Biochemical and ultrastructural studies in the neural retina and retinal pigment epithelium of STZ-diabetic rats: effect of captopril.

We measured the activities of total Na+, K+-ATPase (Na, K-ATPase), its alpha1 and alpha2/alpha3 isoforms and the angiotensin-converting enzyme (ACE) in the microvascular and neural compartments of the retina, and/or retinal pigment epithelium (RPE) of streptozotocin (STZ)-diabetic rats. The effect of captopril, an ACE inhibitor on Na, K-ATPase activities was also determined and correlated to morphological changes. Insulin-dependent diabetes mellitus was induced by a single intraperitoneal injection of STZ (60 mg/kg) in male Long-Evans rats. ACE activity was inhibited by captopril (10 mg/kg given in the drinking water) for 1 month. Na, K-ATPase activity was measured spectrophotometrically or by a radioassay (32P-labeled ATP). The activity of ACE was determined by a radioassay using tritiated benzoyl-gly-gly-gly as substrate. Both the alpha1 and alpha2/alpha3 isoforms of Na, K-ATPase were present in the microvascular and neural compartments of retinas, whereas only one isoform, the alpha2/alpha3, was found in the RPE. In 2-month diabetic rats, the activity of the alpha2/alpha3 isoform was reduced in both the microvascular and neural compartments of retinas, while the activity of the alpha1 isoform was reduced only in the neural isolates. ACE activity was significantly decreased in the retinal neural compartment and unaltered in the microvascular compartment from 2-month diabetic rats. In 5-month diabetic rats, Na, K-ATPase activity was moderately but not significantly reduced in RPE preparations. Ultrastructural studies revealed a significant deepening of basal infoldings in the RPE and a noticeable increase in the size of the extracellular space between the basal infoldings of 5-month diabetic animals. Captopril stimulated Na, K-ATPase activity in the neural retina, but not in the RPE. Diabetes-induced morphological changes in the RPE were not improved by captopril. An enlargement of intercellular space between the RPE cells was a frequent finding in the treated group. In conclusion, captopril stimulated Na, K-ATPase activity in the neural retina of diabetic rats. This stimulation seems to be beneficial to the neural retina. ACE inhibition, however, did not improve RPE morphological changes. Although the clinical significance of increased intercellular spacing between RPE cells in treated animals is not clearly established, we speculate that it might contribute to an increased alteration of their barrier function. Additional studies are necessary to assess both the desirable and adverse effects of captopril and other ACE inhibitors in the retinas of diabetic patients.

Histopathologic and immunocytochemical analysis of the retina and ocular tissues in Batten disease.

PURPOSE: To describe the pathophysiologic features of retinal degeneration in Batten disease (juvenile neuronal ceroid lipofuscinosis [JNCL]) caused by mutations in the CLN3 gene. STUDY DESIGN: Comparative human tissue study. MATERIALS: The retina and other ocular tissues of a 22-year-old man with JNCL were compared with the same tissues of a healthy 30-year-old man. DNA from whole blood and RNA from retina were used for genotype analysis. METHODS: The retinas, corneas, conjunctiva, and ciliary body were processed for histopathologic and immunofluorescence analysis. Genomic DNA was subjected to polymerase chain reaction (PCR) and nucleotide sequence analyses. Reverse transcriptase/PCR and sequence analysis were performed on retinal RNA. RESULTS: The JNCL donor was heterozygous for a approximately 1 kb deletion in CLN3, as found in most JNCL patients. The other allele had a single base pair deletion in exon 6 that resulted in a frame shift. Gross pathology of the JNCL retina resembled that in retinitis pigmentosa, including deposits of bone spicule pigment. Histopathologic studies revealed loss of neurons from all retinal layers. Immunofluorescence labeling with antibodies to rhodopsin, recoverin, and cone opsin demonstrated degenerate rods and cones with short outer segments in the far periphery. Autofluorescent lipopigment granules were prominent in ganglion cells and some cells of the inner nuclear layer, but not in the photoreceptors. The retinal pigment epithelium (RPE) had fewer lipofuscin granules than the control specimen. Increased numbers of lipofuscin granules were found in the epithelia of the ciliary body and conjunctiva, but not in the cornea of the JNCL eye. CONCLUSIONS: Immunofluorescence studies revealed degenerate rods and cones in the far periphery. Lipofuscin granules were decreased in the RPE, consistent with loss of photoreceptor outer segments. The novel finding that degenerate photoreceptors did not contain autofluorescent inclusions suggests that granule accumulation may not precede photoreceptor degeneration in JNCL. The presence of normal photoreceptor proteins in the degenerate rods and cones suggests that these cells may be capable of functional regeneration if a therapy for Batten disease is developed.

Angiotensin-converting enzyme activity in retinas of streptozotocin-induced and Zucker diabetic rats. The effect of angiotensin II on Na+,K(+)-ATPase activity.

PURPOSE: To investigate whether serum and/or retinal angiotensin-converting enzyme (ACE) activity might correlate with the decrease in sodium potassium adenosine triphosphatase (Na,K-ATPase) activity in the retina of experimentally diabetic rats. METHODS: Insulin-dependent diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) in male Sprague-Dawley rats. Male Zucker fatty diabetic (ZDF/Gmifa) rats were used as models of non-insulin-dependent diabetes mellitus. ACE activity in the serum and retina of diabetic rats (1 through 5 months) and age-matched control animals was measured by radioimmunoassay using benzoyl-gly-gly-gly as substrate. The activity of total Na,K-ATPase was determined spectrophotometrically. The alpha 1 and alpha 3 isozymes of Na,K-ATPase were distinguished pharmacologically by their differential sensitivity to ouabain and were measured in the retina. RESULTS: Serum ACE activity was significantly increased in rats with STZ-induced diabetes at 3 weeks through 4 months of diabetes (28% to 32%) but was significantly decreased in ZDF rats after 2 to 5 months of diabetes (-9% to -16%). The activity of ACE in retinas obtained from the same groups of STZ and ZDF rats was significantly reduced at all time points examined in both models (-43% and -55%, respectively). The effect of angiotensin II (AngII) on the activity of Na,K-ATPase in retinas from normal rats was also studied in vitro. AngII significantly lowered the activities of total Na,K-ATPase (-16%) and its alpha 1 and alpha 3 isozymes. The inhibitory effect of AngII was abolished completely by losartan (0.1 microM), a specific antagonist of the AT1 receptor-subtype of AngII, and by nordihydroguaiaretic acid (50 microM), which at this concentration inhibits the lipoxygenase and cytochrome P-450-dependent pathways of arachidonic acid metabolism. The inhibitory effect of AngII on the Na,K-ATPase activity was not altered significantly by NG-iminoethyl ornithine (10 microM), an irreversible nitric oxide synthase inhibitor. CONCLUSIONS: The authors suggest that systemic ACE probably is not involved in the mechanisms responsible for the reduced activity of Na,K-ATPase in diabetes. Although AngII inhibits retinal Na,K-ATPase by a mechanism possibly involving arachidonic acid metabolites, it is unlikely that AngII contributes to the decreased Na,K-ATPase activity because of its reduced formation by retinal ACE in diabetes. The possible importance of reduced retinal ACE activity in diabetes warrants further investigation.

Captopril ameliorates the decreased Na+,K(+)-ATPase activity in the retina of streptozotocin-induced diabetic rats.

PURPOSE: To examine the effect of captopril, an angiotensin-converting enzyme (ACE) inhibitor, on the activity of retinal sodium-potassium ATPase (Na,K-ATPase) and the activity of ACE in the serum and retina of streptozotocin (STZ)-induced diabetic rats. METHODS: Experimental diabetes was induced in male Long-Evans rats by a single intraperitoneal injection of STZ (55 mg/kg body weight). Some groups of normal and diabetic animals were treated with captopril (10 mg/kg per day) added to the drinking water for either a week or a month. After 2 and 4 months of diabetes, the specific activity of retinal total Na,K-ATPase was determined. The components of the activity of Na,K-ATPase caused by the alpha 1 and alpha 3 isoforms were pharmacologically separated by their different sensitivity to ouabain. The activity of ACE in the serum and retina was measured by radioassay using benzoyl-gly-gly-gly as substrate (10(5) cpm, 5 mM). RESULTS: The total Na,K-ATPase activity was decreased significantly after 2 (16%, P < 0.02) and 4 months (15%, P < 0.02) of diabetes. At both time points examined, the activities of the alpha 1-low-ouabain-affinity isoform and the alpha 3-high-ouabain-affinity isoform of retinal Na,K-ATPase were significantly reduced compared to those of age-matched controls (alpha 1, 9% to 14%, P < 0.05; alpha 3, 14% to 19%, P < 0.05 and P < 0.02 respectively). After 1 month of captopril administration, the activities of both Na,K-ATPase isoforms were at control level in 2-month diabetic rats, whereas they were restored only partially in 4-month diabetic rats. In age-matched normal animals, 1 month of captopril treatment did not alter the specific activities of either Na,K-ATPase isoform. One week or 1 month of captopril administration to diabetic rats did not change the activities of retinal Na,K-ATPase isoforms. Serum ACE activity was elevated significantly in both groups of untreated STZ rats (55% and 40%, respectively). One month of captopril administration further increased the ACE levels in 2- and 4-month diabetic rats (101% and 94%, respectively) and also enhanced significantly the serum ACE activity in normal animals (131%) versus the basal values. In contrast, retinal ACE activity was decreased significantly in both groups of untreated STZ rats (approximately 37%). Captopril exerted a significant inhibitory effect on the retinal ACE activity in 2- and 4-month diabetic rats (37% and 31%, respectively) compared to untreated diabetic animals as well as in normal rats (29%). CONCLUSIONS: These data suggest that stimulation of retinal Na,K-ATPase activity in diabetes is most likely one of the mechanisms through which captopril can improve retinal complications. The effect of captopril seems to be related to local effects in the retina. Whether the inhibition of retinal ACE is part of the mechanism of action of captopril requires further study.

Electron-microscopic study of iris nerves and muscles in diabetes.

The ultrastructure of the iris muscles and innervating nerves in patients with diabetes mellitus and approximately age-matched controls was examined by electron-microscopy. The specimens were obtained during cataract surgery. There were definite histopathological alterations at the nerve terminal innervating the dilator muscle, e.g. the presence of mitochondrial abnormalities, dense bodies and lamellar structures. No change was noted at the nerve terminal to the sphincter muscle. Moderate involvement of the muscle, especially at the sphincter, was observed in the specimens of diabetics. The control specimens had no change at the nerve terminals, while moderate change was observed at the sphincter muscle. Therefore, the change in the sphincter seen in the specimens of diabetics seemed to be mainly the result of the aging process, though the existence of early sympathetic neuropathy was confirmed.