Inhibition of Protein Kinase C Protects against Diabetes-Induced Impairment in Arachidonic Acid Dilation of Small Coronary Arteries

To test the hypothesis that protein kinase C (PKC) -induced reactive oxygen species (ROS) underlie the vascular dysfunction in diabetes, we examined the effects of (S)-13[(dimethylamino)methyl]-10,11–14,15-tetrahydro-4,9:16,21-dimetheno-1H,13Hdibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadi-azacyclohexadecene1,3(2H)-dione (LY333531; LY), a specific PKC inhibitor, on arachidonic acid (AA)-mediated dilation in small coronary arteries from streptozotocin-induced diabetic rats. This study was designed to determine whether diabetes impairs AA-induced vasodilation of small coronary arteries and whether this defect could be blunted by dietary treatment with LY. Coronary diameter was measured using videomicroscopy in isolated pressurized vessels. In controls, AA dose dependently dilated coronary arteries, with 1 M producing 54.7 3.1% and 30 M producing 72.0 3.0% dilation (n 9). In diabetic rats, 1 M AA only produced 31.4 3.8% (n 8; p 0.01 versus control) and 30 M 43.8 3.7% dilation (n 8; p 0.001 versus control). Nitroprusside-mediated vasodilations were similar in control and diabetic rats. In contrast, in diabetic rats receiving LY, AA-mediated coronary dilations were normal. In controls, AA-mediated vasodilation was inhibited by miconazole (an inhibitor of cytochrome P450 epoxygenase) and by iberiotoxin (IBTX, an inhibitor of the large conductance Ca activated K channel), but miconazole and IBTX had no effects in diabetic vessels. In diabetic rats receiving LY, the effects of miconazole and IBTX were similar to control. Superoxide dismutase restored responses to AA in diabetic vessels but had no effect in vessels from control or diabetic rats on LY. These results suggest that AA-mediated vasodilation in rat coronary arteries are impaired in diabetic rats due to increases in generation of ROS. LY protects against these defects in diabetes through inhibition of PKC mediated production of ROS. Diabetes mellitus has become a disease of epidemic proportions with cardiovascular disease the leading cause of death in diabetic patients (Geiss et al., 1995), and patients with diabetes have a 2to 4-fold increase in the risk of coronary artery disease (Beckman et al., 2002a). Metabolic, humoral, and hemodynamic factors all contribute to the development of vascular dysfunction (Cooper et al., 2001). Endothelial dysfunction with impaired activity of various endothelial-derived factors plays an integral role in diabetic vasculopathy (De Vriese et al., 2000; Brownlee, 2001). The process by which hyperglycemia produces endothelial and vascular dysfunction is complex, but several major mechanisms have been proposed (Nishikawa et al., 2000; Brownlee, 2001; Cooper et al., 2001; Gutterman, 2002), including overactivity of the polyol pathway, accumulation of advanced glycation end products, and activation of protein kinase C (PKC). Each of these would result in an enhanced generation of ROS. Activated PKC mechanisms have received increasing attention (Way et al., 2001). Hyperglycemia stimulates PKC through the action of diacylglycerol and nonesterified fatty acids (Cooper et al., 2001; Egan et al., 2001). In particular, the isoform is increased in diabetic vascular tissues (Inoguchi et al., 1992), and administration of LY333531 (LY), a highly specific inhibitor of PKC , attenuates the various vascular abnormalities in streptozotocin-induced diabetic rats (Inoguchi et al., 1992; Ishii et al., 1996). Recently, oral administration of LY in humans has been shown to prevent impaired endothelium-dependent vasodilation caused by hyperglycemia (Beckman et al., 2002b). These findings position This study was supported in part by National Institute of Health Grants HL-74180 and HL-63754 and the Mayo Foundation. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.106666. ABBREVIATIONS: PKC, protein kinase C; ROS, reactive oxygen species; LY333531 (LY), (S)-13[(dimethylamino)methyl]-10,11–14,15-tetrahydro4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione; AA, arachidonic acid; P450, cytochrome P450; LOX, lipoxygenase; HETE, hydroxyeicosatetraenoic acid; ACh, acetylcholine; IBTX, iberiotoxin; BK, channel, large conductance Ca -activated K ; SOD, superoxide dismutase; DHE, dihydroethidium; ecSOD, extracellular superoxide dismutase. 0022-3565/06/3191-199–207 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 1 U.S. Government work not protected by U.S. copyright 106666/3142738 JPET 319:199–207, 2006 Printed in U.S.A. 199 at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from PKC as a key participant in the development of vascular dysfunction in diabetes mellitus. Arachidonic acid (AA) is an important precursor for many vasoactive metabolites that are crucial for the regulation of vascular function. AA is metabolized by cyclooxygenase into prostaglandins and thromboxane; by lipoxygenase (LOX) into leukotrienes, lipoxins, and intrachain hydroxyeicosatetraenoic acids (HETEs); and by cytochrome P450 (P450) epoxygenase into epoxyeicosatrienoic acids and chain terminal HETEs (Foegh and Pamwell, 2002). AA produces potent dilation in human coronary arterioles that is dependent on the P450 pathway (Miura and Gutterman, 1998), whereas the dilation produced in rat mesenteric microvessels is mediated mainly through the LOX pathway (Miller et al., 2003; Zhou et al., 2005). However, the role of AA in the vascular dysfunction of diabetes mellitus is not fully known. Enhanced PKC activities could produce vascular dysfunction through different mechanisms but the common denominator seems to be increase in ROS (Gutterman, 2002). PKC could induce ROS production through activation of NAD(P)H in vascular endothelial cells (Inoguchi et al., 2003). In addition, nitric-oxide synthase in diabetic vessels may become uncoupled, resulting in the generation of superoxide rather than NO (Hink et al., 2001). Increased ROS is known to affect the cyclooxygenase (Zou et al., 2002), LOX (Zhou et al., 2005), and P450 (Lin et al., 2005) enzymes, and it could significantly modulate AA metabolism and the vascular effects of its bioactive products. The goal of this study is to determine whether the AA-mediated dilation of small coronary arteries is impaired in streptozotocin-induced diabetic rats, and to determine the role of PKC in such impairment. Materials and Methods Animals. Diabetes mellitus was produced in male SpragueDawley rats (200–250 g) by injection of streptozotocin (60 mg/kg i.p.). Control rats received vehicle injection. Blood glucose levels in excess of 300 mg/dl were considered diabetic. At induction of diabetes, control and diabetic rats received regular rat chow, but some animals in each group received chow containing LY (10 mg/kg/day) (Inoguchi et al., 1992; Ishii et al., 1996). LY is a highly specific inhibitor of PKC , and the LY-containing chow was specially prepared by Eli Lilly & Co. (Indianapolis, IN). Handling and care of animals, and all animal procedures were approved by the Institutional Animal Care and Use Committee, Mayo Foundation (Rochester, MN). Vasoreactivity Measurements. Two to 4 weeks following induction of diabetes and administration of LY, rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). Hearts were rapidly excised and placed in ice-cold Krebs’ solution that contained 118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11.1 mM dextrose. The secondary and tertiary branches (50–200 m in intraluminal diameter) of the right and left coronary arteries from the epicardial surface as well as branches of the septal coronary arteries were carefully dissected and isolated free of surrounding myocardium and connective tissue under a dissecting microscope (Olympus SZ4045 stereo microscope, Olympus America Inc., Melville, NY). Isolated small coronary arteries (1–2 mm in length) were transferred to a custom-made vessel chamber filled with Krebs’ solution. The arteries were mounted and secured between two borosilicate glass micropipettes (30m-diameter tips) with 10-0 ophthalmic suture. The lumen of the vessel was filled with Krebs’ solution through the micropipettes and maintained at a constant pressure (no flow) of 60 mm Hg. The vessel chamber was transferred to an inverted light microscope stage (Olympus CK40) coupled to a video measurement system (VIA-100; Boeckeler Instruments, Inc., AZ) equipped with a videocamera, monitor, and calibrated video calipers for visualization and recording the intraluminal diameter as described previously (Zhou et al., 2005). Vessels were equilibrated for at least 30 min in oxygenated (20% O2, 5% CO2, balanced with N2, 37°C) Krebs’ solution, which was continuously circulated through the vessel bath. Responses to cumulative additions of each compound were determined at 5-min intervals. The average diameter of the vessels used was 137 5 m for controls, 137 8 m for control on LY diet, 122 6 m for diabetic rats, and 123 5 m for diabetic rats on LY diet (p N.S. among groups). Vessels were unacceptable for experiments if they demonstrated leaks, failed to produce 30% constriction to 60 mM KCl or to graded doses of endothelin-1, or failed to produce an 80% dilation with nitroprusside (10 4 M). To assess the role of endothelium in responses, endothelium was removed by passing an air bubble (1-ml volume) through the isolated vessels. Vessels were used only if they did not relax with acetylcholine (10 4 M; 10% relaxation) but had normal response to nitroprusside (10 4 M; 80% dilation of constriction by endothelin-1) and to KCl (60 mM; 30% constriction of baseline resting diameter). Pharmacological Interventions. All compounds were added abluminally, and the cumulative concentration responses were determined at 3to 5-min intervals between doses. Vessels were constricted to 30 to 60% of baseline diameter with endothelin-1 (doses used were 3.6 0.3 to 6.6 0.6 nM). Concentration-response curves to acetylcholine (ACh; 10 –10 4 M, endothelium-dependent), sodium nitroprusside (10 –10 4 M, endothelium-independent), and AA (1 10 –3 10 5 M) were determined. To determine the mechanisms responsible for mediating dilation to AA, small coronary arteries were preincubated for 30 min with 10 5 M miconazole to inhibit the P450 epoxygenase pathway, or with 10 7 M iberiotoxin (IBTX) to block the large conductance Ca activated K (BK) channels, before dose-response experiments. To determine the effects of ROS in vascular dysfunction, vessels were treated with 150 U/ml superoxide dismutase (SOD) for 30 to 45 min before measuring vasodilator response to ACh and AA. To determine the effects of acute PKC inhibition in vascular dysfunction, vessels were treated with 30 nM LY333531 for 30 min before measuring vasodilation response to AA. Fluorescent Microscopy of Oxidative Stress. The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate the production of superoxide in coronary arteries as described previously (Miller et al., 1998). DHE is a chemically reduced ethidium derivative that is permeable to viable cells. DHE exhibits blue fluorescence in cytoplasm but can be oxidized in cells, reacting with superoxide to form ethidium, which intercalates DNA to produce bright red fluorescence (Munzel et al., 2002). Unfixed frozen ring segments of rat coronary arteries from control, diabetic, and LY-treated diabetic animals were cut into 30m-thick sections and placed on a glass slide. DHE (2 M) was topically applied to each tissue section and incubated in a light-protected humidified chamber at 37°C for 30 min. Slides were then coverslipped, and images were obtained with a confocal laser microscope (LSM 510, Zeiss, Germany) with a 63 water immersion lens. DHE was excited at 488 nm and fluorescence emission was detected with a 585to 615-nm band-pass filter. In addition, autofluorescence intrinsic to the internal elastic lamina, which separates the endothelium from smooth muscles and is present in small arteries, was detected using a 505to 550-nm band-pass filter (green fluorescence) (Wong and Langille, 1996; Burnham et al., 2002), and transmitted light micrographs of the same sections were also obtained. Laser settings were identical for acquisition of images, and vessels from control, diabetic, and diabetic rats on LY were processed in parallel. The light micrograph and fluorescent images for DHE signals and internal elastic lamina were digitally merged to demonstrate anatomical distribution of ROS. The DHE signals were further analyzed densitometrically using Scion Image software (Scion Corporation, Frederick, MD), and the results were expressed as relative densitometric units per unit area. 200 Zhou et al. at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from Materials. DHE was purchased from Invitrogen (Carlsbad, CA). LY333531 was a generous gift from Eli Lilly & Co. and was solubilized in dimethyl sulfoxide as a 20 mM stock solution. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). AA, ACh, and nitroprusside were solubilized in deionized water and stored under nitrogen at 20°C. Iberiotoxin was freshly prepared in Krebs’ solution at 10 7 M. Streptozotocin was freshly prepared in sterile water before injection into the animals. Statistical Analysis. Data are presented as mean S.E.M. n represents the number of vessels used in each experiment. All concentration-response relationships were analyzed using one-way analysis of variance with repeated measures. Pairwise comparisons among the groups were performed using Tukey test with SigmaStat software (Systat Software, Inc., Point Richmond, CA). Statistical significance was defined as p 0.05.