Retinal angiotensin II and angiotensin-(1-7) response to hyperglycemia and an intervention with captopril.

HYPOTHESIS: Hyperglycemia decreases angiotensin-(1-7), the endogenous counter-regulator of angiotensin II in the retina. MATERIALS AND METHODS: The distribution and levels of retinal angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) were evaluated by confocal imaging and quantitative immunohistochemistry during the development of streptozotocin-induced diabetes in rats. RESULTS: In the nondiabetic eye, Ang II was localized to the endfeet of Müller cells, extending into the cellular processes of the inner plexiform layer and inner nuclear layer; Ang-(1-7) showed a wider distribution, extending from the foot plates of the Müller cells to the photoreceptor layer. Eyes from diabetic animals showed a higher intensity and extent of Ang II staining compared with nondiabetic eyes, but lower intensity with a reduced distribution of Ang-(1-7) immunoreactivity. Treatment of the diabetic animals with the angiotensin-converting enzyme inhibitor (ACEI) captopril showed a reduced intensity of Ang II staining, whereas increased intensity and distribution were evident with Ang-(1-7) staining. CONCLUSIONS: These studies reveal that pharmacological inhibition with ACEIs may provide a specific intervention for the management of the diabetes-induced decline in retinal function, reversing the profile of the endogenous angiotensin peptides closer to the normal condition.

Tissue-specific expression of transgenic secreted ACE in vasculature can restore normal kidney functions, but not blood pressure, of Ace-/- mice.

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.

Angiotensin II and its receptor subtypes in the human retina.

PURPOSE: To quantify and evaluate the distribution of angiotensin II (Ang II) and its receptors in the human retina. METHODS: Donor eyes were obtained within 12 hours postmortem and classified as hypertensive or normotensive and diabetic or nondiabetic, based on the donors' medical histories. Ang II in retina and vitreous was quantified by RIA. Ang II receptors were characterized and quantified by competitive membrane-binding assays. Ang II, its heptapeptide metabolite Ang-(1-7), and AT1 and AT2 receptors were localized by immunohistochemistry and confocal imaging. RESULTS: Levels of Ang II in the retina were significantly higher than in vitreous (P < 0.05). Ang II in the diabetic retina had a higher median compared with that in the nondiabetic retina. Ang II and Ang-(1-7) colocalized in retinal Müller cells. The retina had the highest levels of Ang II receptors that were significantly higher than the optic nerve, retinal pigment epithelium-choroid complex, and ciliary body-iris complex (P < 0.05). AT1 receptors were more abundant than AT2 receptors in the retina. Immunoreactivity for AT1 was detected in Müller cells and on blood vessels. AT2 receptors were localized throughout the Müller cells and nuclei of ganglion cells and neurons in the inner nuclear layer. CONCLUSIONS: In the human retina, identification of Ang II and its bioactive metabolite Ang-(1-7) in Müller cells suggests that these glial cells are able to produce and process Ang II. Ang receptors were localized in the blood vessels and neural cells. Local Ang II signaling may thus allow for autoregulation of neurovascular activity. Such an autonomous system could modulate the onset and severity of retinovascular disease.

Vascular expression of germinal ACE fails to maintain normal blood pressure in ACE-/- mice.

Maintenance of normal blood pressure is critical for preserving the integrity of the cardiovascular system. Angiotensin 1-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-aldosterone system (RAAS) and the renal tubuloglomerular feedback response. Although the two structurally related isozymic forms of ACE both generate the vasoactive octapeptide angiotensin II (Ang II) with equal efficiency, both are expressed in a nonoverlapping tissue-restricted fashion. To discriminate the precise physiological role of each ACE in its requisite tissue in vivo, we expressed one ACE isoform exclusively in a single cell type of an Ace null mouse. Previously, we demonstrated that vascular endothelial cell-specific expression of transgenic somatic ACE (sACE) could restore normal blood pressure of Ace-null mice. In this current study, we expressed germinal ACE (gACE) in the vascular endothelial cells of the Ace null mouse. These mice exhibited correct renal structure, renal function, and normal growth rates. Although the mice had elevated levels of gACE bound to vascular endothelial cells and high levels of gACE and Ang II in the circulating serum, blood pressure was restored only partially. This study demonstrated that gACE, even when expressed in the vasculature, could not functionally substitute for sACE.

Glucose utilization by the retinal pigment epithelium: evidence for rapid uptake and storage in glycogen, followed by glycogen utilization.

Glucose utilization and glycogen metabolism by human retinal pigment epithelium (RPE) cultures with high transepithelial resistance maintained on porous Millicell polycarbonate filters, were quantified by fluorophore-assisted carbohydrate electrophoresis (FACE). Glucose uptake was more efficient at the apical surface of the RPE. The utilization of glucose when restricted to either the apical or basal medium was also evaluated. Under both conditions, glucose was quickly transported to the opposite compartment and rapidly utilized. However, glucose from the apical compartment was depleted to a greater extent than from the basal compartment. The de novo synthesis and accumulation of glycogen accompanied glucose utilization. This was paralleled by a concomitant increase in lysosomal glycogen degradation measured as an increase in cell-associated maltodextrins. The highest levels of glucose in glycogen and maltodextrins occurred at 24 h, declining to basal levels at 72 h. Glucose transporter expression in the RPE cultures was evaluated with the reverse transcriptase-polymerase chain reaction. Glucose transporter-1 (GLUT 1) was the isoform expressed in these cells. GLUT 1 localization was determined by immunocytochemistry. GLUT 1 localizes to the apical and basolateral border of the RPE. The intensity of fluorescence was higher on the apical border. The rapid depletion of medium glucose suggests that RPE culture studies should replenish medium glucose more frequently than every 72 h to maintain physiologically relevant glucose concentrations. These studies are the first to demonstrate glucose, glycogen and maltodextrin metabolism by RPE cells, and their detection and quantitation by FACE.