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Role of altered signalling pathways in abnormal vasoconstrictor responses in mesenteric arteries from STZ-diabetic rats Irem, Mueed


Cardiovascular complications are recognized to be the major cause of morbidity and mortality associated with diabetes mellitus. One of the most common features of vascular dysfunction in well-established diabetes is the enhanced reactivity of blood vessels to vasoconstrictors. We and others have consistently found that contractile responses of arteries from rats with well-established diabetes to stimulation of G-protein coupled receptors (GPCRs), such as ai-adrenoceptor (α₁-AR) and endothelin-1 (ET-1) receptors, are enhanced. Previous studies from this lab have demonstrated that the increased contractile responses of arteries from chronic (12-14 weeks) streptozotocin- (STZ-) diabetic rats to α₁-AR stimulation result from a change in the signal transduction process downstream from the receptor. Protein kinase C (PKC) has been suggested to contribute to enhanced contractile responses of arteries from STZ-diabetic rats to stimulation of GPCRs. This was investigated in the present study by comparing the effects of the PKC inhibitors, Ro-318220 and calphostin C on contractile responses of mesenteric arteries from diabetic and age-matched control rats to the α₁-AR agonist, norepinephrine (NE) and to ET-1. Since translocation of PKC to the membrane is considered a hallmark of its activation, the effects of NE and ET-1 on particulate (membrane) levels of three isoforms of PKC (PKCα, δ and ε) that have been implicated in contraction were determined. The effect of NE on phosphorylation of CPI-17, a substrate for PKC, was also investigated. Contractile responses of endothelium-denuded arteries from diabetic rats to NE were enhanced, but were normalized by the PKC inhibitors. In contrast, no change in contractile responses of diabetic arteries to ET-1 could be detected, and PKC inhibition attenuated ET-1 responses to a similar extent in both control and diabetic tissues. NE produced a small translocation of PKCε in control arteries, but a significant translocation of PKCα and a much larger translocation of PKCε in diabetic arteries. ET-1 increased translocation of PKCα, δ and ε to the same extent in both control and diabetic arteries. NE significantly enhanced CPI-17 phosphorylation in diabetic, but not control, arteries and this was blocked by calphostin C. In addition to PKC, the RhoA/Rho kinase (RhoK) signaling pathway is also believed to play an important role in vasoconstriction. In the next part of this study, the role of RhoK in enhanced contractile responses of diabetic mesenteric arteries to stimulation of α₁-ARs was investigated. The selective RhoK inhibitors, Y-27632 and H-1152, produced greater inhibition of contractile responses to phenylephrine (PE) in diabetic than control mesenteric arteries and normalized the difference between them. Contractile responses to ET-1 were not different between control and diabetic arteries, and were not affected by RhoK inhibition. Since translocation of RhoK from the cytosolic to the membrane fraction is considered a marker of enzyme activity, the effects of PE on particulate levels of the two RhoK isoforms (ROCK I and II) were determined. A maximum concentration of PE produced significant translocation of ROCK I and II that was inhibited by Y-27632 in both control and diabetic arteries. The PE-induced translocation of ROCK II was significantly greater in diabetic tissues. PE also produced significant translocation of PKCα, δ and ε in diabetic but not control arteries. Y-27632 blocked the translocation of these isoforms in diabetic arteries but had no effect on ET-1 induced translocation of PKC isoforms in control arteries. These data suggest that increased activation of the PKC/CPI-17 as well as the RhoK pathway contribute to the enhanced contractile responses of diabetic mesenteric arteries to α₁-AR stimulation. They further suggest that there is an interaction between the RhoK and PKC pathways on stimulation of α₁-ARs in diabetic mesenteric arteries, and that RhoK may be upstream of PKC. On the other hand, RhoK does not appear to contribute to contractile responses to ET-1 in either control or diabetic mesenteric arteries; this may be the reason why vasoconstrictor responses to this agonist are not altered in diabetic arteries.

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