Kathleen M. MacLeod

Influence of the endothelium on contractile responses of arteries from diabetic rats

Contractile responses of aortas and mesenteric arteries from control and 3 month streptozotocin-diabetic rats to alpha-adrenoceptor agonists were compared in the presence and absence of endothelium. In the presence of endothelium, responses of both arteries from diabetic animals to norepinephrine and methoxamine were enhanced compared to control, although no response to clonidine could be detected in arteries from either control or diabetic animals. Following endothelium removal, no significant differences were found between control and diabetic arteries in maximum contractile responses to noradrenaline or methoxamine. However, the sensitivity (pD2) of diabetic aortas to these two agonists was significantly increased, while maximum responses of diabetic aortas and mesenteric arteries to clonidine were much greater than control. In addition, no differences between control and diabetic aortas were detected when cGMP levels were measured in the absence and presence of acetylcholine. These results suggest that enhanced responsiveness of arteries from diabetic animals to alpha-adrenoceptor stimulation is not the result of a decrease in endothelium-derived relaxing factor (EDRF) release in diabetic blood vessels.

Protein kinase C‐mediated contractile responses of arteries from diabetic rats

1 The role of protein kinase C (PKC) in mediating enhanced contractile responses of aortae and mesenteric arteries from male rats with 12–14 week streptozotocin‐induced diabetes to noradrenaline (NA) was investigated using the PKC activator, phorbol 12,13‐dibutyrate (PDB), and the PKC inhibitor, staurosporine. 2 Maximum contractile responses of aortae and mesenteric arteries from diabetic rats to NA were significantly enhanced compared with responses of arteries from age‐matched control animals. The maximum NA responses were increased by 59.6 ± 7.9% in aortae and by 54.9 ± 7.4% in mesenteric arteries from diabetic animals, compared to their respective controls. 3 Pretreatment of aortae and mesenteric arteries from both control and diabetic animals with staurosporine (5 × 10−8 m) caused marked inhibition of contractile responses to a maximum concentration of NA (10−5 m in aortae; 3 × 10−5 m in mesenteric arteries). In the presence of staurosporine, no difference was observed in the magnitude of contractile responses of arteries from control and diabetic rats to NA. 4 Maximum contractile responses of mesenteric arteries from diabetic rats to PDB were significantly increased (by 45.0 ± 4.9%) compared to responses of arteries from control animals. In contrast, no significant difference was found in the magnitude of contractile responses of aortae from control and diabetic rats to PDB. 5 Staurosporine (5 × 10−8 m) caused marked attenuation of contractile responses of arteries from control and diabetic rats to a maximum concentration of PDB (3 × 10−6 m). In the presence of staurosporine, the difference in magnitude of contractile responses of mesenteric arteries from control and diabetic rats to PDB was abolished. 6 Contractile responses of aortae and mesenteric arteries from control and diabetic rats to PDB were reduced in the absence of extracellular Ca2+, and in the presence of the Ca2+ channel blockers, nifedipine (3 × 10−6 m) or verapamil (3 × 10−6 m). Under these conditions, no difference was found in the magnitude of contractile responses of mesenteric arteries from control and diabetic rats to PDB. 7 These data suggest that enhanced contractile responses of aortae and mesenteric arteries from streptozotocin‐induced diabetic rats to NA may result, at least in part, from increased activation of PKC. In addition, increased activation of PKC‐mediated processes, which are dependent on the presence of extracellular Ca2+, may further contribute to the enhanced contractile responses of diabetic mesenteric arteries to NA.

Role of inducible nitric oxide synthase in induction of RhoA expression in hearts from diabetic rats

AIMS Recent studies from our laboratory demonstrated that increased expression of the small GTP-binding protein RhoA and activation of the RhoA/rho kinase (ROCK) pathway play an important role in the contractile dysfunction associated with diabetic cardiomyopathy in hearts from streptozotocin (STZ)-induced diabetic rats. Nitric oxide (NO) has been reported to be a positive regulator of RhoA expression in vascular smooth muscle, and we have previously found that the expression of inducible NO synthase (iNOS) is increased in hearts from STZ-diabetic rats. Therefore, in this study, we investigated the hypothesis that induction of iNOS positively regulates RhoA expression in diabetic rat hearts. METHODS AND RESULTS To determine whether NO and iNOS could increase RhoA expression in the heart, cardiomyocytes from non-diabetic rats were cultured in the presence of the NO donor sodium nitroprusside (SNP) or lipopolysaccharide (LPS) in the absence and presence of the selective iNOS inhibitor, N(6)-(1-iminoethyl)-l-lysine dihydrochloride (L-NIL). In a second study, 1 week after induction of diabetes with STZ, rats were treated with L-NIL (3 mg/kg/day) for 8 more weeks to determine the effect of iNOS inhibition in vivo on RhoA expression and cardiac contractile function. Expression of iNOS was elevated in cardiomyocytes isolated from diabetic rat hearts. Both SNP and LPS increased RhoA expression in non-diabetic cardiomyocytes. The LPS-induced elevation in RhoA expression was accompanied by an increase in iNOS expression and prevented by L-NIL. Treatment of diabetic rats with L-NIL led to a significant improvement in left ventricular developed pressure and rates of contraction and relaxation concomitant with normalization of total cardiac nitrite levels, RhoA expression, and phosphorylation of the ROCK targets LIM (Lin-11, Isl-1, Mec-3) kinase and ezrin/radixin/moesin. CONCLUSION These data suggest that iNOS is involved in the increased expression of RhoA in diabetic hearts and that one of the mechanisms by which iNOS inhibition improves cardiac function is by preventing the upregulation of RhoA and its availability for activation.

Selective Inhibition of Protein Kinase C β2 Attenuates Inducible Nitric Oxide Synthase–Mediated Cardiovascular Abnormalities in Streptozotocin-Induced Diabetic Rats

OBJECTIVE Impaired cardiovascular function in diabetes is partially attributed to pathological overexpression of inducible nitric oxide synthase (iNOS) in cardiovascular tissues. We examined whether the hyperglycemia-induced increased expression of iNOS is protein kinase C-β2 (PKCβ2) dependent and whether selective inhibition of PKCβ reduces iNOS expression and corrects abnormal hemodynamic function in streptozotocin (STZ)-induced diabetic rats. RESEARCH DESIGN AND METHODS Cardiomyocytes and aortic vascular smooth muscle cells (VSMC) from nondiabetic rats were cultured in low (5.5 mmol/l) or high (25 mmol/l) glucose or mannitol (19.5 mmol/l mannitol + 5.5 mmol/l glucose) conditions in the presence of a selective PKCβ inhibitor, LY333531 (20 nmol/l). Further, the in vivo effects of PKCβ inhibition on iNOS-mediated cardiovascular abnormalities were tested in STZ-induced diabetic rats. RESULTS Exposure of cardiomyocytes to high glucose activated PKCβ2 and increased iNOS expression that was prevented by LY333531. Similarly, treatment of VSMC with LY333531 prevented high glucose–induced activation of nuclear factor κB, extracellular signal–related kinase, and iNOS overexpression. Suppression of PKCβ2 expression by small interference RNA decreased high-glucose–induced nuclear factor κB and extracellular signal–related kinase activation and iNOS expression in VSMC. Administration of LY333531 (1 mg/kg/day) decreased iNOS expression and formation of peroxynitrite in the heart and superior mesenteric arteries and corrected the cardiovascular abnormalities in STZ-induced diabetic rats, an action that was also observed with a selective iNOS inhibitor, L-NIL. CONCLUSIONS Collectively, these results suggest that inhibition of PKCβ2 may be a useful approach for correcting abnormal hemodynamics in diabetes by preventing iNOS mediated nitrosative stress.

N-acetylcysteine prevents nitrosative stress-associated depression of blood pressure and heart rate in streptozotocin diabetic rats.

Previous studies have indicated that cardiovascular abnormalities such as depressed blood pressure and heart rate occur in streptozotocin (STZ) diabetic rats. Chronic diabetes, which is associated with increased expression of inducible nitric oxide synthase (iNOS) and oxidative stress, may produce peroxynitrite/nitrotyrosine and cause nitrosative stress. We hypothesized that nitrosative stress causes cardiovascular depression in STZ diabetic rats and therefore can be corrected by reducing its formation. Control and STZ diabetic rats were treated orally for 9 weeks with N-acetylcysteine (NAC), an antioxidant and inhibitor of iNOS. At termination, the mean arterial blood pressure (MABP) and heart rate (HR) were measured in conscious rats. Nitrotyrosine and endothelial nitric oxide synthase (eNOS) and iNOS expression were assessed in the heart and mesenteric arteries by immunohistochemistry and Western blot experiments. Untreated diabetic rats showed depressed MABP and HR that was prevented by treatment with NAC. In untreated diabetic rats, levels of 15-F(2t)-isoprostane, an indicator of lipid peroxidation increased, whereas plasma nitric oxide and antioxidant concentrations decreased. Furthermore, decreased eNOS and increased iNOS expression were associated with elevated nitrosative stress in blood vessel and heart tissue of untreated diabetic rats. N-acetylcysteine treatment of diabetic rats not only restored the antioxidant capacity but also reduced the expression of iNOS and nitrotyrosine and normalized the expression of eNOS to that of control rats in heart and superior mesenteric arteries. The results suggest that nitrosative stress depress MABP and HR following diabetes. Further studies are required to elucidate the mechanisms involved in nitrosative stress mediated depression of blood pressure and heart rate.

Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart

Abstract.Mitochondrial dysfunction and protein kinase C (PKC) activation are consistently found in diabetic cardiomyopathy but their relationship remains unclear. This study identified mitochondrial aconitase as a downstream target of PKC activation using immunoblotting and mass spectrometry, and then characterized phosphorylation-induced changes in its activity in hearts from type 1 diabetic rats. PKCβ2 co-immunoprecipitated with phosphorylated aconitase from mitochondria isolated from diabetic hearts. Augmented phosphorylation of mitochondrial aconitase in diabetic hearts was found to be associated with an increase in its reverse activity (isocitrate to aconitate), while the rate of the forward activity was unchanged. Similar results were obtained on phosphorylation of mitochondrial aconitase by PKCβ2in vitro. These results demonstrate the regulation of mitochondrial aconitase activity by PKC-dependent phosphorylation. This may influence the activity of the tricarboxylic acid cycle, and contribute to impaired mitochondrial function and energy metabolism in diabetic hearts.

Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKCβ2

We previously reported that acute inhibition of the RhoA/Rho kinase (ROCK) pathway normalized contractile function of diabetic rat hearts, but the underlying mechanism is unclear. Protein kinase C (PKC) β(2) has been proposed to play a major role in diabetic cardiomyopathy at least in part by increasing oxidative stress. Further evidence suggests that PKC positively regulates RhoA expression through induction of inducible nitric oxide synthase (iNOS) in diabetes. However, in preliminary studies, we found that inhibition of ROCK itself reduced RhoA expression in diabetic hearts. We hypothesized that there is an interaction between RhoA/ROCK and PKCβ(2) in the form of a positive feedback loop that sustains their activation and the production of reactive oxygen species (ROS). This was investigated in cardiomyocytes isolated from diabetic and control rat hearts, incubated with or without cytochalasin D or inhibitors of ROCK, RhoA, PKCβ(2), or iNOS. Inhibition of RhoA and ROCK markedly attenuated the diabetes-induced increases in PKCβ(2) activity and iNOS and RhoA expression in diabetic cardiomyocytes, while having no effect in control cells. Inhibition of PKCβ(2) and iNOS also normalized RhoA expression and ROCK overactivation, whereas iNOS inhibition reversed the increase in PKCβ(2) activity. Each of these treatments also normalized the diabetes-induced increase in production of ROS. Actin cytoskeleton disruption attenuated the increased expression and/or activity of all of these targets in diabetic cardiomyocytes. These data suggest that, in the diabetic heart, the RhoA/ROCK pathway contributes to contractile dysfunction at least in part by sustaining PKCβ(2) activation and ROS production via a positive feedback loop that requires an intact cytoskeleton.

GPCR agonist-induced transactivation of the EGFR upregulates MLC II expression and promotes hypertension in insulin-resistant rats

AIMS The presence of metabolic abnormalities such as insulin resistance and elevated levels of various vasoconstrictor G-protein-coupled receptor (GPCR) agonists contributes to the development of hypertension. Recent studies have suggested a link between disease progression and activation of growth factor receptor signalling pathways such as the epidermal growth factor receptor (EGFR) by matrix metalloproteinases (MMPs). We hypothesized that excessive stimulation of GPCRs such as alpha(1)-adrenergic receptors activates MMP-dependent EGFR transactivation and contributes to the development of hypertension by promoting increased synthesis of contractile proteins in vascular smooth muscle (VSM). METHODS AND RESULTS We tested this concept in experiments using insulin-resistant VSM cells (VSMCs) and fructose hypertensive rats (FHRs), a model of acquired systolic hypertension and insulin resistance. We found that insulin resistance and agonist stimulation increased the expression and activity of MMPs (MMP-2 and MMP-7), the EGFR, contractile proteins such as myosin light chain kinase and MLC II, and their transcriptional activators including P90 ribosomal kinase (P90RSK) and serum response factor, possibly via the activation of extracellular signal-regulated kinase (ERK1/2) in VSMCs. Further, in insulin-resistant VSMCs and arteries from FHRs, disruption of MMP-EGFR signalling either by a pharmacological or small interfering RNA approach normalized the increased expression and activity of contractile proteins and their transcriptional activators and prevented the development of hypertension in FHRs. CONCLUSION Our data suggest that the MMP-EGFR pathway could be a potential target in the treatment of hypertension in insulin resistance and/or hyperglycaemic conditions such as type 2 diabetes.

Role of the PKC/CPI-17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to α-adrenoceptor stimulation

Protein kinase C (PKC) may contribute to enhanced contractile responses of arteries from streptozotocin‐diabetic rats to stimulation of G‐protein coupled receptors. This was investigated by comparing the effects of PKC inhibitors on contractile responses of mesenteric arteries from diabetic and age‐matched control rats to noradrenaline (NA) and endothelin‐1 (ET‐1). The effects of NA and ET‐1 on the distribution of three isoforms of PKC implicated in contraction were also determined. In addition, the effect of NA on phosphorylation of CPI‐17, a substrate for PKC, was investigated. Contractile responses of endothelium‐denuded arteries from diabetic rats to NA were enhanced, but were normalized by PKC inhibition. In contrast, contractile responses to ET‐1 were not significantly different, and were blocked to a similar extent by PKC inhibition, in arteries from control and diabetic rats. NA produced only a small increase in particulate levels of PKCɛ in control arteries (to 125±8% of levels in untreated arteries), but a significant increase in particulate PKCα (to 190±22%) and a much greater increase in particulate PKCɛ (to 230±19%) in arteries from diabetic rats. ET‐1 increased particulate PKCα and ɛ to a similar extent in arteries from control and diabetic rats. NA significantly enhanced CPI‐17 phosphorylation from a basal level of 22±10 to 71±7% of total in arteries from diabetic rats, and this was prevented by PKC inhibition. NA had no detectable effect on CPI‐17 phosphorylation in arteries from control rats. These data suggest that NA‐induced activation of PKC and CPI‐17, its downstream target, is selectively enhanced in arteries from diabetic rats, and mediates the enhanced contractile responses to this agonist.

Maintenance of adrenergic vascular tone by MMP-transactivation of the EGFR requires PI3K and mitochondrial ATP synthesis

AIMS G-protein-coupled receptors (GPCRs) modulate vascular tone, at least in part, via matrix metalloproteinase (MMP) transactivation of the epidermal growth factor receptor (EGFR). We previously have identified novel signalling pathways downstream of the EGFR suggestive of mitogen-activated protein kinase and mitochondrial redox control of vascular tone. In the present study, we examined whether MMP modulation of vascular tone involves phosphoinositide 3-kinase (PI3K) and mitochondrial ATP synthesis. METHODS AND RESULTS To determine whether PI3K is required for the maintenance of adrenergic vascular tone, we first constricted rat small mesenteric arteries with phenylephrine (PE) and then perfused with PI3K inhibitors, LY294002 and wortmannin, both of which produced a dose-dependent vasodilatation. Next, to investigate whether MMPs modulate PI3K activity, we cultured rat aortic vascular smooth muscle cells (VSMCs) and stimulated them with GPCR agonists such as PE and angiotensin II. Inhibition of MMPs (by GM6001) or EGFR (by AG1478) or suppressing the expression of MMP-2 or MMP-7 or the EGFR by small interfering RNA blunted the PI3K phosphorylation of Akt induced by PE. Further, in VSMCs, PI3K inhibitors reduced the PE-induced increase in ATP synthesis and glucose transporter-4 translocation, an effect that was also observed with MMP and the EGFR inhibitors. Further, the PE-induced increase in ATP synthesis activated MMP-7 by mechanisms involving purinergic (P2X) receptors and calcium. CONCLUSION These data suggest that the maintenance of adrenergic vascular tone by the MMP-EGFR pathway requires PI3K activation and ATP synthesis. Further, our data support the view that elevated levels of GPCR agonists exaggerate the MMP transactivation of EGFR response and contribute to enhanced vascular tone and development of cardiovascular disease such as hypertension.