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Am J Physiol Heart Circ Physiol 295: H2135-H2142, 2008. First published September 26, 2008; doi:10.1152/ajpheart.00704.2008
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High glucose increases the expression of Gq/11{alpha} and PLC-β proteins and associated signaling in vascular smooth muscle cells

Magda Descorbeth and Madhu B. Anand-Srivastava

Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada

Submitted 9 July 2008 ; accepted in final form 18 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The levels and activity of protein kinase C and diacylglycerol were shown to be upregulated in diabetes/hyperglycemia; however, studies on the expression of upstream signaling molecules of phosphatidylinositol turnover were lacking. The present study was therefore undertaken to examine whether hyperglycemia/diabetes could also modulate the expression of Gq{alpha} and phospholipase C-β (PLC-β) proteins and associated phosphatidylinositol turnover signaling in aortic vascular smooth muscle cells (VSMCs) and A10 VSMCs exposed to high glucose. Aortic VSMCs from streptozotocin-diabetic rats exhibited an increased expression of Gq{alpha} and PLC-β1 proteins (60% and 30%, respectively) compared with control cells as determined by Western blot analysis. The pretreatment of A10 VSMCs with high glucose (26 mM) for 3 days also augmented the levels of Gq{alpha}, G11{alpha}, PLC-β1 and -β2 proteins by about 50, 35, 30, and 30%, respectively, compared with control cells that were restored to control levels by endothelin-1 (ET-1), ET types A and B (ETA and ETB) receptors, and angiotensin II type 1 (AT1) receptor antagonists. In addition, ET-1-stimulated inositol triphosphate formation was also significantly higher in VSMCs exposed to high glucose, whereas the basal levels of inositol triphosphate were not different between the two groups. Furthermore, the treatment of A10 VSMCs with angiotensin II and ET-1 also significantly increased the levels of Gq/11{alpha} and PLC-β proteins that were restored toward control levels by ETA/ETB and AT1 receptor antagonists. These results suggest that high glucose augments the expression of Gq/11{alpha}, PLC-β, and mediated signaling in VSMCs, which may be attributed to AT1, ETA, and ETB receptors.

Gq{alpha} protein; phospholipase C-β; phosphatidylinositol turnover

VASCULAR COMPLICATIONS including increased contractility and cell proliferation are most common complications in diabetes, and chronic hyperglycemia seems to be an important contributing factor in this process. Various signaling mechanisms such as adenylyl cyclase/cAMP, phosphatidylinositol (PI) turnover, and mitogen-activated protein kinase have been shown to be implicated in the regulation of vascular tone as well as proliferation, and the aberration of these mechanisms may contribute to vascular complications in hyperglycemia/diabetes. Guanine nucleotide regulatory proteins (G proteins) play an important role in the regulation of these signaling mechanisms. Adenylyl cyclase activity is regulated by inhibitory (Gi) and stimulatory (Gs) guanine nucleotide regulatory proteins, whereas the Gq family of G proteins regulates PI turnover signaling. The G proteins are heterotrimeric proteins and are composed of {alpha}-, β-, and {gamma}-subunits. The activation of Gq{alpha} by a G protein-coupled receptor (GPCR) stimulates phospholipase C-β (PLC-β), which hydrolyzes inositol biphosphates (PIP2) to produce the intracellular second messengers; inositol triphosphates (IP3); and diacylglycerol (DAG) (5, 33). The IP3 stimulates the release of the calcium from the intracellular stores, and DAG activates protein kinase C (PKC). The release of intracellular calcium activates the calcium channels localized in the cell membrane and allows the uptake of extracellular calcium inside the cell (27). There exist four distinct genes in the Gq{alpha} subgroup to encode Gq{alpha}, G11{alpha}, G14{alpha}, and G16{alpha}. Gq{alpha}, and G11{alpha} are 88% identical and are expressed ubiquitously (36).

Alterations in the levels of Gq{alpha} and associated signaling components result in impaired cellular functions, resulting in various pathological states including diabetes, hypertrophy, etc. (8). A genetic ablation of Gq{alpha} in mice has been shown to result in a cardiac malformation and craniofacial defects (28), whereas an overstimulation of the Gq pathway in mice was shown in the development of hypertrophic cardiomyopathy (8). An enhanced expression of Gq{alpha}, PKC, and DAG was reported in different tissues from streptozotocin (STZ)-induced diabetic rats as well as in Bio-Breeding (BB) rats (12, 18, 32, 37, 40). In addition, the high-glucose treatment of cultured aortic endothelial and vascular smooth muscle cells (VSMCs) has also been reported to enhance the activity of PKC and DAG (18, 30, 39). However, the studies on the levels of Gq{alpha}, an important upstream signaling molecule of PI turnover in diabetic aorta or in VSMCs under hyperglycemic conditions, are lacking.

Vasoactive peptides such as ANG II and endothelin-1 (ET-1), which can be synthesized locally in the vasculature, have been implicated in diabetes-associated vascular dysfunctions, including vascular remodeling, hypertrophy, and proliferation of VSMCs (10, 13, 20, 25, 31, 35), leading to an impaired relaxation to vasodilators or an exacerbated response to vasoconstrictors. The levels of ET-1 and ANG II have been shown to be elevated in plasma from both type 1 and type 2 diabetes and also in experimental models (7, 15, 22), as well as in aortic endothelial and smooth muscle cells in the presence of high glucose (19, 21, 29), which may contribute to the vascular complications of diabetes.

The present study was undertaken to examine whether high glucose could also increase the expression of Gq{alpha} proteins and mediated signaling in A10 VSMCs and whether the increased expression of Gq{alpha} is attributed to increased levels of vasoactive peptides. We have provided the first evidence that high glucose increases the expression of Gq/11{alpha} in A10 VSMCs, which may be due to the increased levels of ANG II and ET-1 induced by high glucose.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ET-1, ANG II, BQ123, BQ788, and losartan were purchased from Sigma. The antibodies Gq{alpha} (K-17), G11{alpha} (D-17), PLC-β1 (D-8), PLC-β2 (Q-15), and PLC-β3 (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [myo-2-3H]inositol was purchased from Perkin Elmer (Boston, MA). All other chemical were purchased from Sigma Chemical (St. Louis, MO).

Animal preparation. Male Sprague-Dawley rats (200 g; 6–8 wk old) were maintained on standard rat chow and tap water ad libitum with a 12-h:12-h light-dark cycles in a quiet environment. Diabetes was induced by an intraperitoneal injection of STZ (60 mg/kg body wt), dissolved in sodium citrate buffer (pH 4.5). Age-matched control rats were injected with an equal volume of buffer solution. Blood glucose levels were monitored from day 1 to day 5 after the injection using a dextrometer (Ames). STZ-injected rats with blood glucose levels in excess of 26 mM were considered to be diabetic rats (STZ) and used in the study. The blood glucose level of control rats was 5.5 mM. The rats after 5 days of treatment were euthanized, and the aortas were dissected out and used for cell culture. All the protocols used in the present study were approved by the Comité de déontologie de l'experimenttation sur les animaux (CDEA) in Canada.

Cell culture and incubation. A10 VSMC line from embryonic thoracic aorta of rat was obtained from American Type Culture Collection (Manassas, VA). VSMCs from the aorta of the control and diabetic rats were cultured as described previously (4). The cells were cultured in Dulbecco's modified Eagle's medium containing normal glucose (5.5 mM), 10% FBS, and 1% antibiotic-antimycotic (containing penicillin, streptomycin, and amphotrecin B) at 37°C in 95% room air-5% CO2 as described previously (17). The cells were passaged upon reaching confluence with 0.5% trypsin and used between passages 5 and 20. The confluent VSMCs after 24 h of incubation in a serum-free Dulbecco's modified Eagle's medium were exposed to normal glucose (5.5 mM) or high glucose (26 mM) or 26 mM mannitol for different time periods. For the receptor antagonist studies, the cells were incubated in the absence or presence of 10–6 M BQ123, BQ788, or losartan for the last 24 h of the treatment. After incubation, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a 200-µl buffer containing 25 mmol/l Tris·HCl (pH 7.5), 25 mmol/l NaCl, 1 mmol/l sodium orthovanadate, 10 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 2 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 0.5 µg/ml leupeptin on ice. The cell lysates were centrifuged at 12,000 g for 10 min at 4°C, and the supernatants were used for Western blot analysis.

Western blot analysis. Western blot analysis of Gq{alpha}, G11{alpha}, PLC-β1, -β2, and -β3 proteins were performed as described previously (3). After SDS-PAGE, the separated proteins were electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell) with a semidry transblot apparatus (Bio-Rad) at 15 V for 45 min. After transfer, the membranes were washed twice in PBS and were incubated in PBS containing 3% skim milk at room temperature for 2 h. The blots were then incubated with specific antibodies against different proteins: Gq{alpha} (K-17) against Gq{alpha}, G11{alpha} (D-17) against G11{alpha}, PLC-β1 (D-8) against PLC-β1, PLC-β2 (Q-15) against PLC-β2, and PLC-β3 (C-20) against PLC-β3 in PBS containing 1.5% skim milk and 0.1% Tween-20 at room temperature overnight. The antigen-antibody complexes were detected by incubating the blots with goat anti-rabbit IgG (Bio-Rad) conjugated with horseradish peroxidase for 1 h at room temperature. The blots were then washed three times with PBS before the reaction with enhanced-chemiluminescence, Western blotting detection reagents (Amersham). A quantitative analysis of the protein was performed by densitometric scanning of the autoradiographs employing the enhanced laser densitometer, LKB Ultroscan XL, and quantified using the gel-scan XL evaluation software (version 2.1) from Pharmacia (Baie d'Urfé, Québec, Canada).

Determination of IP3 levels. The IP3 levels in VSMCs were determined as described earlier (26). The confluent VSMCs were incubated for 24 h in a serum-free Dulbecco's modified Eagle's medium and were then exposed to high glucose (26 mmol/l) or normal glucose (5.5 mmol/l) for 3 days. Twenty-four hours before the termination of the treatment, the cells were incubated with 5 µCi/ml [myo-2-3H]inositol (specific activity, 25 Ci/mmol). The cells were washed three times with warm (37°C) Earle's balanced salt solution buffer after removing the cultured medium containing the unincorporated isotope. The cells were further incubated for 30 min in the same buffer containing 20 mM lithium chloride to inhibit the conversion of the inositol phosphates to inositol so that the radiolabeled inositol phosphates could accumulate within the cells. The agonists were added for 2 h, and the reaction was terminated by adding 0.9 ml of methanol-chloroform-HCl (40:20:1) as described previously (26). The cells were scraped using a rubber policeman and were then homogenized. Chloroform (0.5 ml) and 0.5 ml distilled water were added to the homogenates, and the samples were then centrifuged to separate lipid and aqueous phases. The aqueous phase was transferred to a column containing 0.8 ml of AG1-X8 resin (200–400 mesh, formate form; Bio-Rad, Hercules, CA) from which inositol phosphates were eluted sequentially with ammonium formate buffers of increasing molarity (9). The radioactivity was measured in a liquid scintillation counter. The lipid phase was counted to measure the phosphatidylinositol pool. The accumulation of inositol phosphate was expressed as the ratio of IP3 to PI x 103 to correct for the variation in the labeling of the lipid pool.

Statistical analysis. Data are expressed as means ± SE and were analyzed by ANOVA in conjunction with a Bonferroni test where applicable. Comparisons between groups (control and high glucose-treated cells) were made with Student's t-test for unpaired samples. A difference between groups was considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The expression of Gq{alpha} and PLC-β proteins in aortic VSMCs from STZ-diabetic rats. Since an enhanced expression of PKC and DAG was reported in different tissues from STZ-induced diabetic rats, as well as in BB rats (12, 18, 32, 37, 40), it was of interest to investigate whether the diabetic state could also contribute to the increased expression of Gq{alpha} and PLC-β proteins, the upstream signaling components of DAG and PKC. To investigate this, we examined the levels of Gq{alpha} and PLC-β1 proteins in aortic VSMCs from day 5 STZ-diabetic rats. As shown in Fig. 1A, the antibodies K-17 against Gq{alpha} recognized a single protein of 42 kDa in the VSMCs from both control and STZ-diabetic rats; however, the expression of Gq{alpha} protein was significantly (~60%) increased in the VSMCs from STZ-diabetic rats compared with control cells. In addition, the antibodies D-8 against PLC-β1 also recognized a single protein of 150 kDa in both groups; however, the expression of PLC-β1 was significantly (~30%) increased in VSMCs from STZ-diabetic rats compared with control cells (Fig. 1B). In addition, ET-1-induced IP3 formation was significantly enhanced in aortic VSMCs from STZ-diabetic rats compared with control rats (data not shown).


Figure 1
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  		Fig. 1. The expression of Gq{alpha} and phospholipase C-β (PLC-β) proteins in aortic vascular smooth muscle cells (VSMCs) from streptozotocin (STZ)-diabetic rats. Cells lysates of VSMCs from STZ-diabetic rats and control (CTL) rats were prepared and subjected to Western blot analysis using specific antibodies against Gq{alpha} (A, top) and PLC-β1 (B, top) as described in MATERIALS AND METHODS. The proteins were quantified by densitometric scanning (A and B, bottom). The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05 and **P < 0.01 vs. CTL.

 
Effect of high glucose on the expression of Gq{alpha} and G11{alpha} proteins in A10 VSMCs. Since hyperglycemia simulates the diabetic state, it was of interest to investigate whether high glucose could also mimic the effect of diabetes on Gq{alpha} protein expression. To test this, the effect of high glucose on the expression of Gq{alpha} and PLC-β proteins was examined in A10 VSMCs. As shown in Fig. 2A, the antibodies K-17 against Gq{alpha} recognized a single protein of 42 kDa in both control and A10 cells exposed to high glucose for 1 and 3 days; however, the relative amounts of immunodetectable Gq{alpha} were not significantly different in cells exposed to high glucose for 1 day compared with control cells exposed to normal glucose (5.5 mM). On the other hand, the expression of Gq{alpha} protein was significantly (~50%) increased in cells exposed to high glucose for 3 days compared with control cells. In addition, the antibodies D-17 against G11{alpha} also recognized a single protein of 42 kDa in control and high glucose-treated cells; however, the expression of G11{alpha} was significantly (~40%) increased in cells exposed to high glucose compared with control cells (Fig. 2B). On the other hand, the treatment of cells with 26 mM of mannitol for 3 days did not have any significant effect on the levels of Gq{alpha} and G11{alpha} proteins compared with control cells (Fig. 2, C and D).


Figure 2
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  		Fig. 2. Effect of high glucose on the expression of Gq/11{alpha} proteins in A10 VSMCs. A10 VSMCs were incubated in the presence of 5.5 mM (CTL) and 26 mM glucose (high glucose) for 1 and 3 days or 26 mM mannitol for 3 days at 37°C. Membranes were prepared and subjected to Western blot analysis using specific antibodies against Gq{alpha} (A and C, top) and G11{alpha} (B and D, top) as described in MATERIALS AND METHODS. The proteins were quantified by densitometric scanning (A–D, bottom). The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05 and ***P < 0.001 vs. CTL.

 
Effect of high glucose on the expression of PLC-β isoforms in A10 VSMCs. To investigate whether high glucose could also increase the expression of PLC-β, a downstream signaling molecule of Gq{alpha} that catalyses the formation of IP3, we examined the effect of high glucose on the expression of different isoforms of PLC-β using specific antibodies. As shown in Fig. 3, the expression of PLC-β1 (Fig. 3A) and PLC-β2 (Fig. 3B) proteins was significantly (~30%) increased in cells treated with high glucose compared with control cells, whereas the expression of PLC-β3 (Fig. 3C) protein was not altered by this treatment. On the other hand, 26 mM mannitol was ineffective in altering the expression of PLC-β proteins (Fig. 3, D and E).


Figure 3
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  		Fig. 3. Effect of high glucose on the expression of PLC-β isoforms in A10 VSMCs. A10 VSMCs were incubated in the presence of 5.5 mM (CTL), 26 mM glucose (high glucose), or 26 mM mannitol for 3 days at 37°C. Membranes were prepared and subjected to Western blot analysis using specific antibodies against PLC-β1 (A and D, top), PLC-β2 (B and E, top), and PLC-β3 (C, top) as described in MATERIALS AND METHODS. The proteins were quantified by densitometric scanning (A–E, bottom). The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05 and ***P < 0.001 vs. CTL.

 
Effect of high glucose on ET-1-induced IP3 formation in A10 VSMCs. To examine whether the increased expression of Gq/11{alpha} and PLC-β1/2 by high glucose was also reflected in functions, the effect of high glucose on IP3 formation was investigated in A10 VSMCs, and the results are shown in Fig. 4. ET-1 increased the formation of IP3 in control and high glucose-treated cells; however, the increase was significantly (~25%) greater in high glucose-treated cells compared with control cells, whereas the basal IP3 levels were not significantly different in two groups of cells.


Figure 4
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  		Fig. 4. Effect of high glucose on endothelin-1 (ET-1)-induced inositol triphosphate (IP3) formation in A10 VSMCs. A10 VSMCs were incubated in the presence of 5.5 mM (CTL) or 26 mM glucose (high glucose) for 3 days at 37°C. [myo-2-3H]inositol (5 µCi/ml) was added on the 3rd day for the last 24 h of the treatment as described in MATERIALS AND METHODS. The cells were further incubated in the absence or presence of ET-1 (10–7 M) for 2 h, and IP3 formation was determined as described in MATERIALS AND METHODS. PIP2, inositol biphosphate. The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 to 4 separate experiments performed in triplicate. ***P < 0.001 vs. CTL; ##P < 0.01.

 
Implication of ETA/ETB and AT1 receptors in high glucose-induced enhanced expression of Gq{alpha}, G11{alpha}, and PLC-β proteins in A10 VSMCs. Since hyperglycemia/diabetes has been shown to exhibit an increased level of vasoactive peptides such as ANG II and ET-1 (7, 15, 19, 21, 22, 29), it was of interest to investigate whether the high glucose-induced enhanced production of ET-1 and ANG II in VSMCs contributes to the increased expression of Gq{alpha} and G11{alpha} proteins. To test this, we examined the effect of the antagonists of ETA and ETB receptor, BQ123, and BQ788, respectively, as well as losartan, AT1 receptors antagonist, in high glucose-induced increased expression of Gq{alpha} and G11{alpha} proteins. The results shown in Fig. 5 indicate that BQ123, BQ788, and losartan restored the high glucose-induced increased levels of Gq{alpha} (Fig. 5A) and G11{alpha} (Fig. 5B) to control levels, suggesting the implication of ETA, ETB, and AT1 receptors in the high glucose-induced increased expression of Gq{alpha} and G11{alpha} proteins in A10 VSMCs.


Figure 5
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  		Fig. 5. Effect of endothelin types A and B (ETA/ETB) and ANG II type 1 (AT1) receptor antagonists on high glucose-induced enhanced expression of Gq{alpha}, G11{alpha}, and PLC-β proteins in A10 VSMCs. A10 VSMCs were incubated in the presence of 5.5 mM (CTL) or 26 mM glucose (high glucose) for 3 days at 37°C. The antagonists BQ123 (10–6 M), BQ788 (10–6 M), or losartan (10–6 M) were added on the 3rd day for the last 24 h of the treatment. Membranes were prepared and subjected to Western blot analysis using specific antibodies against Gq{alpha} (A, top), G11{alpha} (B, top), PLC-β1 (C, top), and PLC-β2 (D, top) as described in MATERIALS AND METHODS. The proteins were quantified by densitometric scanning (A–D, bottom). The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. *P < 0.05 and ***P < 0.001 vs. CTL; #P < 0.05 and ###P < 0.001 vs. high glucose.

 
The effect of ETA, ETB, and AT1 receptor antagonists in high glucose-induced increased expression of PLC-β1 and -β2 proteins in A10 cells was also examined, and the results are shown in Fig. 5, C and D. The increased expression of PLC-β1 (Fig. 5C) and -β2 (Fig. 5D) proteins by high glucose was restored to control levels by BQ123, BQ788, and losartan, suggesting the contribution of ETA, ETB, and AT1 receptors in high glucose-induced enhanced expression of PLC-β1 and PLC-β2 proteins in A10 VSMCs.

Effect of ET-1 and ANG II on the expression of Gq{alpha}, G11{alpha}, and PLC-β proteins in A10 VSMCs. Since AT1, ETA, and ETB receptors appear to be implicated in high glucose-induced increased levels of Gq/11{alpha} and PLC-β, it was of interest to investigate whether the in vitro treatment of A10 VSMCs with ANG II and ET-1 could also result in the enhanced expression of Gq/11{alpha} and PLC-β. The results shown in Fig. 6 indicate that the pretreatment of A10 VSMCs with ET-1 (10–7 M) or ANG II (10–6 M) for 24 h increased the levels of Gq{alpha} by about 70% and 85%, respectively, whereas about a 90% and 160% increase in the levels of G11{alpha} was observed by ET-1 and ANG II, respectively. In addition, the levels of PLC-β proteins were also increased by about 55% and 90% by ET-1 and ANG II, respectively, whereas about a 60% increase in the levels of PLC-β2 was observed by ET-1 and 80% by ANG II (Fig. 7). Furthermore, the enhanced levels of Gq/11{alpha} and PLC-β1/2 induced by ANG II and ET-1 were restored toward control levels by losartan, BQ123, and BQ788, suggesting the implication of AT1 and ETA/ETB receptors in ANG II and ET-1-induced enhanced expression of Gq/11{alpha} and PLC-β proteins in A10 VSMCs.


Figure 6
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  		Fig. 6. Effect of ETA/ETB and AT1 receptor antagonists on ET-1 and ANG II-induced enhanced expression of Gq{alpha} and G11{alpha} protein in A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with BQ123 (10–6 M), BQ788 (10–6 M), or losartan (10–6 M) for 3 h and challenged with ET-1 (10–7 M) or ANG II (10–6 M) for 24 h. Membranes were prepared and subjected to Western blot analysis using specific antibodies against Gq{alpha} (A and C, top) and G11{alpha} (B and D, top) as described in MATERIALS AND METHODS. The proteins were quantified by densitometric scanning (A–D, bottom). The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. **P < 0.01 and ***P < 0.001 vs. CTL; #P < 0.05 and ##P < 0.01 vs. ET-1 or ANG II.

 

Figure 7
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  		Fig. 7. Effect of ETA/ETB and AT1 receptor antagonists on ET-1 and ANG II-induced enhanced expression of PLC-β1 and PLC-β2 protein in A10 VSMCs. A10 VSMCs were pretreated without (CTL) or with BQ123 (10–6 M), BQ788 (10–6 M), or losartan (10–6 M) for 3 h and challenged with ET-1 (10–7 M) or ANG II (10–6 M) for 24 h. Membranes were prepared and subjected to Western blot analysis using specific antibodies against PLC-β1 (A and C, top) and PLC-β2 (B and D, top) as described in MATERIALS AND METHODS. The proteins were quantified by densitometric scanning (A–D, bottom). The results are expressed as a percentage of CTL taken as 100%. Values are means ± SE of 3 separate experiments. **P < 0.01 and ***P < 0.001 vs. CTL; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. ET-1 or ANG II.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously reported that A10 VSMCs exposed to high glucose (26 mM) decreased the expression of Gi{alpha} protein (16). In addition, aortic VSMCs from STZ-diabetic rats as well as STZ-diabetic aorta were also shown to exhibit a decreased expression of Gi{alpha}. (17). DAG/PKC, downstream signaling molecules in the Gq{alpha} pathway, have been shown to be upregulated in hyperglycemia and diabetes (12, 18, 30, 32, 37, 39, 40). An enhanced expression of PKC and DAG has been reported in different tissues from STZ-induced diabetic as well as BB rats (12, 18, 32, 37, 40). In addition, the high glucose treatment of cultured aortic endothelial and VSMCs has also been reported to enhance the activity of PKC and DAG (18, 30, 39); however, the studies on the levels of Gq{alpha}, an important upstream signaling molecule of PI turnover in diabetic aorta or in VSMCs under hyperglycemic conditions, are lacking. In the present study, we demonstrate for the first time that the treatment of A10 VSMCs with high glucose significantly increased the expression of Gq/11{alpha}. The upregulation of Gq{alpha} and G11{alpha} proteins was not due to increased osmolarity because mannitol (26 mM) did not have any effect on the expression of Gq/11{alpha} proteins. In addition, we also report that aortic VSMCs from STZ-diabetic rats also exhibit increased expression of Gq{alpha} proteins, suggesting that hyperglycemia could mimic the diabetic state. An increased expression of Gq{alpha} in sciatic nerves (12) and hearts (37, 40) from STZ-diabetic rats has also been reported. However, our results are in contrast with the studies of Ceccarelli et al. (6) who showed a decreased expression of Gq{alpha} in bovine retinal pericites exposed to 25 mmol/l of glucose (6). In addition, an unaltered or a decreased expression of Gq/11{alpha} protein was also shown in gastric VSMCs from 10-wk STZ-diabetic rats and a genetic model of non-insulin-dependent diabetes (11–12 mo) WBN/Kob diabetic rats (24), respectively. The apparent discrepancies may be attributed to the difference in the cell type used and its origin.

We showed for the first time that the treatment of A10 VSMCs with high glucose increased the expression of PLC-β1 and -β2 but not -β3 proteins. In addition, aortic VSMCs from STZ-diabetic rats also exhibited an increased expression of PLC-β1 proteins. Our results are in accordance with the studies of Frecker et al. (11) who also did not observe any changes in the levels of PLC-β3. We also showed that high glucose enhanced ET-1-induced formation of IP3 to a greater extent compared with that in control cells without increasing basal IP3. In support of this notion, Abebe and MacLeod (1) using aorta from STZ-diabetic rats have also reported that norepinephrine-induced, but not basal, formation of IP3 was greater compared with control rats during contraction. In addition, the arteries from STZ-induced diabetic rats were shown to be more responsive to the contractile effects of norepinephrine than the arteries from control animals (2), which may also be due to the increased formation of IP3 induced by norepinephrine. Taken together, it may be suggested that the increased level of IP3 induced by ET-1 in our studies and the norepinephrine-induced enhanced contraction in STZ rats (1, 2) may be attributed to the increased levels of Gq/11{alpha} and PLC-β1/2 proteins which, after the activation by ET-1 or norepinephrine, contribute to the enhanced production of IP3. On the other hand, the inability of high glucose to increase the levels of IP3, despite increasing the levels of Gq{alpha} and PLC-β, may be due to the fact that the Gq{alpha} needs to be activated to form IP3, and this activation occurs by G protein-coupled receptor activation and not by glucose.

The mechanism by which high glucose increases the levels of Gq/11{alpha}, PLC-β, and mediated signaling is not clear. However, we showed for the first time that ANG II and ET-1, the levels of which are augmented in hyperglycemia (19, 21, 29), may contribute to the enhanced levels of Gq/11{alpha} and PLC-β proteins and mediated signaling observed in the present studies. This notion is supported by our results showing that losartan, a selective AT1 receptor antagonist, BQ123 as well as BQ788, ETA and ETB receptor antagonists, respectively, completely prevented high glucose-induced enhanced levels of Gq/11{alpha} and PLC-β protein in VSMCs and suggests that these effects may be mediated by an autocrine production of ANG II and ET-1. In this regard, the local production of ANG II and ET-1, as well as the receptor expression of AT1, were shown to be significantly increased in aortic endothelial and VSMCs exposed to high glucose (19, 21, 29, 34). Furthermore, ANG II has also been shown to increase the synthesis of ET-1 in VSMCs (14). Since high glucose increases the levels of ANG II in VSMCs (21), it may be possible that the high glucose-induced enhanced production of ANG II may contribute to the increased synthesis of ET-1 in VSMCs. In addition, our results showing that the treatment of A10 VSMCs with ANG II and ET-1 augmented the expression of Gq/11{alpha}, as well as PLC-β proteins that was reversed by losartan, BQ123, and BQ788, further strengthen the implication of AT1 and ETA/ETB receptors in the enhanced expression of Gq{alpha} and PLC-β proteins induced by high glucose. The underlying mechanism(s) by which AT1 and ETA/ATB receptor activation by high glucose induces vascular dysfunction is not well understood at this time. However, ANG II and ET-1 have been reported to increase oxidative stress by activating NADPH oxidase, an enzyme responsible for the production of superoxide anion and other reactive oxygen species (38). An ANG II-induced increased expression of p47phox and p22phox has also been shown recently in A10 VSMCs (23). In addition, the role of oxidative stress in an ANG II-induced enhanced expression of Gi{alpha} proteins and associated adenylyl cyclase signaling in A10 VSMCs has been recently shown (23). Taken together, it may be possible that ANG II-induced enhanced oxidative stress under hyperglycemic conditions also contributes to the enhanced expression of Gq{alpha}/PLC signaling. However, that possibility needs to be investigated.

In conclusion, we have provided the first evidence that diabetes/hyperglycemia through AT1 as well as ETA/ETB receptors increases the expression of Gq/11{alpha} and PLC-β1/2 proteins that may be responsible for the ET-1-induced increased IP3 turnover in VSMCs (Fig. 8). The increased expression of Gq/11{alpha} and PLC-β results in an enhanced production in IP3, which by increasing the intracellular levels of calcium may contribute to the vascular complications observed in diabetes (Fig. 8). Thus it may be suggested that high glucose-induced increased levels of ANG II and ET-1 could be the important factor responsible for the impaired Gq/11{alpha} signaling and the resultant vascular complications of diabetes.


Figure 8
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  		Fig. 8. Schematic diagram summarizing the possible mechanisms by which hyperglycemia/diabetes increase the expression of Gq{alpha}/PLC-β proteins and result in vascular complications. DAG, diacylglycerol.

 

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This study was supported by a Canadian Institutes of Health Research Grant MOP-53074.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Faculty of Medicine, Univ. of Montreal, C. P. 6128, Succ. Centre-ville, Montreal, QC, H3C 3J7, Canada (e-mail: madhu.anand-srivastava{at}umontreal.ca)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 REFERENCES
 

  1. Abebe W, MacLeod KM. Influence of diabetes on norepinephrine-induced inositol 1,4,5-trisphosphate levels in rat aorta. Life Sci 49: L85–L90, 1991.[CrossRef]
  2. Abebe W, MacLeod KM. Protein kinase C-mediated contractile responses of arteries from diabetic rats. Br J Pharmacol 101: 465–471, 1990.[Web of Science][Medline]
  3. Anand-Srivastava MB, de Champlain J, Thibault C. DOCA-salt hypertensive rat hearts exhibit altered expression of G-proteins. Am J Hypertens 6: 72–75, 1993.[Web of Science][Medline]
  4. Anand-Srivastava MB, Franks DJ, Cantin M, Genest J. Presence of "Ra" and "P"-site receptors for adenosine coupled to adenylate cyclase in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 108: 213–219, 1982.[CrossRef][Web of Science][Medline]
  5. Berridge MJ. Inositol trisphosphate as a second messenger in signal transduction. Ann NY Acad Sci 494: 39–51, 1987.[Web of Science][Medline]
  6. Ceccarelli F, Rosa MM, Chakravarthy U. Binding characteristics of ET receptors in retinal pericytes and effects of high glucose incubation. Curr Eye Res 23: 263–270, 2001.[CrossRef][Web of Science][Medline]
  7. Collier A, Leach JP, McLellan A, Jardine A, Morton JJ, Small M. Plasma endothelinlike immunoreactivity levels in IDDM patients with microalbuminuria. Diabetes Care 15: 1038–1040, 1992.[Abstract]
  8. D'Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW. Transgenic Galphaq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci USA 94: 8121–8126, 1997.[Abstract/Free Full Text]
  9. Dean NM, Beaven MA. Methods for the analysis of inositol phosphates. Anal Biochem 183: 199–209, 1989.[CrossRef][Web of Science][Medline]
  10. Ergul A, Johansen JS, Stromhaug C, Harris AK, Hutchinson J, Tawfik A, Rahimi A, Rhim E, Wells B, Caldwell RW, Anstadt MP. Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes: role of endothelin-1. J Pharmacol Exp Ther 313: 70–77, 2005.[Abstract/Free Full Text]
  11. Frecker H, Munk S, Wang H, Whiteside C. Mesangial cell-reduced Ca2+ signaling in high glucose is due to inactivation of phospholipase C-β3 by protein kinase C. Am J Physiol Renal Physiol 289: F1078–F1087, 2005.[Abstract/Free Full Text]
  12. Goraya TY, Wilkins P, Douglas JG, Zhou J, Berti-Mattera LN. Signal transduction alterations in peripheral nerves from streptozotocin-induced diabetic rats. J Neurosci Res 41: 518–525, 1995.[CrossRef][Web of Science][Medline]
  13. Groop PH, Forsblom C, Thomas MC. Mechanisms of disease: pathway-selective insulin resistance and microvascular complications of diabetes. Nat Clin Pract Endocrinol Metab 1: 100–110, 2005.[CrossRef][Web of Science][Medline]
  14. Hahn AW, Resink TJ, Scott-Burden T, Powell J, Dohi Y, Buhler FR. Stimulation of endothelin mRNA and secretion in rat vascular smooth muscle cells: a novel autocrine function. Cell Regul 1: 649–659, 1990.[Web of Science][Medline]
  15. Hargrove GM, Dufresne J, Whiteside C, Muruve DA, Wong NC. Diabetes mellitus increases endothelin-1 gene transcription in rat kidney. Kidney Int 58: 1534–1545, 2000.[CrossRef][Web of Science][Medline]
  16. Hashim S, Li Y, Nagakura A, Takeo S, Anand-Srivastava MB. Modulation of G-protein expression and adenylyl cyclase signaling by high glucose in vascular smooth muscle. Cardiovasc Res 63: 709–718, 2004.[Abstract/Free Full Text]
  17. Hashim S, Liu YY, Wang R, Anand-Srivastava MB. Streptozotocin-induced diabetes impairs G-protein linked signal transduction in vascular smooth muscle. Mol Cell Biochem 240: 57–65, 2002.[CrossRef][Web of Science][Medline]
  18. Inoguchi T, Battan R, Handler E, Sportsman JR, Heath W, King GL. Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 89: 11059–11063, 1992.[Abstract/Free Full Text]
  19. Keynan S, Khamaisi M, Dahan R, Barnes K, Jackson CD, Turner AJ, Raz I. Increased expression of endothelin-converting enzyme-1c isoform in response to high glucose levels in endothelial cells. J Vasc Res 41: 131–140, 2004.[CrossRef][Web of Science][Medline]
  20. Kyaw M, Yoshizumi M, Tsuchiya K, Kirima K, Suzaki Y, Abe S, Hasegawa T, Tamaki T. Antioxidants inhibit endothelin-1 (1-31)-induced proliferation of vascular smooth muscle cells via the inhibition of mitogen-activated protein (MAP) kinase and activator protein-1 (AP-1). Biochem Pharmacol 64: 1521–1531, 2002.[CrossRef][Web of Science][Medline]
  21. Lavrentyev EN, Estes AM, Malik KU. Mechanism of high glucose induced angiotensin II production in rat vascular smooth muscle cells. Circ Res 101: 455–464, 2007.[Abstract/Free Full Text]
  22. Lewis EJ, Hunsicker LG, Bain RP, Rohde RD. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N Engl J Med 329: 1456–1462, 1993.[Abstract/Free Full Text]
  23. Li Y, Lappas G, Anand-Srivastava MB. Role of oxidative stress in angiotensin II-induced enhanced expression of Gi{alpha} proteins and adenylyl cyclase signaling in A10 vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 292: H1922–H1930, 2007.[Abstract/Free Full Text]
  24. Lin S, Kajimura M, Takeuchi K, Kodaira M, Hanai H, Nishimura M, Kaneko E. Alterations of GTP-binding proteins (Gsalpha and Gq/11alpha) in gastric smooth muscle cells from streptozotocin-induced and WBN/Kob diabetic rats. Dig Dis Sci 45: 1517–1524, 2000.[CrossRef][Web of Science][Medline]
  25. Marrero MB, Schieffer B, Li B, Sun J, Harp JB, Ling BN. Role of Janus kinase/signal transducer and activator of transcription and mitogen-activated protein kinase cascades in angiotensin II- and platelet-derived growth factor-induced vascular smooth muscle cell proliferation. J Biol Chem 272: 24684–24690, 1997.[Abstract/Free Full Text]
  26. Mouawad R, Li Y, Anand-Srivastava MB. Atrial natriuretic peptide-C receptor-induced attenuation of adenylyl cyclase signaling activates phosphatidylinositol turnover in A10 vascular smooth muscle cells. Mol Pharmacol 65: 917–924, 2004.[Abstract/Free Full Text]
  27. Neves SR, Ram PT, Iyengar R. G protein pathways. Science 296: 1636–1639, 2002.[Abstract/Free Full Text]
  28. Offermanns S, Zhao LP, Gohla A, Sarosi I, Simon MI, Wilkie TM. Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice. EMBO J 17: 4304–4312, 1998.[CrossRef][Web of Science][Medline]
  29. Park JY, Takahara N, Gabriele A, Chou E, Naruse K, Suzuma K, Yamauchi T, Ha SW, Meier M, Rhodes CJ, King GL. Induction of endothelin-1 expression by glucose: an effect of protein kinase C activation. Diabetes 49: 1239–1248, 2000.[Abstract]
  30. Ramana KV, Friedrich B, Tammali R, West MB, Bhatnagar A, Srivastava SK. Requirement of aldose reductase for the hyperglycemic activation of protein kinase C and formation of diacylglycerol in vascular smooth muscle cells. Diabetes 54: 818–829, 2005.[Abstract/Free Full Text]
  31. Sachidanandam K, Harris A, Hutchinson J, Ergul A. Microvascular versus macrovascular dysfunction in type 2 diabetes: differences in contractile responses to endothelin-1. Exp Biol Med (Maywood) 231: 1016–1021, 2006.[Abstract/Free Full Text]
  32. Shiba T, Inoguchi T, Sportsman JR, Heath WF, Bursell S, King GL. Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation. Am J Physiol Endocrinol Metab 265: E783–E793, 1993.[Abstract/Free Full Text]
  33. Smrcka AV, Hepler JR, Brown KO, Sternweis PC. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science 251: 804–807, 1991.[Abstract/Free Full Text]
  34. Sodhi CP, Kanwar YS, Sahai A. Hypoxia and high glucose upregulate AT1 receptor expression and potentiate ANG II-induced proliferation in VSM cells. Am J Physiol Heart Circ Physiol 284: H846–H852, 2003.[Abstract/Free Full Text]
  35. Sowers JR, Epstein M. Diabetes mellitus and associated hypertension, vascular disease, and nephropathy. An update. Hypertension 26: 869–879, 1995.[Abstract/Free Full Text]
  36. Strathmann M, Simon MI. G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. Proc Natl Acad Sci USA 87: 9113–9117, 1990.[Abstract/Free Full Text]
  37. Tappia PS, Asemu G, Aroutiounova N, Dhalla NS. Defective sarcolemmal phospholipase C signaling in diabetic cardiomyopathy. Mol Cell Biochem 261: 193–199, 2004.[CrossRef][Web of Science][Medline]
  38. Touyz RM, Yao G, Viel E, Amiri F, Schiffrin EL. Angiotensin II and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells. J Hypertens 22: 1141–1149, 2004.[CrossRef][Web of Science][Medline]
  39. Williams B, Gallacher B, Patel H, Orme C. Glucose-induced protein kinase C activation regulates vascular permeability factor mRNA expression and peptide production by human vascular smooth muscle cells in vitro. Diabetes 46: 1497–1503, 1997.[Abstract]
  40. Yang JM, Cho CH, Kong KA, Jang IS, Kim HW, Juhnn YS. Increased expression of Galphaq protein in the heart of streptozotocin-induced diabetic rats. Exp Mol Med 31: 179–184, 1999.[Web of Science][Medline]




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