W. Daniel Stamer

Forskolin stimulation of water and cation permeability in aquaporin 1 water channels

Aquaporin1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3′,5′-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin1. The cationic permeability of the aquaporin1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

Current understanding of conventional outflow dysfunction in glaucoma

Purpose of review Regulation of intraocular pressure by the conventional (trabecular) outflow pathway is complicated, involving a myriad of mechanical and chemical signals. In most, intraocular pressure is maintained within a tight range over a lifetime. Unfortunately in some, dysfunction results in ocular hypertension and open-angle glaucoma. In the context of established knowledge, this review summarizes recent investigations of conventional outflow function, with the goal of identifying areas for future inquiry and therapeutic targeting. Recent findings Mechanical stimulation of conventional outflow cells due to intraocular pressure fluctuations impacts contractility, gene expression, pore formation, enzyme activity, and signaling. Numerous local signaling mediators in the conventional pathway such as bioactive lipids, cytokines, nitric oxide, and nucleotides participate in the regulation of outflow. Interestingly outflow through the conventional pathway is not uniform, but segmental, with passageways constantly changing due to focal protease activity of trabecular cells clearing extracellular matrix materials. The relationship between extracellular matrix expression and trabecular meshwork contractility appears to coordinately impact outflow resistance and is the target of a new class of drugs, the Rho kinase inhibitors. Summary The conventional outflow pathway is a dynamic, pressure-sensitive tissue that is vulnerable to pathology on many fronts, each representing a therapeutic opportunity.

eNOS, a Pressure-Dependent Regulator of Intraocular Pressure

PURPOSE Pathology in the primary drainage pathway for aqueous humor in the eye is responsible for ocular hypertension, the only treatable risk factor in patients with glaucoma. Unfortunately, the mechanisms that regulate pressure-dependent drainage of aqueous humor and thus intraocular pressure (IOP) are unknown. To better understand one possible underlying molecular factor that regulates IOP, nitric oxide (NO), pressure-dependent drainage in transgenic mice overexpressing endothelial NO synthase (eNOS) was studied. METHODS IOP was measured by rebound tonometry in mice, and pressure versus flow data were measured by ex vivo perfusion at multiple pressures between 8 and 45 mm Hg, using mock AH ±100 μM L-NAME. A subset of eyes was examined histologically using standard techniques or was assayed for fusion protein expression by Western blot analysis. RESULTS IOP was lower (9.6 ± 2.7 vs. 11.4 ± 2.5 mm Hg; mean ± SD; P = 0.04) and pressure-dependent drainage was higher (0.0154 ± 0.006 vs. 0.0066 ± 0.0009 μL/min/mm Hg; P = 0.002) in the transgenic mice than in the wild-type animals; however, pressure-independent drainage was unaffected. The NOS inhibitor L-NAME normalized pressure-dependent drainage in transgenic animals. For IOP >35 mm Hg, the slope of the pressure-flow curve in wild-type mice increased to match that seen in transgenic mice. Shear stress in the pressure-dependent pathway at elevated pressures was calculated to be in a range known to affect eNOS expression and activity in vascular endothelia. CONCLUSIONS Endothelial NOS overexpression lowers IOP by increasing pressure-dependent drainage in the mouse eye. Data are consistent with NOs having a mechanoregulatory role in aqueous humor dynamics, with eNOS induction at elevated IOPs leading to increased pressure-dependent outflow.

L-DOPA Is an Endogenous Ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and three different volumes (1 ml, 500 μl and 100 μl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 μl and 50 μl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40–150 nm). The three kits, though, produced a significantly higher yield (80–300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.

Extracellular Trafficking of Myocilin in Human Trabecular Meshwork Cells

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.

Ion Channel Function of Aquaporin-1 Natively Expressed in Choroid Plexus

Aquaporins are known as water channels; however, an additional ion channel function has been observed for several including aquaporin-1 (AQP1). Using primary cultures of rat choroid plexus, a brain tissue that secretes CSF and abundantly expresses AQP1, we confirmed the ion channel function of AQP1 and assessed its functional relevance. The cGMP-gated cationic conductance associated with AQP1 is activated by an endogenous receptor guanylate cyclase for atrial natriuretic peptide (ANP). Fluid transport assays with confluent polarized choroid plexus cultures showed that AQP1 current activation by 4.5 μm ANP decreases the normal basal-to-apical fluid transport in the choroid plexus; conversely, AQP1 block with 500 μm Cd2+ restores fluid transport. The cGMP-gated conductance in the choroid plexus is lost with targeted knockdown of AQP1 by small interfering RNA (siRNA), as confirmed by immunocytochemistry and whole-cell patch electrophysiology of transiently transfected cells identified by enhanced green fluorescent protein. The properties of the current (permeability to Na+, K+, TEA+, and Cs+; voltage insensitivity; and dependence on cGMP) matched properties characterized previously in AQP1-expressing oocytes. Background K+ and Cl− currents in the choroid plexus were dissected from AQP1 currents using Cs-methanesulfonate recording salines; the background currents recorded in physiological salines were not affected by AQP1–siRNA treatment. These results confirm that AQP1 can function as both a water channel and a gated ion channel. The conclusion that the AQP1-associated cation current contributes to modulating CSF production resolves a lingering concern as to whether an aquaporin ionic conductance can have a physiologically relevant function.

Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line.

BackgroundAquaporin-1 (AQP1) channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM) was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex.ResultsTEA blocked water permeability of AQP1 channels in kidney and kidney-derived cells, demonstrating this effect is not limited to the oocyte expression system. Equivalent inhibition is seen in MDCK cells with viral-mediated AQP1 expression, and in rat renal descending thin limbs of Henles loops which abundantly express native AQP1, but not in ascending thin limbs which do not express AQP1. External TEA (10 mM) does not block the cGMP-dependent AQP1 ionic conductance, measured by two-electrode voltage clamp after pre-incubation of oocytes in 8Br-cGMP (10–50 mM) or during application of the nitric oxide donor, sodium nitroprusside (2–4 mM).ConclusionsTEA selectively inhibits osmotic water permeability through native and heterologously expressed AQP1 channels. The pathways for water and ions in AQP1 differ in pharmacological sensitivity to TEA, and are consistent with the idea of independent solute pathways within the channel structure. The results confirm the usefulness of TEA as a pharmacological tool for the analysis of AQP1 function.

Glucocorticoids with different chemical structures but similar glucocorticoid receptor potency regulate subsets of common and unique genes in human trabecular meshwork cells

BackgroundIn addition to their well-documented ocular therapeutic effects, glucocorticoids (GCs) can cause sight-threatening side-effects including ocular hypertension presumably via morphological and biochemical changes in trabecular meshwork (TM) cells. In the present study, we directly compared the glucocorticoid receptor (GR) potency for dexamethasone (DEX), fluocinolone acetonide (FA) and triamcinolone acetonide (TA), examined the expression of known GRα and GRβ isoforms, and used gene expression microarrays to compare the effects of DEX, FA, and TA on the complete transcriptome in two primary human TM cell lines.MethodsGR binding affinity for DEX, FA, and TA was measured by a cell-free competitive radio-labeled GR binding assay. GR-mediated transcriptional activity was assessed using the GeneBLAzer beta-lactamase reporter gene assay. Levels of GRα and GRβ isoforms were assessed by Western blot. Total RNA was extracted from TM 86 and TM 93 cells treated with 1 μM DEX, FA, or TA for 24 hr and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by Rosetta Resolver Gene Expression Analysis System.ResultsThe GR binding affinity (IC50) for DEX, FA, and TA was 5.4, 2.0, and 1.5 nM, respectively. These values are similar to the GR transactivation EC50 of 3.0, 0.7, and 1.5 nM for DEX, FA, and TA, respectively. All four GRα translational isoforms (A-D) were expressed in TM 86 and TM 93 total cell lysates, however, the C and D isoforms were more highly expressed relative to A and B. All four GRβ isoforms (A-D) were also detected in TM cells, although GRβ-D isoform expression was lower compared to that of the A, B, or C isoforms. Microarray analysis revealed 1,968 and 1,150 genes commonly regulated by DEX, FA, and TA in TM 86 and TM 93, respectively. These genes included RGC32, OCA2, ANGPTL7, MYOC, FKBP5, SAA1 and ZBTB16. In addition, each GC specifically regulated a unique set of genes in both TM cell lines. Using Ingenuity Pathway Analysis (IPA) software, analysis of the data from TM 86 cells showed that DEX significantly regulated transcripts associated with RNA post-transcriptional modifications, whereas FA and TA modulated genes involved in lipid metabolism and cell morphology, respectively. In TM 93 cells, DEX significantly regulated genes implicated in histone methylation, whereas FA and TA altered genes associated with cell cycle and cell adhesion, respectively.ConclusionHuman trabecular meshwork cells in culture express all known GRα and GRβ translational isoforms, and GCs with similar potency but subtly different chemical structure are capable of regulating common and unique gene subsets and presumably biologic responses in these cells. These GC structure-dependent effects appear to be TM cell-lineage dependent.

Outflow physiology of the mouse eye: pressure dependence and washout.

PURPOSE Mice are commonly used in glaucoma research, but relatively little is known about aqueous outflow dynamics in the species. To facilitate future use of the mouse as a model of aqueous humor outflow, several fundamental physiological parameters were measured in the mouse eye. METHODS Eyes from adult mice of either sex (C57BL/6 background) were enucleated, cannulated with a 33-gauge needle, and perfused at constant pressure while inflow was continuously measured. RESULTS At 8 mm Hg, total outflow facility (C(total)) was 0.022 ± 0.005 μL/min/mm Hg (all values mean ± SD; n = 21). The flow-pressure relationship was linear up to 35 mm Hg. The conventional outflow facility (C(conv)) was 0.0066 ± 0.0009 μL/min/mm Hg, and the unconventional outflow (F(u)) was 0.114 ± 0.019 μL/min, both measured at room temperature. At 8 mm Hg, 66% of the outflow was via the unconventional pathway. In a more than 2-hour-long perfusion at 8 mm Hg, the rate of facility change was 2.4% ± 5.4% (n = 11) of starting facility per hour. The ocular compliance (0.086 ± 0.017 μL/mm Hg; n = 5) was comparable to the compliance of the perfusion system (0.100 ± 0.004 μL/mm Hg). CONCLUSIONS Mouse eyes are similar to human eyes, in that they have no detectable washout rate and a linear pressure-flow relationship over a broad range of intraocular pressures. Because of the absence of washout and the apparent presence of a true Schlemms canal, the mouse is a useful model for studying the physiology of the inner wall of Schlemms canal and the conventional outflow tissues.