and protein products for CIC-3 and pI-cln be detected in PE cells? What are the relative abundances of the functional Cl- channels and of these transcripts and protein products in the PE and NPE cells? (iii) What are the functional effects of overexpressing (by transfection) and of down-regulating (by antisense oligodeoxynucleotides and antibodies) the CIC-3 Cl- channel and the pI-cln Cl- channel regulator?

We propose to study the roles of cGMP and cGMP-mediated transmitters in regulating ciliary epithelial function. On a systemic level, the natriuretic peptides (ANP and BNP) and NO on the one hand, and angiotensin II (AII) on the other, form a pair of balancing and opposing transmitters regulating vasomotion; the natriuretic peptides and AII also oppose each other in regulating natriuresis. This integrative balance is partly mediated through changes in the intracellular concentration of cGMP (cyclic guanosine-3',5'-monophosphate), which is increased by natriuretic peptides and NO and decreased by AII. Published data indicate that the ciliary epithelium can respond and/or has receptors to all of these transmitters. Furthermore, on an intracellular level, the two cyclic nucleotides cGMP and cAMP (cyclic adenosine-3',5'-monophosphate) appear to constitute an additional pair of balancing and opposing messengers, regulating the secretion of aqueous humor. On the basis of published data and our unpublished observations, we have formulated a hypothesis concerning the roles of cGMP in regulating the rate of aqueous humor secretion. We propose that the major effect of cGMP is to reverse a cAMP- mediated inhibition of the Na,K-exchange pump, involving both protein kinase A and DARPP-32 of the non-pigmented ciliary epithelial cells. The experimental approach of the current program will be to measure both transepithelial and transmembrane transport by the ciliary epithelium. We shall perform transepithelial measurements and cell-attached patch clamping of the intact ciliary epithelium from both the rabbit (whose characteristics have been well defined) and the cat (whose properties are likely closer to those of primates). We shall also study primary and/or continuous lines of non-pigmented and pigmented ciliary epithelial cells (of human, bovine, rabbit and cat origin) by patch clamping in the conventional whole-cell, perforated whole-cell, cell-attached and excised- patch modes. The specific objectives of the research program are to: (1) determine the effects of each transmitter (ANP, BNP, AII, and NO) separately, and the interactions of these transmitters; (2) test whether the interactions between the transmitters and second messengers (cGMP and cAMP), and between cAMP and cGMP themselves, conform to the hypothesis proposed; and (3) determine whether the cyclic nucleotide-regulated effects are mediated by the transport mechanisms postulated.

DESCRIPTION: The PI proposes that a novel paracrine system based on purinergic signaling allows for intraepithelial communication between the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cell layers. From the work of others, purinergic mechanisms are known to alter aqueous humor inflow and intraocular pressure. He specifically proposes that: (1) ATP (and possibly other nucleotides and adenosine) are released directly by NPE and PE cells; (2) Membrane ecto-enzymes hydrolyse ATP to AMP and extracellular adenosine; (3) Adenosine acts on NPE cells to stimulate aqueous humor formation by activating Cl- channels; (4) Extracellular ATP stimulates aqueous humor reabsorption by activating PE anion channels; and (5) Net aqueous humor formation is regulated by coordinating these opposing purinergic effects (and other less well defined transport effects) on unidirectional secretion and reabsorption. This coordination may reflect synergistic effects of purines and an unidentified intracellular regulator, whose action is mimicked by tamoxifen. The PI will address the following specific aims: (i) What are the characteristic biophysical and pharmacologic properties of the channels targeted by purines, especially the NPE Cl- channels and PE anion channels? (ii) What are the mode and trigger for purine release by the NPE and PE ciliary epithelial cells? (iii) What are the receptors and signaling pathways underlying purinergic stimulation of transport by the NPE and PE cell? (iv) How are these purinergic effects coordinated to modulate net aqueous humor secretion? The proposed interdisciplinary research will combine patch-clamp and volumetric physiologic methods with molecular biological and structural strategies, correlating measurements from isolated-cell, broken-cell and cell-couplet preparations with those from whole tissues. The novel hypothesis (that aqueous humor formation is directly modulated by an interlayer paracrine release of purinergic messengers) can provide the basis for understanding the purinergic effects on aqueous humor dynamics.

DESCRIPTION (provided by applicant): The proposed research involves integrating membrane physiology and a novel method of measuring intraocular pressure in the mouse to address the mechanisms of aqueous humor outflow and intraocular pressure. Cell swelling within the trabecular meshwork (TM) and Schlemm's canal (SC) decreases the aqueous humor outflow facility (C), and cell shrinkage within this small region increases C. Subtype-specific adenosine agonists have also been reported to alter outflow. On the basis of published evidence and our own data, we propose that: (1) TM-cell swelling directly and indirectly modulates outflow; (2) SC-cell swelling also alters the resistance to flow across the SC inner wall; (3) adenosine and ATP receptors modulate cell volume regulation of TM and SC cells; (4) the mechanisms and/or regulation of cell volume control are different in TM and SC cells; and (5) the novel application of servo-null micropipette technology to measure IOP in the mouse will permit direct extension of the membrane physiology to in vivo physiology. The program provides an unusual opportunity for integrating cellular and whole-animal techniques in developing novel pharmacologic interventions for reducing intraocular pressure (IOP) in treating glaucoma and in studying targeted genetic mouse models of aqueous humor outflow. The specific aims are to: (1) Identify the mechanisms and regulation of cell volume control of human TM and SC cells and immortalized mouse TM cells; (2) Determine the actions and identity of the purinergic receptors and ectoenzymes responsible for modulating the cell volume mechanisms and regulations in these cells; and (3) Extend the cell studies to analysis of aqueous humor dynamics with the servo-null micropipette system (SNMS) we have developed, which provides the first and only reliable approach for measuring IOP in the mouse.

Recent advances, including work by us and our collaborators, are leading to an increasingly comprehensive and defining concept of the mechanism of aqueous humor secretion by the ciliary epithelial bilayer, comprising the nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells. We propose to develop these ideas further through an integrated program addressing: membrane physiology with molecular, electrophysiological and videomicroscopic techniques; tissue physiology with the unique approach of electron-probe X-ray microanalysis; and whole organ physiology with assessments of intraocular pressure in living animals. The program offers an unusual opportunity to expand to strategies for developing novel, highly specific and more effective pharmacologic interventions to lower aqueous humor secretion and reduce IOP in glaucomatous patients. Specific aims: (1) For the NPE cells at the aqueous surface, establish the central role of the ClC-3 channel and Cl- release in secretion of aqueous humor. (2) For the PE cells at the opposite (stromal) surface of the tissue, establish the central role of paired Na+/H+ and Cl-/HC03 - exchangers in taking up Cl- from the stroma. (3) For the coordinated bilayer as a whole, establish that the transepithelial balance between secretion and reabsorption varies antero-posteriorly along the surface of the ciliary epithelium, and document that certain drugs can act synergistically both to block secretion at the aqueous surface and to stimulate reabsorption at the stromal surface.