High glucose induces alteration of gap junction permeability and phosphorylation of connexin-43 in cultured aortic smooth muscle cells.

Abstract

Gap junction is thought to have a crucial role in maintaining tissue homeostasis. We examined the effect of a high glucose level on gap junctional intercellular communication (GJIC) activity in cultured vascular smooth muscle cells (VSMCs) using the fluorescent dye transfer method. After a 48-h incubation with 22 mmol/l glucose (high glucose level), GJIC activity of VSMCs was significantly reduced compared with incubation with 5.5 mmol/l glucose (normal glucose level) (P < 0.05). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 5 x 10(-8) mol/l), a protein kinase C (PKC) activator, for 1 h also reduced GJIC activity (P < 0.01). In addition, treatment of the cells with calphostin C, a specific PKC inhibitor, for 3 h completely restored the GJIC activity inhibited by the high glucose level. Western blot analysis showed that connexin 43 (Cx43), which is the major functional protein of gap junction, is present in multiphosphorylated forms: a nonphosphorylated form (P0) and phosphorylated forms (P1, P2, and P3). Incubation of VSMCs with a high glucose level significantly increased the density ratio of P3/P0 compared with a normal glucose level (P < 0.05). Similarly, treatment of the cells with TPA significantly increased the P3/P0 ratio compared with controls (P < 0.01). In addition, the increase in the P3/P0 density ratio induced by a high glucose level was restored to the control level by both staurosporine and calphostin C. These results suggest that the high glucose level induced the inhibition of GJIC activity in cultured VSMCs through excessive phosphorylation of Cx43, mediated by PKC activation. This may contribute to the development of the macroangiopathy associated with diabetes.

High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C--dependent activation of NAD(P)H oxidase in cultured vascular cells.

“…tured vascular cells was significantly inhibited by both GF109203X and calphostin C, PKC-specific inhibitors, suggesting the role of PKC. It is well established that high glucose activates PKC through an increase in de novo DAG synthesis in vascular cells (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Taken together, the present results strongly suggest that high glucose level stimulated ROS production through PKC-dependent activation of NAD(P)H oxidase in cultured vascular cells.…”

“…In phagocytic cells, one of the regulators of NADPH oxidase activity may be protein kinase C (PKC) (13)(14)(15). Numerous studies, including ours, have shown that high glucose levels or diabetes may activate PKC in various vascular cells (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Therefore, high glucose or diabetes might stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in vascular cells.…”

Altered gap junctional communication and renal haemodynamics in Zucker fatty rat model of type 2 diabetes

“…Also in the present study, blockade of Cx43 did not have any significant effects on GFR in ZDF rats, although western blotting showed a significant increase in phosphorylation of Cx43 in ZDF rat kidney. This phosphorylation of Cx43, like that induced by high glucose in vascular smooth muscle cells, would interfere with gap junction expression, distribution, degradation and function [15,17,37]. Indeed, Satriano et al [38] have reported reduced levels of Cx43 in kidney from a streptozotosin-induced rat model of type 1 diabetes.…”

“…Indeed, the phosphorylation of Cx43 modulates channel gating, turnover and distribution [15]. Interestingly, high glucose treatment decreases gap junction function in microvascular endothelial cells and aortic smooth muscle cells either by reducing production or increasing phosphorylation of Cx43 [16,17]. These observations raise the possibility that dysfunction of gap junctions from altered Cxs and also varied abundance of Cxs contribute to diabetic nephropathy, a disease characterised by reduced afferent arteriolar tone and glomerular hyperfiltration [18][19][20].…”

Reduced EDHF responses and connexin activity in mesenteric arteries from the insulin-resistant obese Zucker rat

“…Cell cultures exposed to high glucose levels have been used to study gap junctions and specific connexins in isolation, with results showing attenuation of gap junction activity in bovine aortic endothelial and smooth muscle cells [22,23]. Furthermore, investigations using animal models of type 1 diabetes have shown attenuation of EDHF-mediated responses [24] and gap junction activity in mesenteric [25] arteries from streptozotocin (STZ)-induced diabetic rats.…”