Induction of resistance to endothelin-1's biochemical actions by elevated glucose levels in retinal pericytes

Abstract

Because retinal pericytes have contractile properties and are affected by diabetes, we have studied the responsiveness of pericytes to ET-1, a potent vasoconstrictor, in the presence of various concentrations of glucose. Cultured calf retinal pericytes were exposed to glucose levels of 5.5 or 25 mM for up to 8 days. Radioreceptor studies that used [125I]ET-1 showed that pericytes contained high-affinity binding sites with Kd of 3 x 10(-10) M, and these binding affinities were unaffected by glucose concentration. Receptor number appears to be elevated, but this increase was NS. Responsiveness of pericytes to ET-1 was studied with respect to stimulation of DAG and IP3 levels and PKC activities. In contrast to receptor binding, exposure to 25 mM glucose for > 6 days blunted pericyte responsiveness to ET-1. The time course of ET-1 stimulation as measured by [3H]glycerol labeling, and IP3 level showed a 98% increase in [3H]DAG at 10 min and a fourfold increase for IP3, respectively. Cells exposed to 25 mM glucose only had a 32% increase for DAG, and no increase for IP3 was observed. Dose-response studies on the stimulation of [3H]DAG increase showed the range of ET-1's effect to be between 10(-9) and 10(-7) M. At maximum, cells exposed to 5.5 mM glucose had a 70% increase versus only a 30% increase in those exposed to 25 mM glucose. Similarly, ET-1 only increased the total DAG levels in pericytes exposed to 5.5 mM glucose by 41%. PKC activity also was measured because DAG is one of its cellular activators.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

“…In RPE cells with low-grade intrinsic AR expression (75), d-glucose increases phosphoinositide turnover and DAG levels (19). Analogously divergent effects of d-glucose on DAG and PKC signal transduction have been reported in retinal microvascular endothelial cells (76) and pericytes (39), that also differ in AR expression (77). Although not the focus of this report, the relationship between AR pathway activity and MI in SH-SY5Y cells may be different from either high or low AR-expressing RPE cells.…”

“…1 D) (28). Diminished MARCKS phosphorylation after 6 d could not be attributed to PKC downregulation after a putative transient initial increase in PKC activity induced by d-glucose (as described in various nonneural cells [36][37][38][39][40][41]) since MARCKS phosphorylation was not increased 2 h and 24 h after exposure to 50 mM d-glucose (data not shown). Thus in SH-SY5Y cells cultured in elevated levels of d-glucose, flux through AR was low relative to sorbitol clearance, MI depletion was primarily non-AR mediated perhaps by competitive inhibition by d-glucose of Na-MI cotransport (42)(43)(44), depletion of MI limited basal PI synthesis (7,19,26,45), and basal PKC-mediated MARCKS phosphorylation was reduced (19).…”

“…However, these views are at best complex and fragmentary, if not at times paradoxical and contradictory. Diabetes and/or elevated d-glucose levels have been reported to decrease MI and phosphoinositide-derived arachidonyl-DAG (18), PKC activity (16,39), and/or PKCrelated cellular functions (20, 61) in some but not all tissues prone to diabetic complications. Yet detailed patterns of response may vary even within the same tissue.…”

The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.

“…Nitric oxide deficiency might perturb NCV and nerve (Na+,K+)-ATPase through a nonischemic mechanism, perhaps interacting with other more well established AR2-related defects in signal transduction (1,(47)(48)(49)(50)(51)(52)(53) involved in modulation of (Na+,K+)-ATPase activity (1,2,9,13,54). NO may modulate phosphoinositide turnover by unknown mechanisms (55) such that NO deficiency might act synergistically with MI depletion in altering phosphoinositide signal transduction and (Na+,K+)-ATPase regulation.…”

Ambient glucose and aldose reductase-induced myo-inositol depletion modulate basal and carbachol-stimulated inositol phospholipid metabolism and diacylglycerol accumulation in human retinal pigment epithelial cells in culture.

“…Several independent lines of investigation suggest that this interaction may be quite complex, involving functionally discrete metabolic pools of MI, PPI, and/or PI synthase (1)(2)(3). In the second metabolic response pattern to glucose, exhibited in vitro by retinal endothelial and glomerular cells with relatively low AR2 activity (1,8,9), DAG content, glucose incorporation into DAG, and protein kinase C translocation from the cytosolic to the membrane fraction are all increased after exposure to hyperglycemic concentrations of glucose (10)(11)(12). This …”