Possible Involvement of Phospholipase D and Protein Kinase C in Vascular Growth Induced by Elevated Glucose Concentration
Hyperglycemia is believed to be a major cause of diabetic vascular complications. To elucidate the effect of hyperglycemia on vascular response, we studied hyperproliferation, hypertrophy, and the natriuretic peptide response of vascular smooth muscle cells under high-glucose conditions. We observed that cells cultured in high glucose (22.2 mmol/L) showed hyperproliferation and hypertrophy and that natriuretic peptide receptor responses were suppressed compared with cells cultured in normal glucose (5.6 mmol/L). We also examined phospholipase D and protein kinase C activities and found that in high-glucose conditions such activities are higher than in cells cultured in normal glucose. The activation of phospholipase D was not prevented by coincubation with 1 μmol/L protein kinase C(19-36), a specific protein kinase C inhibitor, but the activation of protein kinase C was. Protein kinase C(19-36) also markedly attenuated vascular hyperproliferation and hypertrophy as well as glucose-induced suppression of natriuretic peptide receptor response. These results show that hyperglycemia may be linked to vascular hyperproliferation, hypertrophy, and a suppressed natriuretic peptide receptor response, which are caused by increased phospholipase D and protein kinase C activities.
Hyperglycemia is probably an important etiologic factor in the development of vascular complications, such as nephropathy1 and accelerated atherosclerosis,2 in diabetic patients. However, the mechanisms for accelerated atherosclerotic disease in diabetes are not clear. VSMC growth and proliferation seem to be important factors in the development of atherosclerosis.3 Although hyperglycemia in diabetes has been suggested to contribute to complications, few studies have focused on the direct effect of elevated glucose on VSMCs.
Phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline, is considered to be activated by growth factors and causes cell proliferation.4 PKC, a member of a family of serine-threonine–specific kinases, plays a key role in a range of signal transduction processes.5 Activation of PKC induces phosphorylation of many proteins in cells, causing alterations in various biological systems thought to underlie several pathological states, including cardiac hypertrophy6 and atherosclerosis.7
At least three receptors of the natriuretic peptide family—the ANP-A receptor (activated by ANP or BNP), ANP-B receptor (activated by CNP), and clearance receptor—have been identified.8 ANP-A and ANP-B receptors are distributed in VSMCs.9 Natriuretic peptides can act not only as vasodilators but also as growth inhibitors of VSMCs.10
Recently, we have shown that the ANP-A receptor response is negatively regulated by PKC.11 The effect of high glucose on endothelial cell and VSMC function in blood vessels that affect PKC activity have been reported.12 13 These findings led us to speculate on the possible involvement of phospholipase D and PKC in high glucose–induced vascular remodeling and receptor dysfunction. Therefore, we designed this study to determine whether high glucose concentrations regulate vascular hyperproliferation and hypertrophy as well as the ANP-A and ANP-B receptor responses on VSMCs and whether PKC inhibitors prevent this process.
Type II collagenase, Nonidet P-40, dithiothreitol, IBMX, and PMA were purchased from Sigma Chemical Co. DMEM, penicillin-streptomycin, trypsin-EDTA (Versine), FCS, the specific PKC inhibitor peptide PKC(19-36), and its control, [Glu27] PKC(19-36), were purchased from GIBCO Laboratories. cGMP radioimmunoassay kits, [3H]thymidine, [3H]leucine, [methyl-3H]choline, [3H]ethanolamine, and the PKC assay system were purchased from Amersham Japan Co. Multiwell pipettes and flasks were purchased from Becton Dickinson. Rat ANP(1-28), rat BNP(1-45), and rat CNP(1-22) were purchased from the Peptide Institute.
VSMCs were grown from explants of renal arteries from 14-week-old normotensive Wistar rats; rats were handled as described previously.14 15 Cells were identified as VSMCs according to their morphological and growth characteristics as previously reported.16 17 VSMCs were grown in DMEM supplemented with 10% FCS. Cells from passages 3 to 5 were used and subcultured after trypsinization on a weekly basis because cells became confluent in 1 week. Each plate was replenished twice a week with fresh medium. For studies of cells under hyperglycemic conditions, the cells were allowed to grow for two passages in high-glucose (22.2 mmol/L) DMEM before use. To more closely simulate chronic hyperglycemia, we passaged cells in high glucose rather than add glucose acutely. For control of osmolarity, cells were grown for two passages in 5.6 mmol/L glucose plus 16.6 mmol/L mannose.
For determination of cell numbers, VSMCs were placed into six-well culture dishes at 2×104/mL and grown in DMEM containing 10% FCS that was changed every 72 hours. After the medium was aspirated, the same medium without FCS was applied for 48 hours. Cultures were washed with calcium- and magnesium-free phosphate-buffered saline [PBS(−)] and harvested with trypsin-EDTA solution. Counts were made with a Coulter counter.18
Determination of DNA and Protein Synthesis
Relative rates of DNA and protein synthesis were assessed by determination of incorporation of [3H]thymidine and [3H]leucine, respectively, into trichloroacetic acid–precipitable material. Quiescent VSMCs grown in 24-well culture dishes were pulsed 4 hours with [3H]thymidine (10 μCi/mL) or [3H]leucine (10 μCi/mL), washed with cold PBS(−), and incubated with 5% trichloroacetic acid at 4°C for 10 minutes. Cells were dissolved in 1N NaOH at 37°C for 30 minutes and neutralized. Radioactivity was determined by liquid scintillation counting.
Measurement of cGMP
After incubation with or without high glucose, cells were washed three times with 2 mL DMEM and stimulated for 10 minutes with different concentrations of ANP, BNP, or CNP dissolved in DMEM with 0.5 mmol/L IBMX. Rapid aspiration and addition of 1.5 mL ice-cold 65% ethanol stopped the reaction. After evaporation by a centrifugal evaporator, the dry residue was dissolved in an assay buffer. cGMP levels were determined by radioimmunoassay with the Amersham cGMP radioimmunoassay kit as previously described.11
Membrane Guanylate Cyclase Activity
After treatment with high glucose (22.2 mmol/L) or normal glucose (5.6 mmol/L) plus 16.6 mol/L mannose for two passages, 175-cm2 culture flasks on ice were washed twice with 5 mL cold HEPES-buffered saline, and cells were scraped into 3 mL of a cold mixture of 20 mmol/L HEPES (pH 7.4), 50 mmol/L NaCl, 5 nmol/L EDTA, and 1 mmol/L dithiothreitol and were homogenized by passage 10 times through a 22-gauge needle. After centrifugation for 15 minutes at 5000g and washing with the same buffer, membrane proteins were solubilized by incubation on ice for 30 minutes in a solution containing 20 mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 10% glycerol, 1% Triton X-100, and 1 mmol/L dithiothreitol. After centrifugation for 15 minutes at 5000g, supernatant fluids were adjusted to equal protein concentrations and incubated for 10 minutes with or without 1 μmol/L CNP. Incubation was continued for 10 minutes at 37°C after dilution of 100 μL of each sample into a solution containing a final concentration of 20 mmol/L HEPES, pH 7.4, with 1 mmol/L GTP, 1 mmol/L MnCl2, and 1 mmol/L IBMX in a total reaction volume of 250 μL. The reaction was terminated with 750 μL of 50 mmol/L sodium acetate, pH 4.0, and then the solution was boiled for 3 minutes. cGMP was assayed with the Amersham radioimmunoassay kit. Protein was measured by the Coomassie blue method of Bradford.19
Flow Cytometric Analysis of Cell Size and Cell Cycle
Quiescent VSMCs grown in flasks were detached with 0.25% trypsin at 37°C for 5 minutes and then pelleted by centrifugation (1000 rpm 5 minutes). The cells were resuspended in DMEM and applied to a flow cytometer (Epics Profile) for measurement of cell size. The cells were resuspended in 200 μL of solution A (30 mg/L trypsin, 3.4 mmol/L citric acid, 1.5 mmol/L spermin, 0.5 mmol/L Tris-HCl, 2 mL/L Nonidet P-40). Ten minutes later, 150 μL of solution B (500 mg/L trypsin inhibitor, 100 mg/L RNase, 3.4 mmol/L citric acid, 1.5 mmol/L spermin, 0.5 mmol/L Tris-HCl, 2 mL/L Nonidet P-40) was added, and the mixture was left for 10 minutes at room temperature. One hundred fifty microliters of solution C (622 μmol/L propidium iodide, 3.0 mmol/L spermin, 3.4 mmol/L citric acid, 0.5 mmol/L Tris-HCl, 2 mL/L Nonidet P-40) was then added, and the mixture was left for more than 10 minutes. All samples for cell cycle analysis20 were analyzed within 3 hours on the flow cytometer. Red blood cells were used as the internal standard for DNA analysis.
Phospholipase D Activity Measured by Choline or Ethanolamine Release
Cells in 35-mm dishes were cultured in a medium containing [methyl-3H]choline (5 μCi/mL per dish) or [3H]ethanolamine (5 μCi/mL per dish) for 24 hours for labeling of cellular phosphatidylcholine and phosphatidylethanolamine, respectively. After the labeling medium was removed, cells were washed twice with buffer A (20 mmol/L HEPES [pH 7.4], 120 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1.5 mmol/L CaCl2, 0.1% [wt/vol] bovine serum albumin, 5.6 mmol/L glucose plus 16.6 mmol/L mannose, or 22.2 mmol/L glucose). After 0.5 to 1 hour of incubation with buffer A, the reaction was terminated by removal of the buffer and addition of 0.75 mL methanol. Cells were harvested by gentle scraping, and the dishes were washed again with 0.75 mL methanol. Distilled water (0.6 mL) and chloroform (0.75 mL) were added to the cells. The aqueous and lipid phases were obtained from the cell extracts by addition of 0.75 mL chloroform and 0.75 mL water followed by centrifugation (3000g, 10 minutes). Fractionation of choline and ethanolamine metabolites from the aqueous phases was performed on Dowex 50w (H+)-packed columns (Bio-Rad Econo columns, 1 mL bed volume) as described by Kiss and Crilly.21 The initial flow-through (5 mL), along with 5 or 3 mL water that followed, contained glycerophosphocholine or glycerophosphoethanolamine, respectively. Choline phosphate and ethanolamine phosphate were eluted by 20 and 15 mL water, respectively. Finally, choline and ethanolamine were eluted by 20 and 8 mL of 1 mol/L HCl, respectively.
Cell Fractionation and PKC
VSMCs were washed twice with an ice-cold assay buffer (50 mmol/L Tris-HCl [pH 7.5] buffer containing 2 mmol/L EDTA, 2 mmol/L EGTA, 0.25 mol/L sucrose, 10 mmol/L 2-mercaptoethanol, 0.21 mmol/L leupeptin, and 0.23 mmol/L phenylmethylsulfonyl fluoride). Cells were scraped and sonicated with three 10-second bursts. The homogenates were centrifuged at 100 000g for 60 minutes at 4°C to separate cytosolic and particulate fractions. The cytosolic fraction was kept on ice with Nonidet P-40 added to a final concentration of 1%. The pellet, resuspended in assay buffer containing 1% Nonidet P-40, was stirred on ice for 1 hour and then centrifuged at 100 000g for 30 minutes. PKC activity was measured by a modification of a method previously reported and with the use of the Amersham PKC assay system.11 In brief, a sample of the reaction mixture (50 mmol/L Tris-HCl [pH 7.5], 3 mmol/L calcium acetate, 100 μmol/l l-α-phosphatidyl-l-serine, 1 μmol/L PMA, 225 μmol/L substrate peptide, 7.5 mmol/L dithiothreitol, and 0.05% [wt/vol] sodium azide) was mixed with magnesium [32P]ATP and incubated at 25°C for 15 minutes. The substrate peptide interacts 8%, 13%, and 38% of PKC activity with cAMP-dependent protein kinase, phosphorylase kinase, and proteolytic PKC fragment, respectively, but it does not interact with hexokinase and myosin light chain kinase. An acidic reaction-quenching reagent was added to stop the reaction. Phosphorylated peptide was separated on binding paper. After the paper was washed, the extent of phosphorylation was detected by scintillation counting. PKC assay was linear for 15 minutes. PKC activity was determined by subtraction of the initial rate of protein kinase activity (in the absence of activators) from the initial rate of protein kinase activity in the presence of phosphatidylserine, calcium acetate, and PMA.
Statistical analysis was performed by ANOVA and Scheffé's modified t test.22 Values of P<.05 were considered to be significant.
Inhibition of High Glucose–Induced Cell Proliferation by PKC Inhibitor
As shown in Fig 1⇓, cell numbers under high-glucose (22.2 mmol/L) conditions were higher than those under normal-glucose (5.6 mmol/L) plus 16.6 mmol/L mannose conditions. The PKC inhibitor PKC(19-36) had an inhibitory effect on this high glucose–induced cell proliferation of VSMCs, although the inactive PKC peptide [Glu27] PKC(19-36) had little effect. Cell numbers between cells treated with 5.6 mmol/L glucose and those treated with 5.6 mmol/L glucose plus 16.6 mmol/L mannose did not differ. In these experiments, once VSMCs reached confluence, they were cultured in FCS-free medium for 48 hours to induce quiescence. PKC(19-36) at 1 and 0.1 μmol/L reduced the number of VSMCs cultured in 22.2 mmol/L glucose 28% and 16%, respectively. Cell viability was checked by trypan blue staining, which confirmed that more than 99% of cells were alive.
Antiproliferative and Antihypertrophic Actions of PKC Inhibitor on Postconfluent VSMCs
Fig 2⇓ shows the effects of the PKC inhibitor PKC(19-36) on the incorporation of [3H]thymidine and [3H]leucine in postconfluent VSMCs stimulated in the normal-glucose (5.6 mmol/L) plus 16.6 mmol/L mannose FCS-free medium or VSMCs stimulated by high glucose (22.2 mmol/L). PKC(19-36) inhibited DNA and protein synthesis of VSMCs in a dose-dependent manner, although the inactive PKC peptide [Glu27] PKC(19-36) had little effect. PKC(19-36) did not cause loss of cells at confluence. Two passages after addition of PKC(19-36), less than 1% of cells was found to be present in the supernatant media. Cell viability was checked by trypan blue staining, confirming that more than 99% of cells were alive.
Effect of Elevated Glucose on Natriuretic Peptide Responses in VSMCs
As shown in Fig 3A⇓, ANP, BNP, and CNP stimulated the formation of cGMP by guanylate cyclase in intact VSMCs. VSMCs in high glucose (22.2 mmol/L) had a suppressed maximal response, generally ranging 27% to 40% below that of cells treated in normal glucose (5.6 mmol/L). This decrease was observed in cells treated in high glucose after the first passage and became more distinct after the second passage (Fig 3B⇓). Cells treated with different concentrations of glucose exhibited a dose-related decrease in cGMP formation by ANP, BNP, or CNP (Fig 3C⇓).
Guanylate Cyclase Activity in VSMCs
Particulate guanylate cyclase activity of VSMCs was 5.0±0.5 pmol/min per milligram in cells treated with normal glucose (5.6 mmol/L) plus 16.6 mmol/L mannose and 4.3±0.5 in those treated with high glucose (22.2 mmol/L). After CNP (1 μmol/L) treatment for 10 minutes, particulate guanylate cyclase activity was 17.3±1.2 pmol/min per milligram in normal glucose–treated cells and 12.0±0.7 in high glucose–treated cells (Fig 3D⇑).
Flow Cytometric Analysis
Fig 4⇓ shows cell size of postconfluent VSMCs defined by flow cytometric analysis. Long-term treatment with 1 μmol/L PKC(19-36) tended to reduce cell size and caused a significant left-hand shift in cell size in cells treated with high glucose (22.2 mmol/L). This result further confirmed the inhibitory effect of the PKC inhibitor on cellular hypertrophy of VSMCs.
VSMCs cultured without FCS for 48 hours showed G0-G1 stage growth. Long-term high glucose (22.2 mmol/L) treatment itself without growth factors did not change the cell cycle (data not shown). As shown in Fig 5⇓ and the Table⇓, PDGF (1 ng/mL) treatment for 24 hours induced changes in the cell cycle from the G0-G1 stage to S (15.8%) and G2-M (14.8%) stages. High-glucose treatment changed the cell cycle from the G0-G1 stage to S (20.0%) and G2-M (17.7%) stages. PKC(19-36) (1 μmol/L) suppressed this high glucose–induced change.
Phospholipase D Activities in Normal and High Glucose
To evaluate one potential mechanism for the accelerated VSMC growth by high glucose, we measured phospholipase D activities in cells treated with normal glucose (5.6 mmol/L) plus 16.6 mmol/L mannose and high glucose (22.2 mmol/L). As shown in Fig 6⇓, phospholipase D activities in high glucose–treated cells were greater than those in normal glucose–treated cells. This increase was not blocked by coincubation of the cells with 1 μmol/L PKC(19-36).
PKC Activities in Normal and High Glucose
To evaluate one potential mechanism for the decreased natriuretic peptide receptor responses, we measured PKC activities in VSMCs treated with normal glucose (5.6 mmol/L) plus 16.6 mmol/L mannose and high glucose (22.2 mmol/L). As shown in Fig 6⇑, membrane-bound (particulate) PKC activities in high glucose–treated cells were greater than those in normal glucose–treated cells, with a corresponding decrease in cytosolic PKC activities. VSMCs were coincubated with 1 μmol/L PKC(19-36) in either normal- or high-glucose medium for two passages before in situ PKC activity was determined. With normal-glucose medium, the PKC inhibitor peptide reduced basal PKC activity. Moreover, when high-glucose medium was coincubated with 1 μmol/L PKC(19-36), glucose-induced PKC activation was completely prevented at this PKC(19-36) concentration (Fig 7⇓).
Role of PKC in Glucose-Induced Natriuretic Peptide Receptor Response
After demonstrating that the PKC activation induced by long-term high-glucose exposure could be prevented by coincubation with a PKC inhibitor peptide, we used the same experimental maneuver to examine specifically the role of PKC in mediating glucose-induced suppressed natriuretic peptide receptor response in VSMCs. Fig 8⇓ shows that in the absence of PKC inhibitor peptide, long-term exposure to high-glucose (22.2 mmol/L) medium resulted in a significant suppression of natriuretic peptide receptor response in VSMCs. In contrast, when the high-glucose medium was supplemented with 1 μmol/L PKC(19-36) to prevent glucose-induced PKC activation, the suppression of the natriuretic peptide response was significantly reduced. The percentage of this reduction of BNP- and CNP-mediated response was significantly greater in the high glucose–treated group (ANP: 22.8±7.0 versus 36.8±13.1, n=6, P=NS; BNP: 10.3±3.4 versus 32.5±10.0, n=6, P<.05; CNP: 17.2±3.4 versus 52.8±8.3, n=6, P<.05).
In the present study, we have shown that high glucose can induce hyperplasia and hypertrophy of VSMCs in culture, confirming the previous report of high glucose–induced mitogenic and hypertrophic actions.23 Furthermore, we have demonstrated that PKC(19-36), a specific PKC inhibitor, prevents high glucose–induced hyperplasia and hypertrophy of VSMCs in a concentration-dependent manner. It has been reported that PKC(19-36) inhibits PKC with an IC50 (inhibitor concentration required to produce 50% inhibition of peptide substance phosphorylation at the Km concentration) of 0.18±0.02 μmol/L although it is a poor inhibitor of cAMP-dependent protein kinase (IC50=423±67 μmol/L) and a moderate inhibitor of myosin light chain kinase (IC50=24±2 μmol/L).24 It has also been reported that plasma glucose levels were 29.8±2.6 mmol/L in streptozotocin-induced diabetic rats.25 Thus, it seems plausible that PKC(19-36) may prevent vascular hypertrophy and hyperplasia in vivo.
We have shown in the present study that long-term treatment with high glucose increases membrane-bound PKC activity in VSMCs. Cultured VSMCs of bovine,12 porcine,23 and rat26 origin grown in the presence of high-glucose media have been shown to exhibit increased activity of membrane-bound PKC. This has been attributed to an increased formation of diacylglycerol from glycolytic intermediates such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.12 In the present study, we have demonstrated for the first time that long-term high-glucose treatment increases phospholipase D activity in VSMCs. It has been reported that phospholipase D activates membrane-bound PKC.27 Therefore, a possible pathway for activated PKC in high glucose–treated cells other than the increased glycolytic metabolites12 has been postulated. It has been reported that PKC activates phospholipase D.28 The fact that the PKC inhibitor peptide PKC(19-36) (1 μmol/L), which completely prevented PKC activation by high glucose (Fig 7⇑), failed to inhibit the increased phospholipase D activity by high glucose suggests that this phospholipase D activation is not secondary to PKC activation. In fact, it has been reported that the α-isoform of PKC is responsible for phospholipase D activation.29 30 It has been also reported that high glucose does not increase the α-isoform of PKC but activates the βII isoform of PKC.31 Therefore, it is possible that the activated βII isoform of PKC does not activate phospholipase D.
We have also demonstrated that elevated glucose decreases ANP-A (stimulated by ANP or BNP) and ANP-B (stimulated by CNP) receptor responses in VSMCs in culture. We have already reported that PKC decreases biologically active ANP-A receptor responses in VSMCs.11 In the present study, the PKC inhibitor peptide significantly reversed glucose-induced suppression of ANP-A and ANP-B receptor responses. Therefore, it is reasonable to assume that high glucose increases PKC activity, which decreases ANP-A and ANP-B receptor responses, as observed in pressor hormone receptors such as angiotensin II and arginine vasopressin.32 This finding may explain the fact that in the vascular bed of individuals with diabetes mellitus, the in vivo vasodilator response to ANP as well as regional production of cGMP is specifically attenuated.33
From the results of the present study, we have postulated involvement of PKC in cell growth and proliferation. However, the role of PKC in VSMC hyperplasia is controversial. It has been reported that PMA stimulated DNA synthesis of quiescent VSMCs from cell line,34 carotid artery,35 pulmonary artery,36 and rat aorta.37 On the other hand, it has been reported that PMA did not stimulate38 or inhibit DNA synthesis of quiescent VSMCs from rabbit aorta.39 This is partly because PMA is used to stimulate PKC. Prolonged incubation with phorbol esters leads to downregulation of PKC activity, making studies with these esters difficult to interpret. However, the published results obtained with synthetic inhibitors of PKC show a clearer picture. H-7 inhibited the restimulation of quiescent rat aortic VSMCs by both PDGF and epidermal growth factor.37 Staurosporine was effective in quiescent rabbit aortic VSMCs restimulated with serum,40 and K252a had similar effects in bovine carotid arterial VSMCs.35 Therefore, it is tempting to assume that long-term PKC activation stimulates hyperplasia of VSMCs. Recently, we have demonstrated that an aldose reductase inhibitor, epalrestat, prevents high glucose–induced hyperproliferation and hypertrophy, possibly through PKC suppression.41 The present study provides the basis of the prevention of high glucose–induced enhancement of vascular growth by aldose reductase inhibitor.
We studied the possible mechanism of this hyperplasia. Glucose itself at 22.2 mmol/L without growth factors did not change the cell cycle (data not shown). However, 1 ng/mL PDGF significantly changed the cell cycle from the G0-G1 stage to S and G2-M stages, suggesting that PDGF is a competent growth factor.42 Glucose at 22.2 mmol/L with 1 ng/mL PDGF significantly increased VSMCs in the S and G2-M stages, suggesting that glucose is not a competent growth factor but a progression growth factor. The PKC inhibitor PKC(19-36) at 1 μmol/L inhibited this increase. Since PKC(19-36) decreases membrane-bound PKC activity, it is plausible to speculate that the high glucose–induced change in the cell cycle is due to PKC activation, which is blocked by a PKC inhibitor.
The physiological significance of this high glucose–induced decrease in ANP receptor responses remains to be elucidated. It has been reported that ANP not only inhibits the action of endogenous vasoconstrictors18 43 but also inhibits hypertrophy and proliferation of VSMCs.10 It has also been reported that high glucose may increase DNA and protein synthesis, which causes vascular hypertrophy and proliferation.21 The fact that the glucose-induced suppression of the natriuretic peptide response was aggravated after the second passage also suggests that high glucose may affect DNA. Indeed, ANP is reported to inhibit both proliferation of VSMCs stimulated by PDGF44 and hypertrophy of VSMCs stimulated by angiotensin II.10 Therefore, it is possible that the suppressed response of ANP receptors may cause vascular hyperproliferation and hypertrophy, although we do not have any direct evidence for this.
In conclusion, a possible involvement of phospholipase D and PKC has been shown in vascular hyperproliferation and hypertrophy induced by high glucose. ANP-A and ANP-B receptor responses are suppressed in VSMCs grown in a high glucose concentration. This suppression may be caused by increased PKC activity and play some role in accelerated vascular growth by high glucose.
Selected Abbreviations and Acronyms
|ANP||=||atrial natriuretic peptide|
|BNP||=||brain natriuretic peptide|
|CNP||=||C-type natriuretic peptide|
|DMEM||=||Dulbecco's modified Eagle's medium|
|FCS||=||fetal calf serum|
|PDGF||=||platelet-derived growth factor|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
|VSMC||=||vascular smooth muscle cell|
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture; Osaka City University Medical Research Foundation; Japan Research Foundation for Chronic Diseases and Rehabilitation (RFCDR-Japan); and ONO Medical Research Foundation. We would like to thank Atsumi Ohnishi for excellent technical assistance.
A part of this study was presented at the 49th Annual Fall Conference of the Council for High Blood Pressure Research, New Orleans, Louisiana, September 19-22, 1995.
- Received January 4, 1996.
- Revision received January 29, 1996.
- Revision received April 10, 1996.
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