Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Predisposition to arrhythmia and autonomic dysfunction in Nhlh1-deficient mice.

Nhlh1 is a basic helix-loop-helix transcription factor whose expression is restricted to the nervous system and which may play a role in neuronal differentiation. To directly study Nhlh1 function, we generated null mice. Homozygous mutant mice were predisposed to premature, adult-onset, unexpected death. Electrocardiograms revealed decreased total heart rate variability, stress-induced arrhythmia, and impaired baroreceptor sensitivity. This predisposition to arrhythmia is a likely cause of the observed death in the mutant mice. Heterozygosity for the closely related transcription factor Nhlh2 increased the severity of the Nhlh1-null phenotype. No signs of primary cardiac structural or conduction abnormalities could be detected upon necropsy of the null mice. The pattern of altered heart rhythm observed in basal and experimental conditions (stress and pharmacologically induced) suggests that a deficient parasympathetic tone may contribute to the arrhythmia in the Nhlh1-null mouse. The expression of Nhlh1 in the developing brain stem and in the vagal nuclei in the wild-type mouse further supports this hypothesis. The Nhlh1 mutant mouse may thus provide a model to investigate the contribution of the autonomic nervous system to arrhythmogenesis.

Glucose-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase-dependent upregulation of the platelet-derived growth factor-B receptor potentiates vascular smooth muscle cell chemotaxis

The aim of this study was to investigate the effects of elevated D-glucose concentrations on vascular smooth muscle cell (VSMC) expression of the platelet-derived growth factor (PDGF) beta receptor and VSMC migratory behavior. Immunoprecipitation, immunofluorescent staining, and RT-PCR of human VSMCs showed that elevated D-glucose induced an increase in the PDGF beta receptor that was inhibited by phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors. Exposure to 25 mmol/l D-glucose (HG) induced increased phosphorylation of protein kinase B (PKB) and extracellular-regulated kinase (ERK). All HG chemotaxis assays (with either 10 days' preincubation in HG or no preincubation) in a FCS or PDGF-BB gradient showed positive chemotaxis, whereas those in 5 mmol/l D-glucose did not. Assays were also run with concentrations ranging from 5 to 25 mmol/l D-glucose. Chemotaxis was induced at concentrations >9 mmol/l D-glucose. An anti-PDGF beta receptor antibody inhibited glucose-potentiated VSMC chemotaxis, as did the inhibitors for the PI3K and MAPK pathways. This study has shown that small increases in D-glucose concentration, for a short period, increase VSMC expression of the PDGF beta receptor and VSMC sensitivity to chemotactic factors in serum, leading to altered migratory behavior in vitro. It is probable that similar processes occur in vivo with glucose-enhanced chemotaxis of VSMCs, operating through PDGF beta receptor-operated pathways, contributing to the accelerated formation of atheroma in diabetes.