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Am J Physiol Heart Circ Physiol 280: H1537-H1546, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 4, H1537-H1546, April 2001

Regulation of B2-kinin receptors by glucose in vascular smooth muscle cells

Julie Christopher1, Victoria Velarde1, Da Zhang1, Douglas Mayfield1, Ronald K. Mayfield1,3, and Ayad A. Jaffa1,2

1 Department of Medicine and 2 Departments of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, and 3 Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The development of vascular disease is accelerated in hyperglycemic states. Vascular injury plays a pivotal role in the progression of atherosclerotic vascular disease in diabetes, which is characterized by increased vascular smooth muscle cell (VSMC) proliferation and extracellular matrix accumulation. We previously reported that diabetes alters the activity of the kallikrein-kinin system and results in the upregulation of kinin receptors in the vessel wall. To determine whether glucose can directly influence the regulation of kinin receptors, the independent effect of high glucose (25 mM) on B2-kinin receptors (B2KR) in VSMC was examined. A threefold increase in B2KR protein levels and a 40% increase in B2KR surface receptors were observed after treatment with high glucose after 24 h. The mRNA levels of B2KR were also significantly increased by high glucose as early as 4 h later. To elucidate the cellular mechanisms by which glucose regulates B2KR, we examined the role of protein kinase C (PKC). High glucose increased total PKC activity and resulted in the translocation of conventional PKC isoforms (beta 1 and beta 2), novel (epsilon ), and atypical (zeta ) PKC isoforms into the membrane. Inhibition of PKC activity prevented the increase in B2KR levels induced by ambient high glucose. These findings provide the first evidence that glucose regulates the expression of B2 receptors in VSMC and provide a rationale to further study the interaction between glucose and kinins on the pathogenesis of atherosclerotic vascular disease in diabetes.

protein kinase C; bradykinin; intracellular calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PROLONGED HYPERGLYCEMIA is known to be an independent risk factor for the development of vascular complications associated with diabetes and contributes significantly to the development of vascular disease (21). Chronic hyperglycemia has been linked to a myriad of diabetic complications, including micro- and macrovascular disease, basement membrane thickening, protein glycosylation, and various cell cycle abnormalities (5, 13, 28, 41). In addition, glucose toxicity has been shown to have a deleterious effect on insulin secretion and action (9). Although it is still debated if glucose directly causes vascular disease in diabetic patients, many potential mechanisms can be demonstrated in vitro. Exposure of rabbit aorta to high glucose levels impairs the endothelium-dependent relaxation evoked by vasoactive mediators, such as acetylcholine and bradykinin (45). Moreover, elevated glucose levels have also been shown to alter calcium homeostasis, to induce a sustained activation of the Na+/H+ antiport, to regulate the expression of both transforming growth factor (TGF-alpha ) and basic fibroblast growth factor in vascular smooth muscle cells (VSMC), and to induce endothelial dysfunction in vivo (2, 31, 32, 50). The mechanisms by which high glucose alters the behavior of VSMC have not been fully elucidated; however, recent studies (6, 17, 25, 27, 36, 43) showed that high glucose concentrations can mediate several cell signaling events in VSMC, including cellular proliferation, increased activation of the mitogen-activated protein kinase (MAPK) pathway [p42-, p44-, and p38-MAPK, and Jun NH2-terminal kinase (JNK)], increased protein kinase C (PKC) activity, induction of oxidative stress, increased extracellular matrix production, and increased activity of the nuclear factor (NF)-kappa B transcription factor.

Bradykinin is generated by kallikreins from their precursor kininogens and is a potent endothelium-dependent vasodilator that increases vascular permeability and plays a primary role in inflammation. Bradykinin causes relaxation of the VSMC through the synthesis and release of nitric oxide from the endothelium (46). In contrast, injury to the integrity of the endothelium enables bradykinin to directly increase intracellular calcium levels and induce VSMC contraction (4). Several studies (11, 15, 49) of VSMC showed that bradykinin, like high glucose, is capable of activating MAPK through a PKC-dependent mechanism, increasing extracellular matrix proteins, such as collagen I and fibronectin, stimulating TGF-beta , and generating reactive oxygen species. Furthermore, Velarde et al. (48) reported that bradykinin and high glucose synergize to stimulate the expression of TGF-beta , collagen I, and fibronectin mRNA levels in VSMC.

The findings of Christopher et al. (7) demonstrate that the expression of B2-kinin receptors (B2KR) is upregulated in the aorta of diabetic rats. To determine whether glucose can directly influence the regulation of kinin receptors, the independent effect of high glucose (25 mM) on B2KR in VSMC was examined. The findings presented in this paper provide the first evidence that high glucose regulates the expression of B2KR in VSMC via a PKC-mediated mechanism and point to an interaction between glucose and kinins to modulate VSMC fibrosis in states of diabetic vascular injury.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VSMC culture. Rat aortic VSMC from male Sprague-Dawley rats (Charles River) were prepared by modification of the method of Majeck and Clowes (30). A 2-cm segment of the artery was cleaned of fat and adventitia and was then incubated in 1 mg/ml collagenase for 3 h at room temperature. The artery was then cut into small sections and fixed to a culture flask for explanation in MEM containing 10% fetal calf serum (FCS), 1% nonessential amino acids, 100 mU/ml penicillin, and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% O2-5% CO2. The medium was changed every 3-4 days, and cells were passaged every 6-8 days by harvesting with trypsin-EDTA. VSMC isolated by this procedure were homogenous and were used in all of the studies in between passages 2 and 6.

RT-PCR. RNA was extracted from cells with the use of Tri-Reagent (Molecular Research Center; Cincinnati, OH) according to the manufacturer's protocol. The RNA was then converted to cDNA by using MLV-RT (Promega; Madison, WI) according to the manufacturer's protocol at 37°C for 1 h. The PCR reaction was carried out in 25 µl total volume containing 1× PCR buffer, 200 µM dNTP, 2 ng/µl of each primer, 5 µl of first-strand cDNA, and 1 unit of Taq (Qiagen; Valencia, CA). The primers used for amplification of the rat B2KR were 5'-AAGACAGCAGTCACCATC-3' and 5'-GACAAACACCAGATCGGA-3'. The cycling conditions were an initial denaturation at 95°C for 5 min, followed by 40 cycles of 94°C for 45 s, 56°C for 45 s, and 72°C for 2 min. As a control for quantification, all of the samples were also amplified with the use of beta -actin-specific primers, 5'-GAACCCTAAGGCCAACCGTG-3' and 5'-TGGCTATAGAGGTCTTTACGG-3'. The cycling conditions for beta -actin included an initial denaturation at 95°C for 5 min, followed by 25 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s. PCR reactions were visualized on a 1% agarose gel, photographs were taken, and densitometric analysis was performed with the use of the National Institutes of Health (NIH) Image software program.

Western blotting. VSMC were washed 2× in PBS containing 2 mM of sodium orthovanadate. The cells were subsequently scraped in 100 µl of SDS sample buffer. Samples were then boiled for 5 min and analyzed on a 12% SDS-PAGE. Separated proteins were transferred to 0.45-µm polyvinylidene difluoride membranes at 300 mA for 2 h. The membranes were blocked for 30 min in 1% BSA-Tween 20 (TTBS) at 37°C. Blocked membranes were then blotted overnight at 4°C with the appropriate antibody diluted in 1% BSA-TTBS and then washed with TTBS. Finally, the membranes were incubated in a secondary antibody conjugated to horseradish peroxidase, and immunoreactive bands were visualized with the use of a chemiluminiscence reagent (Renaissance, New England Nuclear; Boston, MA) according to the manufacturer's protocol. Densitometric analysis was performed by using the NIH Image program.

Flow cytometry. VSMC were washed 1× in PBS containing 2% FCS. The cells were subsequently placed in 2-3 ml of disassociation buffer (Sigma; St. Louis, MO). Cells were centrifuged at 2,000 g for 5 min and resuspended in 50 µl of PBS containing 2% FCS. Primary antibody was added, and cells were vortexed and incubated on ice for 30 min. The cells were then washed 1× in PBS containing 2% FCS. The pellet was resuspended in 50 µl of PBS containing 2% FCS and 2 µl (1:25 dilution) of secondary antibody conjugated to FITC (Sigma) was added. The tubes were vortexed and incubated on ice for 30 min, and then washed 1× with PBS containing 2% FCS. Finally, the cells were resuspended in 200 µl of PBS containing 2% FCS, placed on ice, and counted on a flow cytometer (Becton-Dickinson).

Binding assay. To determine the effects of glucose on B2KR number and binding affinity, VSMC grown in six-well plates were washed with and allowed to equilibrate in 1 ml of binding buffer (25 mM TES, 1 mM o-phenanthroline, 15 µM captopril, and 0.1% BSA, pH 7.0). To assess the effect of glucose (25 mM) on bradykinin receptor binding, saturation binding experiments were performed by incubating cells with [3H]bradykinin (100,000 dpm/well) and in varying concentrations of unlabeled bradykinin in binding buffer for 2 h at 4°C. Cells were washed 3× with 2 ml of ice-cold binding buffer and solubilized for 10 min in 1% SDS, 0.1 M NaOH, and 0.1 M Na2CO3 solution and counted for radioactivity. Data were analyzed by using the Ligand program to determine the affinity constants and the receptor number.

Intracellular calcium determination. Intracellular calcium release was assessed by using a fluorimetric imaging plate reader (FLIPR) system (Molecular Devices; Sunnyvale, CA), a high throughput optical screening system for cell based fluorometric assays. VSMC were grown to confluence in 96-well clear bottom black microplates (Corning Costar; Cambridge, MA). Six wells were used for each condition evaluated. Cells were dye loaded with 4 µM fluo 3-acetoxymethyl ester (excitation 488 nm, emission 540 nm; Molecular Probes; Eugene, OR) in a loading buffer (1× Hanks' balanced salt solution buffer, 20 mM HEPES, and 2.5 mM probenecid, pH 7.4) for 1 h at 37°C. After washing four times with loading buffer, cells were exposed in the FLIPR by automatic simultaneous addition of varying concentrations of bradykinin. Intracellular calcium release was monitored over 6 min.

Cytosol versus membrane extraction. VSMC were suspended in 10 mM Tris · HCl (pH 7.5) and 0.25 M sucrose (0.5 ml of solution per 150-mm plate) and homogenized with a polytron on setting 7 for two times for 5 s, followed by setting 10 for four times for 5 s. Samples were centrifuged at 3,000 rpm for 5 min, and the supernatant was centrifuged again at 19,000 rpm for 15 min. The supernatant from this spin was harvested as the cytosolic fraction. The membrane pellet was resuspended in 50 µl of lysis buffer and incubated on ice for 30 min, vortexing every 10 min. Finally, samples were centrifuged at 12,000 g for 20 min, and the supernatant was retained as the membrane fraction. Proteins from both fractions were quantified by using a Bradford assay (3). Protein (15 µg) was run on an SDS-PAGE for Western blot analysis as previously described.

PKC activity. VSMC were washed 2× in PBS containing 2 mM sodium orthovanidate. The cells were subsequently scraped in PBS containing orthovanidate and centrifuged at 5,000 rpm for 5 min. Pellets were resuspended in 200 µl of lysis buffer and incubated on ice for 30 min. Finally, the samples were centrifuged at 15,000 rpm for 5 min, and supernatants were quantified by using a Bradford assay. Proteins were then measured for PKC activity using a PKC assay kit (Upstate Biotechnology) according to the manufacturer's protocol. This assay kit is designed on the basis of the phosphorylation of a specific substrate peptide (QKRPSQRSKYL) by using the transfer of [gamma -32P]ATP by PKC kinase. The phosphorylated substrate is then separated from the residual gamma -32P by using P81 phosphocellulose paper and quantitated by using a scintillation counter.

Statistical analysis. All of the data are expressed as means ± SE and analyzed by ANOVA or Student's t-test for unpaired analysis. Values were considered significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of glucose on the expression of B2KR. The B2KR is constitutively expressed in a wide range of tissues and mediates most of the in vivo effects of bradykinin. To examine whether high-glucose conditions could regulate the expression of B2KR in VSMC, quiescent VSMC were treated with 25 mM glucose for 2, 4, 6, and 24 h. After treatment, RNA was isolated from the cells, and RT-PCR was performed by using rat B2KR and beta -actin-specific primers. Our results show a time-dependent increase in B2KR expression over time with a peak response at 4 h of exposure to high glucose (Fig. 1). The beta -actin mRNA levels remained unchanged by high glucose concentrations.


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  		Fig. 1.   Glucose (Glu)-induced expression of B2-kinin receptors (B2KR) mRNA in vascular smooth muscle cells (VSMC). Quiescent VSMC were treated with 25 mM glucose for 2, 4, 6, and 24 h as indicated. RNA was isolated and RT-PCR was performed by using B2KR-specific primers and/or beta -actin-specific primers as outlined in METHODS. Photographs are representative of at least 3 separate experiments. Bar graph represents means ± SE of the intensities of the bands and is expressed as fold increase above control. *P < 0.05 vs. control.

We also examined the effects of high glucose on the regulation of angiotensin (ANG) II receptor (AT2) mRNA levels, another vasoactive G protein-coupled receptor. Incubation of VSMC with high glucose for 4 h significantly reduced the mRNA levels of AT2 by 20% compared with unstimulated control cells, and this effect was maintained for 24 h [1 vs. 0.8 ± 0.25, 0.8 ± 0.15 (*P < 0.005), 1 ± 0.3, and 0.8 ± 0.35 AT2 mRNA/beta -actin mRNA, % of control vs. 2, 4, 6, and 24 h glucose, respectively, n = 3 experiments]. Our finding supports previous published data showing that high glucose concentration downregulates the number of AT2 receptors in VSMC by a similar extent (51).

To demonstrate that the increase in B2KR mRNA we observed corresponds to an increase in protein levels, quiescent VSMC were stimulated with either D-glucose or L-glucose (25 mM) for 24 h, and the receptor protein levels were measured by Western blot analysis by using an anti-B2 receptor-specific antibody (1:4,000). The B2KR antibody was raised against the second extracellular loop (169) of the B2 receptor and has the following amino acid sequence: RTMKEYSDEGH (Zymed Lab). Probing of VSMC lysate with the B2 receptor antibody showed a single immunoreactive band at 60 kDa. The results shown in Fig. 2 indicate that the B2KR protein levels were increased approximately threefold above control levels after treatment with 25 mM D-glucose (control vs. 25 mM glucose, P < 0.05, n = 7 experiments). The levels of B2KR remained unchanged after treatment with 25 mM L-glucose. These results indicate that the increase in B2KR protein is due to the effects of high glucose and not due to the osmotic load of glucose.


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  		Fig. 2.   Glucose-induced increase in B2KR protein levels in VSMC. Quiescent VSMC were treated with either 25 mM D-glucose or 25 mM L-glucose for 24 h. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes as described in METHODS. B2KR proteins were visualized by using an anti-B2KR-specific antibody (1:4,000 dilution). Blots are representative of 7 experiments. Bar graph represents means ± SE of the intensities of the bands and is expressed as fold increase above control. *P < 0.01 vs. control.

To demonstrate whether protein synthesis is required for the maintenance of the glucose-induced increase in B2KR levels, VSMC were pretreated for 6 h with 2 ng/µl of cyclohexamide, an inhibitor of protein synthesis, followed by stimulation with 25 mM glucose for 24 h. Receptor protein levels were measured by Western analysis by using an anti-B2 receptor-specific antibody (1:4,000). The results shown in Fig. 3 reveal that the threefold increase in B2KR protein levels induced by high glucose was completely attenuated by treatment with cyclohexamide (25 mM glucose vs. cyclohexamide plus glucose, P < 0.05, n = 4 experiments). The effects of cyclohexamide on glucose-induced increases in B2KR mRNA levels were also studied. Treatment of VSMC with cyclohexamide for 24 h, followed by glucose (25 mM) for 4 h, did not significantly alter the mRNA levels of B2KR in response to high glucose (0.60 ± 0.18 vs. 0.66 ± 0.2 B2KR mRNA levels/beta -actin mRNA levels, glucose vs. glucose plus cyclohexamide, respectively, n = 3 experiments). These results indicate that sustained increase in B2KR levels in response to high glucose requires translational regulation of the B2KR gene.


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  		Fig. 3.   Glucose-induced increase in B2KR protein levels is attenuated by cyclohexamide. Quiescent VSMC were treated with 25 mM D-glucose in the presence or absence of 2 ng/µl cyclohexamide. Cells were pretreated with cyclohexamide for 6 h before the addition of 25 mM glucose for 24 h. Proteins were separated by SDS-PAGE and transferred to PVDF membranes as described in METHODS. B2KR proteins were visualized by using an anti-B2KR-specific antibody (1:4,000 dilution). Blots are representative of 7 experiments. Bar graph represents means ± SE of the intensities of the bands and is expressed as fold increase above control. *P < 0.05 vs. control. dagger P < 0.05 vs. D-glucose (25 mM).

To further explore the regulation of B2KR by glucose, we examined the effects of high glucose on B2-kinin surface receptors. Quiescent VSMC were treated with 25 mM glucose for 24 h and analyzed by flow cytometry. The results from these experiments are shown in Fig. 4. Figure 4A shows control VSMC with no anti-B2KR antibody as a negative control. Figure 4B shows control VSMC incubated with anti-B2KR antibody as a positive control to determine the basal number of B2 surface receptors. Figure 4C shows VSMC stimulated with 25 mM glucose for 24 h and incubated with anti-B2KR antibody. As shown in the representative bar graph (Fig. 4D), there is a 40% increase in B2-kinin surface receptors after treatment with high glucose for 24 h. Taken together, these results provide the first evidence that hyperglycemia can upregulate the expression of B2KR in VSMC.


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  		Fig. 4.   Upregulation B2KR surface receptors in VSMC by high glucose. Quiescent VSMC were treated with 25 mM glucose for 24 h, and B2 surface receptors were measured by flow cytometry. An anti-B2KR-specific antibody was used to identify B2KR present at the cell surface. Scans are representative of 3 experiments. A: control VSMC with no anti-B2KR antibody as a negative control. B: control VSMC incubated with anti-B2KR as a positive control. C: VSMC stimulated with 25 mM glucose for 24 h and incubated with anti-B2KR. D: bar graph represents means ± SE of the increase in receptors and is expressed as percent above control. *P < 0.05 vs. control. FL1-H, fluorescence intensity; FSC-H, cell number.

To determine the effects of high glucose on B2KR number and binding affinity, VSMC were treated with either 5 or 25 mM glucose, and binding studies were preformed. The data indicate that high ambient glucose concentrations resulted in a 62% increase in receptor number (maximal binding, Bmax), and about a twofold increase in the affinity of bradykinin for its receptor (dissociation constant, Kd) compared with VSMC treated with normal glucose (5 mM) concentrations (Bmax: 537 ± 187 vs. 331 ± 88 fmol/mg protein; Kd: 4.5 ± 2.3 vs. 2.2 ± 0.6 nM/mg protein; 25 vs. 5 mM glucose, respectively, n = 3).

Furthermore, to demonstrate that this glucose-induced increase in B2KR number was related to an increase in function of B2KR, we studied the effect of glucose on bradykinin-induced intracellular calcium release. VSMC were pretreated with either 5 or 25 mM glucose, followed by stimulation with varying concentrations of bradykinin. For these experiments, we used FLIPR to measure intracellular calcium release in response to bradykinin. As shown in Fig. 5A, bradykinin (10-10 - 10-6 M) evoked a rapid and transient rise in intracellular calcium in a concentration-dependent manner. We next sought to determine whether glucose would influence the response of bradykinin on the release of intracellular calcium. The results are shown in Fig. 5B. Bradykinin (10-8 M) produced a rapid and transient rise in intracellular calcium. However, in the presence of glucose (25 mM), the peak response of bradykinin to stimulate intracellular calcium release was increased by 20%. Glucose (25 mM) had no significant effect on basal release of intracellular calcium (Fig. 5B). These findings indicate that not only does glucose increase the number of functional B2KR, but also the response of bradykinin to stimulate the release of intracellular calcium.


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  		Fig. 5.   Effect of high glucose on bradykinin (BK)-induced intracellular calcium concentration ([Ca2+]i) responses in VSMC. VSMC were grown to confluence in 96-well microplates and [Ca2+]i response was monitored by a fluorimetric imaging plate reader (FLIPR) over a 6-min time interval. A: cells were exposed in the FLIPR by automatic simultaneous addition to varying concentrations of BK (10-10 - 10-6 M) to determine an optimum dose. B: cells were pretreated with glucose (25 mM) for 24 h, followed by exposure in the FLIPR to BK (10-8 M). Graphs represent mean peak heights; n = 6 experiments.

Effects of glucose on PKC activity in VSMC. Several investigators (25, 35) showed that one key pathway through which high glucose concentrations elucidate cellular response is through the activation of PKC. Therefore, we sought to examine whether 25 mM glucose, at time intervals between 30 min and 3 h, could activate PKC in VSMC. For these experiments, quiescent VSMC were treated with 25 mM glucose for 30 min, 1 h, and 3 h, and total cell lysates were isolated. PKC activity was measured by using a kit from Upstate Biotechnology according to the manufacturer's protocol. The results shown in Fig. 6 indicate that high glucose significantly increased the activity of PKC as early as 1 h and remained elevated at 3 h poststimulation (0 vs. 1 or 3 h, P < 0.02, n = 5 experiments). Mannitol (20 mM) or L-glucose did not influence PKC activity, indicating that the increase in PKC activity that we observed is due to glucose rather than osmotic load (14).


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  		Fig. 6.   Activation of protein kinase C (PKC) by hyperglycemia. Quiescent VSMC were treated with 25 mM glucose for various times (0, 0.5, 1, and 3 h). PKC activity was measured in the cell lysate. Bar graph represents the means ± SE of the increase of PKC activity and is expressed as fold increase above control (0 h). Results are representative of five experiments. *P < 0.02 vs. control.

Role of PKC in glucose regulation of B2KR. Recent studies (12) showed that PKC may play a role in regulating the number or mRNA expression of cell surface receptors. Because glucose promotes PKC activation in VSMC, we sought to determine whether PKC is essential for the regulation of B2KR by high glucose. Quiescent VSMCs were pretreated with the specific cell-permeable broad-spectrum PKC inhibitor GF109203X (GFX; 2 µM) for 2 h, followed by stimulation with 25 mM glucose for 24 h. The results are shown in Fig. 7A. High glucose levels stimulated the increase in B2 receptor levels; however, in the presence of the PKC inhibitor, this increase in B2 receptors induced by high glucose was completely eliminated (P < 0.02, glucose vs. glucose plus PKC inhibitor, n = 5 experiments). We also studied the effects of another PKC inhibitor, H7 (10 µM), on glucose-induced B2 receptor regulation. We found that H7 also reduced the increase in B2 receptors in response to glucose in a similar manner to that of GFX (data not shown).


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  		Fig. 7.   Role of PKC in the regulation of B2KR by high glucose. A: quiescent VSMC were pretreated with a PKC inhibitor GF109203X (GFX; 2 µM) for 2 h, followed by stimulation with 25 mM glucose for 24 h. Proteins were separated by SDS-PAGE and transferred to PVDF membrane, and immunoblotted with specific anti-B2KR antibody (1:4,000 dilution). B: quiescent VSMC were pretreated with GFX (2 µM) for 2 h, followed by stimulation with 25 mM glucose for 4 h. RNA was isolated and RT-PCR was performed by using B2KR and/or beta -actin-specific primers. Bar graph represents means ± SE of the intensities of the bands and is expressed as fold increase above control. All blots are representative of at least 3 separate experiments. *P < 0.01 vs. control. #P < 0.05 vs. glucose.

To further explore the role of PKC in modulating the glucose-induced increase in B2KR mRNA levels, VSMC were pretreated with the PKC inhibitor GFX (2 µM) for 2 h, followed by stimulation with 25 mM glucose for 4 h, because this time point showed the peak increase in B2KR mRNA levels (Fig. 1). B2KR mRNA levels were measured by RT-PCR. As shown in Fig. 7B, treatment of VSMC with 25 mM glucose once again produced a significant increase in B2KR mRNA levels compared with unstimulated cells (P < 0.05, control vs. 25 mM glucose, n = 3 experiments). However, in the presence of the PKC inhibitor GFX, the increase in B2KR mRNA levels in response to glucose stimulation was completely eliminated (P < 0.05, glucose vs. glucose plus GFX, n = 3 experiments). Neither the PKC inhibitor nor high glucose concentration influenced the mRNA levels of beta -actin. Taken together, these studies implicate that PKC is a key player in regulating the expression of B2KR in VSMC by high glucose.

To determine the isoforms of PKC that are activated in response to glucose stimulation, we used Western blots to measure the translocation of PKC isoforms from the cytosol to the membrane. The family of PKCs includes at least 11 isoforms categorized into three groups: conventional, novel, and atypical. For these studies, we elected to measure representatives from each of the three categories that are known to be present in VSMC, including conventional PKC isoforms (beta 1 and beta 2), a novel isoform (epsilon ), and an atypical isoform (zeta ). VSMC were treated with 25 mM glucose for 1 and 3 h, respectively, and the cytosol and membrane proteins were extracted. Western blot analysis was performed by using specific anti-PKC isoform antibodies (1:6,000 × dilution, Santa Cruz, CA). Addition of 25 mM glucose for 3 h significantly increased PKC-beta 1 and PKC-beta 2 immunoreactivity in the membrane fraction compared with unstimulated cells (Fig. 8, A and B). The effect of 25 mM glucose on PKC-epsilon and PKC-zeta are shown in Fig. 9, A and B. Glucose significantly increased the immunoreactivity of PKC-epsilon in the membrane fraction 3 h after glucose stimulation, whereas the cytosolic fraction was unaltered (Fig. 9A). With regard to PKC-zeta , 25 mM glucose significantly increased the translocation from the cytosol to the membrane fraction as early as 1 h, and this effect was maintained at 3 h of postglucose stimulation (Fig. 9B).


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  		Fig. 8.   Response of PKC isoforms (beta 1 and beta 2) to high glucose. Quiescent VSMC were treated with 25 mM glucose for 1 and 3 h and cytosolic and membrane fractions were separated. Proteins from each fraction were resolved by SDS-PAGE and transferred to PVDF membranes and were immunoblotted with either anti-PKC-beta 1-specific antibody (1:6,000 dilution) or with anti-PKC-beta 2-specific antibody (1:6,000 dilution). Bar graphs represent the means ± SE of the intensities of the bands and are expressed as fold increase above control. All of the blots are representative of at least 3 separate experiments. *P < 0.05 vs. control.



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  		Fig. 9.   Response of PKC isoforms (epsilon  and zeta ) to high glucose. Quiescent VSMC were treated with 25 mM glucose for 1 and 3 h, respectively, and cytosolic and membrane fractions were separated. Proteins from each fraction were resolved by SDS-PAGE and transferred to PVDF membranes and were immunoblotted with either anti-PKC-epsilon -specific antibody (1:6,000 dilution) or with anti-PKC-zeta -specific antibody (1:6,000 dilution). Bar graphs represent means ± SE of the intensities of the bands and are expressed as fold increase above control. All blots are representative of at least 3 separate experiments. *P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrate that high glucose exerts a significant effect on the expression of B2KR in VSMC. We showed that hyperglycemia increases the mRNA levels, protein levels, and surface receptors for the B2KR in vascular tissue. This effect of high glucose is specific for D-glucose and unrelated to changes in osmotic load. In addition, the upregulation of B2KR mRNA and protein levels in response to high glucose is mediated via activation of PKC.

The development of vascular lesions is accelerated in the diabetic state, and evidence indicates that diabetes per se is a powerful, independent risk factor for the development of vascular disease (8, 21). The mechanisms by which diabetes accelerates vascular damage are not fully elucidated. Diabetes is associated with endothelial denudation, VSMC proliferation, basement membrane changes, and impaired endothelium-dependent relaxation of blood vessels (47). Many studies (i.e., Ref. 42) demonstrated an association between circulating high glucose levels and the development of diabetic complications in human and animal models. The mechanisms by which hyperglycemia alters vascular function continue to be investigated, and recent studies show that glucose can influence VSMC function in a variety of ways. Stimulation of VSMC with high glucose leads to an increase in intracellular calcium, activation of the MAPK (p42, p44, and p38) pathway, PKC, and generation of reactive oxygen species. These second messengers have been implicated in mediating the vascular complications associated with high glucose, such as cellular proliferation and extracellular matrix accumulation (6, 17, 36, 43). In addition, high glucose has been shown to influence the regulation of vascular receptors of vasoactive peptides that are important modulators of vascular tone and structure. Glucose has been shown to downregulate ANG II and arginine vasopressin receptors in VSMC, both of which are pressor hormones (51), whereas in the present study we found that high glucose levels increase the expression of B2KR in VSMC. The upregulation of B2 receptors by high glucose was observed by using glucose concentrations equivalent to those achieved in poorly controlled type I diabetic patients. Moreover, our (7) recent studies demonstrate that the expression of B2KR mRNA levels in the aorta of diabetic rats was induced as early as 3 days and remained elevated at 7 days after the induction of diabetes, compared with nondiabetic control rats.

The localization of kinin receptors within the vascular wall and their activation by bradykinin implies that this system has a role in the regulation of vascular function (33). The vasoactive nonpeptide bradykinin, which is the principal effector of the kallikrein-kinin system, can be generated both systemically and locally within the vascular wall (22). Recent studies in our lab and by others provide some insights as to how a change in kinin receptors could alter VSMC function. We (34, 49) showed that activation of B2KR in VSMC by bradykinin elicits a rise in intracellular calcium, increases proliferation, stimulates MAPK activation and nuclear translocation, and induces expression of c-fos and c-jun genes and the formation of the activator protein-1 complex. The cellular mechanism through which bradykinin stimulates MAPK activation and c-fos mRNA expression in VSMCs involves the activation of calcium-calmodulin pathway, SRC kinase, PKC, and MAPK kinase (34, 49). More recently, we showed (11, 48) that bradykinin, like glucose, is capable of stimulating the expression of extracellular matrix proteins via autocrine activation of TGF-beta and that bradykinin and glucose synergize to stimulate collagen I and fibronectin mRNA levels in VSMC. In addition, the results of the present study demonstrate that not only does high ambient glucose result in the increase of B2KR number and affinity, but also the functional responses of bradykinin to evoke rises in intracellular calcium. Furthermore, exposure of endothelial cells to high glucose significantly enhanced the potency of bradykinin to stimulate intracellular calcium and cGMP levels (14). Taken together, our findings demonstrate that bradykinin and glucose share common signaling pathways leading to vascular dysfunction. Therefore, we speculate that the upregulation of B2 receptors and their activation by high glucose may provide a mechanism through which glucose could alter VSMC function.

It is widely accepted that glucose, through the activation of diacylglycerol (DAG), increases the activity of PKC in a variety of cell types (10, 20, 24). This activation is known to be important in vascular cells to regulate permeability, contractility, extracellular matrix, cell growth, angiogenesis, cytokine actions, and leukocyte adhesions, all of which are functionally altered in the diabetic state (37, 38). Our findings are consistent with previous reports (16) showing that high glucose levels increase the activity of PKC in VSMC. However, in the present study, we demonstrated that activation of PKC by high glucose also regulates the expression of B2 receptors. Inhibition of PKC activity eliminated the increase in B2KR induced by high glucose.

Eleven characterized isoforms of PKC have been classified into three groups: 1) conventional PKCs (alpha , beta 1, beta 2, and gamma ) are calcium dependent and DAG sensitive, 2) novel PKCs (delta , epsilon , eta , theta , and µ) are also DAG sensitive, but are calcium independent, and 3) atypical PKCs (zeta  and lambda ) are insensitive to DAG, but can be activated by phosphotidylserine (25). To elucidate which isoforms of PKC may be responsible for the glucose-induced increase in B2KR, we chose members from each of the three groups, previously shown to be present in VSMC, to measure glucose-induced translocation from the cytosol to the membrane. Our data are consistent with other observations showing that high glucose concentrations stimulate the activation and membrane translocation of PKC-beta 1, -beta 2, and -epsilon in VSMC (16, 19, 39). However, the findings of the present study also demonstrate that high glucose concentrations induced activation and membrane translocation of PKC-zeta . The activation of the zeta -isoform by high glucose in VSMC has not been reported, and, interestingly, the zeta -isoform is insensitive to DAG activation. Although the mechanism of its activation by glucose is not yet defined, Amiri and Garcia (1) also showed that activation of PKC-zeta in response to phorbol ester phorbol 12-myristate 13-acetate only occurs in the presence of hyperglycemia. Thus it is possible that, under hyperglycemic conditions, there is cross-talk between different PKC isoforms such that DAG-sensitive PKC isoforms could activate atypical PKC isoforms (23).

The changes we observed in kinin receptor regulation by glucose are not unprecedented. Glucose has been shown (18, 40, 44, 51) to regulate the expression of other receptor genes such as the glucagon receptor, the platelet-derived growth factor-beta receptor, the insulin-like growth factor-1 receptor, AT2 receptors, and arginine vasopressin receptors. Although the cellular mechanism by which glucose regulates the expression of certain genes has not been fully elucidated, several possibilities are known. Glucose response elements have been characterized and found to be present in the promoters of some genes, such as the glucagon receptor (44). In addition, high glucose concentration has been shown to activate transcription factors such as NF-kappa B and to induce phosphorylation of cAMP response element binding protein (26, 27). Interestingly, two putative cAMP response element sites have been identified on the B2KR gene promoter (29). Thus one possible mechanism through which high glucose could regulate the expression of B2 receptors is by enhancing the activity of these transcription factors, which in turn may induce transcription of the B2KR gene.

In summary, we showed that the expression of B2KR in VSMC is upregulated by high glucose concentration, and this effect is specific to glucose and not due to osmolarity. The results also demonstrate that glucose induces the activation and membrane translocation of PKC-beta 1, -beta 2, -epsilon , and -zeta and implicate PKC as a key player in modulating the expression of B2KR in VSMC under hyperglycemic conditions. These data provide further rationale for studying the interaction between glucose and kinins in the progression and pathogenesis of vascular disease in diabetes.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-55782 and Training Fellowship HL-07260 (to J. Christopher), National Institutes of Health Grant DK-46543, a Research Award from the American Diabetes Association (to A. A. Jaffa), a Merit Review Grant from the Research Service of the Department of Veterans Affairs (to R. K. Mayfield), a Postdoctoral Research Fellowship Award from the Juvenile Diabetes Foundation (to V. Velarde), and Public Health Service Shared Equipment Grant S10RR13005.


    FOOTNOTES

Address for reprint requests and other correspondence: A. A. Jaffa, Dept. of Medicine, Endocrinology-Diabetes-Medical Genetics, Medical Univ. of South Carolina, 114 Doughty St., PO Box 250776, Charleston, SC 29425 (E-mail: jaffaa{at}musc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 31 July 2000; accepted in final form 14 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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