all subjects gave written informed consent , and the study was approved by the local ethics committee . monocytes were separated using superparamagnetic polystyrene beads coated with a primary monoclonal antibody specific for the cd14 membrane antigen expressed on human monocytes ( invitrogen , groningen , germany ) and resuspended in hanks balanced solution containing ( in mmol / l ) nacl 136 , kcl 5.40 , cacl2 1 , kh2po4 0.44 , na2hpo4 0.34 , hepes 10 , ph 7.4 . monocytes were counted in neubauer chamber and adjusted in each experiment . to evaluate the effects of high glucose and oxidative stress on trp channel expression , monocytes were exposed to 5.6 mmol / l d - glucose ( control ) , 30 mmol / l d - glucose , or 100 mol / l peroxynitrite ( onoo ) for 4 h. additional experiments were performed using 30 mmol / l l - glucose or 30 mmol / l d - glucose in the presence of the superoxide dismutase mimetic tempol ( tmp , 100 mol / l ) . we also evaluated the effects of lipopolysaccharide - induced oxidative stress , tumor necrosis factor- ( tnf-)-induced activation of nadph oxidase , and production of superoxide radicals as well as reduction of superoxide radicals using diphenylene iodonium . next , the effects of selective inhibition of phosphatidylcholine - specific phospholipase c by tricyclodecan-9-yl xanthogenate ( d609 ) or the inhibition of phospholipase c activation by 1-[6-[((17)-3-methoxyestra-1,3,5 -trien-17-yl)amino[hexyl]-1h - pyrrole-2,5-dione ( u73122 ) on trp expression in monocytes . total rna was isolated from monocytes using the rneasy mini kit including rnase - free dnase set ( qiagen , hilden , germany ) . using the transcriptor first - strand cdna synthesis kit ( roche diagnostics , mannheim , germany ) , cdna was synthesized from 2 g of total rna using oligo dt ( 1218 ) and 5 units avian myeloblastosis virus reverse transcriptase at 50c for 60 min , followed by heating to 85c for 5 min . quantitative ( q ) real - time rt - pcrs for trpc1 , trpc3 , trpc5 , trpc6 , trp melastatin type 6 ( trpm6 ) , trpm7 , tnf- , and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) were performed using a lightcycler - faststart dna master sybr green i kit ( roche diagnostics ) . the primers were as follows : trpc1 ( reference sequence [ refseq ] database accession number : nm_003304.4 ) , forward , 5tgcttaccaaactgctggtg3 ; reverse , 5aactgttttgccgtttgacc3. trpc3 ( nm_003305 ) , forward , 5gactttgggatgctgtccat3 ; reverse , 5gtacgcaatccgagagaagc3. trpc5 ( nm_012471.2 ) , forward , 5ccaccagctatcagataagg3 ; reverse , 5cgaaacaagccacttatacc3. trpc6 ( nm_004621 ) , forward , 5gccaatgagcatctggaaat3 ; reverse , 5tggagtcacatcatgggaga3. trpm6 ( nm_017662.4 ) , forward , 5tgattcctctcggagtgaacagcac3 ; reverse , 5ttatgattggcacctggagtccttg3. trpm7 ( nm_017672.3 ) , forward , 5cttatgaagaggcaggtcatgg3 ; reverse , 5catcttgtctgaaggactg3. tnf- ( nm_000594.2 ) , forward , 5cccagggacctctctctaatc3 ; reverse , 5atgggctacaggcttgtcact3. gapdh ( nm_002046 ) , forward , 5aactgcttagcacccctggc3 ; reverse , 5atgaccttgcccacagcctt3. the expected and observed sizes of the pcr products were ( in bp ) trpc1 243 ; trpc3 249 ; trpc5 161 ; trpc6 243 ; trpm6 347 ; trpm7 214 ; tnf- 84 ; and gapdh 200 . lightcycler - faststart dna master sybr green i kit and 500 nmol / l of each primer were used in a final volume of 20 l . the reaction was initiated at 95c for 10 min , followed by 40 cycles of denaturation at 95c for 10 s ; annealing for 10 s at 68c ( trpm6 ) , at 60c ( trpc3 , trpm7 , tnf- ) , at 57c ( trpc1 , trpc6 ) , or at 54c ( trpc5 , gapdh ) ; and extension at 72c for 15 s. melting curve analysis was performed from 65c to 95c with a heating rate of 0.1c / s . data were recorded on a lightcycler 2.0 instrument using lightcycler software version 4.0 ( roche diagnostics ) . the relative quantification method was used whereby the change in expression of the target genes ( trpc1 , trpc3 , trpc5 , trpc6 , trpm6 , trpm7 , tnf- ) relative to the housekeeping gene ( gapdh ) was calculated . for the identification of trpc channel proteins , quantitative in - cell western assays of human monocytes were performed using the odyssey infrared imaging system ( licor biosciences , bad homburg , germany ) . human monocytes in 96-well plates were incubated with rabbit anti - human trpc3 or trpc6 antibodies ( 1:1,000 ; alomone labs , jerusalem , israel ) for 2 h , washed , and incubated with irdye800-infrared fluorescent dye - conjugated sheep anti - rabbit antibodies ( 1:1,000 ; biomol , hamburg , germany ) overnight at 4c . conventional immunoblotting was performed as described previously by our group ( 13 ) . for ratiometric imaging experiments , mol / l of the calcium - sensitive , cell - permeable , intracellular fluorescence dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methyl - phenoxy)-ethane - n , n , nn-tetra acetic acid penta acetoxymethyl ester ( fura-2/am ; merck biosciences , darmstadt , germany ) at room temperature for 60 min and washed to remove extraneous dye . fluorescence measurements were performed in a temperature - controlled 96-well fluorescent plate reader at 37c ( fluoroskan ascent fluorometer ; thermo labsystems oy , helsinki , finland ) at 510-nm emission with excitation wavelengths of 340 and 380 nm . baseline fluorescence was measured for 10 min , and stable fluorescence readings were obtained throughout . calcium influx was activated after administration of membrane - permeable 1-oleoyl-2-acetyl - sn - glycerol . we used the membrane - permeable 2-aminoethoxydiphenylborane ( 2-apb ) to inhibit calcium influx through trpc channels as previously described ( 15,16 ) . monocytes were incubated with the dye 2,7-dichlorofluorescein diacetate ( dcf - da , 50 mol / l ) for 60 min and then were washed and resuspended in hanks ' balanced salt solution . dcf - da is a nonpolar compound that readily diffuses into cells , where it is hydrolyzed to the nonfluorescent polar derivative dcfh and thereby trapped within the cells . in the presence of reactive oxygen species , dcfh is oxidized to the highly fluorescent dcf , which was monitored spectrophotometrically at 530 nm with an excitation wavelength of 485 nm . comparisons between groups were analyzed using nonparametric mann - whitney test ( graphpad prism 5.0 ; graphpad software , la jolla , ca ) . data from multiple groups were analyzed using the nonparametric kruskal - wallis and dunn multiple comparison post hoc test . where error bars do not appear on the figure , error was within the symbol size . all subjects gave written informed consent , and the study was approved by the local ethics committee . monocytes were separated using superparamagnetic polystyrene beads coated with a primary monoclonal antibody specific for the cd14 membrane antigen expressed on human monocytes ( invitrogen , groningen , germany ) and resuspended in hanks balanced solution containing ( in mmol / l ) nacl 136 , kcl 5.40 , cacl2 1 , kh2po4 0.44 , na2hpo4 0.34 , hepes 10 , ph 7.4 . monocytes were counted in neubauer chamber and adjusted in each experiment . to evaluate the effects of high glucose and oxidative stress on trp channel expression , monocytes were exposed to 5.6 mmol / l d - glucose ( control ) , 30 mmol / l d - glucose , or 100 mol / l peroxynitrite ( onoo ) for 4 h. additional experiments were performed using 30 mmol / l l - glucose or 30 mmol / l d - glucose in the presence of the superoxide dismutase mimetic tempol ( tmp , 100 mol / l ) . we also evaluated the effects of lipopolysaccharide - induced oxidative stress , tumor necrosis factor- ( tnf-)-induced activation of nadph oxidase , and production of superoxide radicals as well as reduction of superoxide radicals using diphenylene iodonium . next , the effects of selective inhibition of phosphatidylcholine - specific phospholipase c by tricyclodecan-9-yl xanthogenate ( d609 ) or the inhibition of phospholipase c activation by 1-[6-[((17)-3-methoxyestra-1,3,5 -trien-17-yl)amino[hexyl]-1h - pyrrole-2,5-dione ( u73122 ) on trp expression in monocytes . total rna was isolated from monocytes using the rneasy mini kit including rnase - free dnase set ( qiagen , hilden , germany ) . using the transcriptor first - strand cdna synthesis kit ( roche diagnostics , mannheim , germany ) , cdna was synthesized from 2 g of total rna using oligo dt ( 1218 ) and 5 units avian myeloblastosis virus reverse transcriptase at 50c for 60 min , followed by heating to 85c for 5 min . quantitative ( q ) real - time rt - pcrs for trpc1 , trpc3 , trpc5 , trpc6 , trp melastatin type 6 ( trpm6 ) , trpm7 , tnf- , and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) were performed using a lightcycler - faststart dna master sybr green i kit ( roche diagnostics ) . the primers were as follows : trpc1 ( reference sequence [ refseq ] database accession number : nm_003304.4 ) , forward , 5tgcttaccaaactgctggtg3 ; reverse , 5aactgttttgccgtttgacc3. trpc3 ( nm_003305 ) , forward , 5gactttgggatgctgtccat3 ; reverse , 5gtacgcaatccgagagaagc3. trpc5 ( nm_012471.2 ) , forward , 5ccaccagctatcagataagg3 ; reverse , 5cgaaacaagccacttatacc3. trpc6 ( nm_004621 ) , forward , 5gccaatgagcatctggaaat3 ; reverse , 5tggagtcacatcatgggaga3. trpm6 ( nm_017662.4 ) , forward , 5tgattcctctcggagtgaacagcac3 ; reverse , 5ttatgattggcacctggagtccttg3. trpm7 ( nm_017672.3 ) , forward , 5cttatgaagaggcaggtcatgg3 ; reverse , 5catcttgtctgaaggactg3. tnf- ( nm_000594.2 ) , forward , 5cccagggacctctctctaatc3 ; reverse , 5atgggctacaggcttgtcact3. gapdh ( nm_002046 ) , forward , 5aactgcttagcacccctggc3 ; reverse , 5atgaccttgcccacagcctt3. the expected and observed sizes of the pcr products were ( in bp ) trpc1 243 ; trpc3 249 ; trpc5 161 ; trpc6 243 ; trpm6 347 ; trpm7 214 ; tnf- 84 ; and gapdh 200 . lightcycler - faststart dna master sybr green i kit and 500 nmol / l of each primer were used in a final volume of 20 l . the reaction was initiated at 95c for 10 min , followed by 40 cycles of denaturation at 95c for 10 s ; annealing for 10 s at 68c ( trpm6 ) , at 60c ( trpc3 , trpm7 , tnf- ) , at 57c ( trpc1 , trpc6 ) , or at 54c ( trpc5 , gapdh ) ; and extension at 72c for 15 s. melting curve analysis was performed from 65c to 95c with a heating rate of 0.1c / s . data were recorded on a lightcycler 2.0 instrument using lightcycler software version 4.0 ( roche diagnostics ) . the relative quantification method was used whereby the change in expression of the target genes ( trpc1 , trpc3 , trpc5 , trpc6 , trpm6 , trpm7 , tnf- ) relative to the housekeeping gene ( gapdh ) was calculated . for the identification of trpc channel proteins , quantitative in - cell western assays of human monocytes were performed using the odyssey infrared imaging system ( licor biosciences , bad homburg , germany ) . human monocytes in 96-well plates were incubated with rabbit anti - human trpc3 or trpc6 antibodies ( 1:1,000 ; alomone labs , jerusalem , israel ) for 2 h , washed , and incubated with irdye800-infrared fluorescent dye - conjugated sheep anti - rabbit antibodies ( 1:1,000 ; biomol , hamburg , germany ) overnight at 4c . for ratiometric imaging experiments , monocytes were loaded with 2 mol / l of the calcium - sensitive , cell - permeable , intracellular fluorescence dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methyl - phenoxy)-ethane - n , n , nn-tetra acetic acid penta acetoxymethyl ester ( fura-2/am ; merck biosciences , darmstadt , germany ) at room temperature for 60 min and washed to remove extraneous dye . fluorescence measurements were performed in a temperature - controlled 96-well fluorescent plate reader at 37c ( fluoroskan ascent fluorometer ; thermo labsystems oy , helsinki , finland ) at 510-nm emission with excitation wavelengths of 340 and 380 nm . baseline fluorescence was measured for 10 min , and stable fluorescence readings were obtained throughout . calcium influx was activated after administration of membrane - permeable 1-oleoyl-2-acetyl - sn - glycerol . we used the membrane - permeable 2-aminoethoxydiphenylborane ( 2-apb ) to inhibit calcium influx through trpc channels as previously described ( 15,16 ) . monocytes were incubated with the dye 2,7-dichlorofluorescein diacetate ( dcf - da , 50 mol / l ) for 60 min and then were washed and resuspended in hanks ' balanced salt solution . dcf - da is a nonpolar compound that readily diffuses into cells , where it is hydrolyzed to the nonfluorescent polar derivative dcfh and thereby trapped within the cells . in the presence of reactive oxygen species , dcfh is oxidized to the highly fluorescent dcf , which was monitored spectrophotometrically at 530 nm with an excitation wavelength of 485 nm . comparisons between groups were analyzed using nonparametric mann - whitney test ( graphpad prism 5.0 ; graphpad software , la jolla , ca ) . data from multiple groups were analyzed using the nonparametric kruskal - wallis and dunn multiple comparison post hoc test . where error bars do not appear on the figure , error was within the symbol size . the induction of reactive oxygen species by high glucose was investigated using the dye dcf - da . compared with control conditions ( d - glucose , 5.6 mmol / l ) , the administration of 30 mmol / l d - glucose for 90 min significantly increased reactive oxygen species from 1.0  0.01 to 1.84  0.17 arbitrary units [ au ] ( p < 0.01 ) . furthermore , the effect of d - glucose was blocked by the concurrent administration of the superoxide dismutase mimetic tmp ( 100 mol / l ) to 0.78  0.07 au ( p < 0.01 compared with d - glucose alone ) . to investigate whether high glucose  induced oxidative stress may affect trp channels , we first analyzed trp mrna in human monocytes from healthy control subjects using qrt - pcr . the melting curve analysis showed the presence of one single peak in monocytes after 4-h treatment under control conditions , with high glucose or onoo . the expected and observed product sizes using gel electrophoresis were ( in bp ) trpc1 243 ; trpc3 249 ; trpc5 161 ; trpc6 243 ; trpm6 347 ; trpm7 214 ; and gapdh 200 . we determined the effects of high glucose and oxidative stress on trpc3 and trpc6 mrna in human monocytes . as shown in fig . 1 , compared with control conditions with 5.6 mmol / l d - glucose , administration of high d - glucose ( 30 mmol / l ) significantly increased trpc3 mrna by 1.55-fold ( p < 0.01 ; n = 6 ) and trpc6 mrna by 2.58-fold ( p < 0.01 ; n = 12 ) . the administration of high l - glucose ( 30 mmol / l ) did not significantly affect trpc3 or trpc6 mrna . concurrent administration of superoxide dismutase mimetic tmp ( final concentration 100 mol / l ) blocked the stimulating effect of high d - glucose on trpc3 and trpc6 mrna expression , indicating that high d - glucose causes its effects by increased oxidative stress . compared with control conditions , oxidative stress induced by 100 mol / l onoo also significantly increased the trpc3 mrna by 1.77-fold ( p < 0.01 ; n = 15 ) and trpc6 mrna by 3.47-fold ( p < 0.01 ; n = 11 ) . changes of mrna expression of ( a ) trpc3 , ( b ) trpc6 , ( c and d ) trpc1 , trpc5 , trpm6 , trpm7 , and tnf- induced by high glucose and onoo . exposure of monocytes to high glucose ( hg ; 30 mmol / l d - glucose ) or onoo ( 100 mol / l ) for 4 h significantly increased the trpc3 and trpc6 mrna expression . the effect of high glucose is attenuated by superoxide dismutase mimetic tmp ( 100 mol / l ) . control ( open bar ) indicates control glucose ( 5.6 mmol / l d - glucose ) . data are mean  sem from at least four to six independent experiments . * p < 0.05 ; * * p < 0.01 compared with control conditions . e : bar graph showing trpc3 mrna and trpc6 mrna expression ( normalized ratio ) in monocytes from 28 age - matched control subjects ( open bars ) and from 18 patients with type 2 diabetes ( filled bars ) . * p < 0.05 compared with control . the administration of high d - glucose significantly increased mrna from trpc1 , trpc5 , trpm6 , or trpm7 by 1.81- , 3.10- , 7.22- , or 7.99-fold compared with control conditions , respectively ( each p < 0.05 ; n = 4 ) . the administration of onoo significantly increased mrna from trpc1 , trpc5 , trpm6 , or trpm7 by 4.57- , 3.53- , 12.91- , or 3.51-fold , respectively ( each p < 0.05 ; n = 4 ) . we now investigated whether upregulated trp expression was associated with elevated monocyte inflammatory gene expression . indeed , after the administration of high d - glucose or onoo and upregulation of trp channels , mrna of the inflammatory cytokine tnf- was also significantly increased by 6.11- or 2.32-fold ( each p < 0.05 ; next , we evaluated whether induction of oxidative stress by several pathways may affect trpc expression . induction of oxidative stress using lipopolysaccharide significantly increased trpc3 mrna expression in monocytes from 1.00  0.48 to 9.60  0.40 ( each n = 5 ; p < 0.05 ) and trpc6 mrna expression from 1.00  0.23 to 3.90  0.11 ( each n = 5 ; p < 0.05 ) . it is known that tnf-induced activation of nadph oxidase increases superoxide radicals ( 17 ) . tnf- significantly increased trpc3 mrna expression in monocytes from 1.00  0.01 to 1.58  0.21 ( each n = 4 ; p < 0.05 ) and trpc6 mrna expression from 1.00  0.00 to 1.32  0.08 ( each n = 4 ; p < 0.05 ) . in agreement with these findings , the reduction of superoxide radicals using diphenylene iodonium ( 18 ) significantly reduced trpc3 mrna expression in monocytes from 1.00  0.06 to 0.52  0.05 ( each n = 5 ; p < 0.05 ) and trpc6 mrna expression from 1.00  0.12 to 0.36  0.03 ( each n = 5 ; p < 0.05 ) . these data indicate that trpc3 and trpc6 mrna are regulated by oxidative stress , in particular by superoxide radicals . we also investigated a possible role of phospholipase c on glucose - induced trpc expression . compared with control conditions , the inhibition of phosphatidylcholine - specific phospholipase c by d609 , or the inhibition of phospholipase c activation by u73122 , did not significantly affect high d - glucose  induced trpc3 mrna expression ( control 1.00  0.01 ; high d - glucose 1.57  0.10 [ p < 0.05 compared with control ] ; high d - glucose plus d609 1.47  0.03 [ not significant compared with high d - glucose alone ] ; high d - glucose plus u73122 1.95  0.03 [ not significant compared with high d - glucose alone ] ; each n = 4 ) . it is known that trpc - induced calcium influx is regulated by phospholipase c. however , phospholipase c seems not to be involved in glucose - induced upregulation of trpc mrna expression . to evaluate whether increased trpc mrna is a feature of diabetes , we compared trpc1 , trpc3 , trpc5 , and trpc6 mrna in monocytes from 18 patients with type 2 diabetes ( 10 women , 8 men ; mean age 57  5 years ; systolic blood pressure 133  3 mmhg ; diastolic blood pressure 79  2 mmhg ; serum sodium 135  1 mmol / l ; serum potassium 4.4  0.1 mmol / l ; hemoglobin 11.2  0.5 g / dl ; a1c 7.5  0.3% ) and from 28 age - matched control subjects ( 10 women , 18 men ; mean age 56  4 years ; systolic blood pressure 132  4 mmhg ; diastolic blood pressure 81  2 mmhg ; serum sodium 136  1 mmol / l ; serum potassium 4.4  0.2 mmol / l ; hemoglobin 12.3  0.5 g / dl ; a1c 5.6  0.1% ) . trpc6 mrna was significantly higher in monocytes from patients with type 2 diabetes compared with control subjects ( trpc6 normalized ratio 0.028  0.014 [ n = 18 ] vs. 0.015  0.006 [ n = 28 ] ; p < 0.05 ; fig . on the other hand , trpc1 , trpc3 , or trpc5 mrna was not significantly different between the two groups ( trpc1 0.025  0.012 vs. 0.025  0.006 [ p = 0.62 ] ; trpc3 0.010  0.005 vs. 0.003  0.001 [ p = 0.40 ] ; trpc5 0.007  0.004 vs. 0.003 these data support the view that increased trpc6 expression is a characteristic feature of peripheral blood cells from patients with type 2 diabetes . trpc protein expression in monocytes was quantified using a quantitative in - cell western assay . as shown in fig . 2 , compared with control conditions with 5.6 mmol / l d - glucose , administration of high d - glucose ( 30 mmol / l ) significantly increased trpc3 protein expression by 1.58-fold ( p < 0.01 ; n = 10 ) and trpc6 protein expression by 1.57-fold ( p < 0.01 ; n = 8) . the administration of high l - glucose ( 30 mmol / l ) did not significantly affect trpc3 or trpc6 protein expression . concurrent administration of tmp ( 100 mol / l ) blocked the stimulating effect of high d - glucose on trpc3 and trpc6 protein expression . quantitative in - cell western assay of trpc3 and trpc6 channel protein expression in monocytes treated with hg ( 30 mmol / l d - glucose ) in the absence and presence of tmp ( 100 mol / l ) , onoo ( 100 mol / l ) , l - glucose ( 30 mmol / l ) , or control ( 5.6 mmol / l d - glucose ) . a : representative conventional western assay ( trpc3 protein and gapdh protein for loading control with top values indicating densitometric analysis of blots ) . b and d : representative in - cell western assays and ( c and e ) summary data are shown . data are means  sem from at least eight independent experiments . * * p < 0.01 compared with control conditions . compared with control conditions , oxidative stress induced by 100 mol / l onoo also significantly increased the trpc3 protein expression by 1.70-fold ( p < 0.01 ; n = 9 ) and trpc6 protein expression by 1.8-fold ( p < 0.01 ; n = 9 ) . 1-oleoyl-2-acetyl - rac - glycerol ( oag ) increased cytosolic calcium to 2.41  0.01 ( n = 15 ) , whereas oag in the presence of high d - glucose increased cytosolic calcium to 2.79  0.03 ( n = 14 ; p < 0.01 ) . furthermore , it should be noted that concurrent preincubation of cells with tmp ( 100 mol / l ) and high d - glucose normalized oag - induced calcium influx ( 2.49  0.02 ; n = 13 ; not significant compared with control ) . these data indicate that high d - glucose  associated oxidative stress promotes increased trpc - mediated calcium influx . recordings of fura-2 fluorescence in human monocytes . a : oag - induced calcium influx was measured under control conditions ( 5.6 mmol / l d - glucose ,  ) and in the presence of high glucose ( 30 mmol / l d - glucose ) without (  ) and with (  ) tmp ( 100 mol / l ) . b : summary data showing intracellular calcium levels after treatment of monocytes with high d - glucose for 4 h ( control ) stimulated with onoo in the absence and presence of the membrane - permeable trpc blocker , 2-apb . * * p < 0.01 . after treatment of monocytes with high d - glucose for 4 h , the administration of onoo subsequently increased intracellular calcium levels . in the presence of a membrane - permeable trpc blocker , 2-apb , the onoo - induced calcium increase was significantly attenuated ( 3.99  0.05 vs. 4.56  0.04 ; each n = 15 ; p < 0.01 compared with onoo alone ; fig . the induction of reactive oxygen species by high glucose was investigated using the dye dcf - da . compared with control conditions ( d - glucose , 5.6 mmol / l ) , the administration of 30 mmol / l d - glucose for 90 min significantly increased reactive oxygen species from 1.0  0.01 to 1.84  0.17 arbitrary units [ au ] ( p < 0.01 ) . furthermore , the effect of d - glucose was blocked by the concurrent administration of the superoxide dismutase mimetic tmp ( 100 mol / l ) to 0.78  0.07 au ( p < 0.01 compared with d - glucose alone ) . induced oxidative stress may affect trp channels , we first analyzed trp mrna in human monocytes from healthy control subjects using qrt - pcr . the melting curve analysis showed the presence of one single peak in monocytes after 4-h treatment under control conditions , with high glucose or onoo . the expected and observed product sizes using gel electrophoresis were ( in bp ) trpc1 243 ; trpc3 249 ; trpc5 161 ; trpc6 243 ; trpm6 347 ; trpm7 214 ; and gapdh 200 . we determined the effects of high glucose and oxidative stress on trpc3 and trpc6 mrna in human monocytes . as shown in fig . 1 , compared with control conditions with 5.6 mmol / l d - glucose , administration of high d - glucose ( 30 mmol / l ) significantly increased trpc3 mrna by 1.55-fold ( p < 0.01 ; n = 6 ) and trpc6 mrna by 2.58-fold ( p < 0.01 ; n = 12 ) . the administration of high l - glucose ( 30 mmol / l ) did not significantly affect trpc3 or trpc6 mrna . concurrent administration of superoxide dismutase mimetic tmp ( final concentration 100 mol / l ) blocked the stimulating effect of high d - glucose on trpc3 and trpc6 mrna expression , indicating that high d - glucose causes its effects by increased oxidative stress . compared with control conditions , oxidative stress induced by 100 mol / l onoo also significantly increased the trpc3 mrna by 1.77-fold ( p < 0.01 ; n = 15 ) and trpc6 mrna by 3.47-fold ( p < 0.01 ; n = 11 ) . changes of mrna expression of ( a ) trpc3 , ( b ) trpc6 , ( c and d ) trpc1 , trpc5 , trpm6 , trpm7 , and tnf- induced by high glucose and onoo . exposure of monocytes to high glucose ( hg ; 30 mmol / l d - glucose ) or onoo ( 100 mol / l ) for 4 h significantly increased the trpc3 and trpc6 mrna expression . the effect of high glucose is attenuated by superoxide dismutase mimetic tmp ( 100 mol / l ) . control ( open bar ) indicates control glucose ( 5.6 mmol / l d - glucose ) . data are mean  sem from at least four to six independent experiments . * p < 0.05 ; * * p < 0.01 compared with control conditions . e : bar graph showing trpc3 mrna and trpc6 mrna expression ( normalized ratio ) in monocytes from 28 age - matched control subjects ( open bars ) and from 18 patients with type 2 diabetes ( filled bars ) . * p < 0.05 compared with control . the administration of high d - glucose significantly increased mrna from trpc1 , trpc5 , trpm6 , or trpm7 by 1.81- , 3.10- , 7.22- , or 7.99-fold compared with control conditions , respectively ( each p < 0.05 ; n = 4 ) . the administration of onoo significantly increased mrna from trpc1 , trpc5 , trpm6 , or trpm7 by 4.57- , 3.53- , 12.91- , or 3.51-fold , respectively ( each p < 0.05 ; n = 4 ) . we now investigated whether upregulated trp expression was associated with elevated monocyte inflammatory gene expression . indeed , after the administration of high d - glucose or onoo and upregulation of trp channels , mrna of the inflammatory cytokine tnf- was also significantly increased by 6.11- or 2.32-fold ( each p < 0.05 ; n = 4 ) , respectively . next , we evaluated whether induction of oxidative stress by several pathways may affect trpc expression . induction of oxidative stress using lipopolysaccharide significantly increased trpc3 mrna expression in monocytes from 1.00  0.48 to 9.60  0.40 ( each n = 5 ; p < 0.05 ) and trpc6 mrna expression from 1.00  0.23 to 3.90  0.11 ( each n = 5 ; p < 0.05 ) . it is known that tnf-induced activation of nadph oxidase increases superoxide radicals ( 17 ) . tnf- significantly increased trpc3 mrna expression in monocytes from 1.00  0.01 to 1.58  0.21 ( each n = 4 ; p < 0.05 ) and trpc6 mrna expression from 1.00  0.00 to 1.32  0.08 ( each n = 4 ; p < 0.05 ) . in agreement with these findings , the reduction of superoxide radicals using diphenylene iodonium ( 18 ) significantly reduced trpc3 mrna expression in monocytes from 1.00  0.06 to 0.52  0.05 ( each n = 5 ; p < 0.05 ) and trpc6 mrna expression from 1.00  0.12 to 0.36  0.03 ( each n = 5 ; p < 0.05 ) . these data indicate that trpc3 and trpc6 mrna are regulated by oxidative stress , in particular by superoxide radicals . we also investigated a possible role of phospholipase c on glucose - induced trpc expression . compared with control conditions , the inhibition of phosphatidylcholine - specific phospholipase c by d609 , or the inhibition of phospholipase c activation by u73122 , did not significantly affect high d - glucose induced trpc3 mrna expression ( control 1.00  0.01 ; high d - glucose 1.57  0.10 [ p < 0.05 compared with control ] ; high d - glucose plus d609 1.47  0.03 [ not significant compared with high d - glucose alone ] ; high d - glucose plus u73122 1.95  0.03 [ not significant compared with high d - glucose alone ] ; each n = 4 ) . it is known that trpc - induced calcium influx is regulated by phospholipase c. however , phospholipase c seems not to be involved in glucose - induced upregulation of trpc mrna expression . to evaluate whether increased trpc mrna is a feature of diabetes , we compared trpc1 , trpc3 , trpc5 , and trpc6 mrna in monocytes from 18 patients with type 2 diabetes ( 10 women , 8 men ; mean age 57  5 years ; systolic blood pressure 133  3 mmhg ; diastolic blood pressure 79  2 mmhg ; serum sodium 135  1 mmol / l ; serum potassium 4.4  0.1 mmol / l ; hemoglobin 11.2  0.5 g / dl ; a1c 7.5  0.3% ) and from 28 age - matched control subjects ( 10 women , 18 men ; mean age 56  4 years ; systolic blood pressure 132  4 mmhg ; diastolic blood pressure 81  2 mmhg ; serum sodium 136  1 mmol / l ; serum potassium 4.4  0.2 mmol / l ; hemoglobin 12.3  0.5 g / dl ; a1c 5.6  0.1% ) . trpc6 mrna was significantly higher in monocytes from patients with type 2 diabetes compared with control subjects ( trpc6 normalized ratio 0.028  0.014 [ n = 18 ] vs. 0.015  0.006 [ n = 28 ] ; p < 0.05 ; fig . on the other hand , trpc1 , trpc3 , or trpc5 mrna was not significantly different between the two groups ( trpc1 0.025  0.012 vs. 0.025  0.006 [ p = 0.62 ] ; trpc3 0.010  0.005 vs. 0.003  0.001 these data support the view that increased trpc6 expression is a characteristic feature of peripheral blood cells from patients with type 2 diabetes . trpc protein expression in monocytes was quantified using a quantitative in - cell western assay . as shown in fig . 2 , compared with control conditions with 5.6 mmol / l d - glucose , administration of high d - glucose ( 30 mmol / l ) significantly increased trpc3 protein expression by 1.58-fold ( p < 0.01 ; n = 10 ) and trpc6 protein expression by 1.57-fold ( p < 0.01 ; n = 8) . the administration of high l - glucose ( 30 mmol / l ) did not significantly affect trpc3 or trpc6 protein expression . concurrent administration of tmp ( 100 mol / l ) blocked the stimulating effect of high d - glucose on trpc3 and trpc6 protein expression . quantitative in - cell western assay of trpc3 and trpc6 channel protein expression in monocytes treated with hg ( 30 mmol / l d - glucose ) in the absence and presence of tmp ( 100 mol / l ) , onoo ( 100 mol / l ) , l - glucose ( 30 mmol / l ) , or control ( 5.6 mmol / l d - glucose ) . a : representative conventional western assay ( trpc3 protein and gapdh protein for loading control with top values indicating densitometric analysis of blots ) . the trpc3/gapdh ratio ( fold over control ) is also indicated . b and d : representative in - cell western assays and ( c and e ) summary data are shown . data are means  sem from at least eight independent experiments . * * p < 0.01 compared with control conditions . compared with control conditions , oxidative stress induced by 100 mol / l onoo also significantly increased the trpc3 protein expression by 1.70-fold ( p < 0.01 ; n = 9 ) and trpc6 protein expression by 1.8-fold ( p < 0.01 ; n = 9 ) . 1-oleoyl-2-acetyl - rac - glycerol ( oag ) increased cytosolic calcium to 2.41  0.01 ( n = 15 ) , whereas oag in the presence of high d - glucose increased cytosolic calcium to 2.79  0.03 ( n = 14 ; p < 0.01 ) . furthermore , it should be noted that concurrent preincubation of cells with tmp ( 100 mol / l ) and high d - glucose normalized oag - induced calcium influx ( 2.49  0.02 ; n = 13 ; not significant compared with control ) . these data indicate that high d - glucose  associated oxidative stress promotes increased trpc - mediated calcium influx . a : oag - induced calcium influx was measured under control conditions ( 5.6 mmol / l d - glucose ,  ) and in the presence of high glucose ( 30 mmol / l d - glucose ) without (  ) and with (  ) tmp ( 100 mol / l ) . b : summary data showing intracellular calcium levels after treatment of monocytes with high d - glucose for 4 h ( control ) stimulated with onoo in the absence and presence of the membrane - permeable trpc blocker , 2-apb . * * p < 0.01 . after treatment of monocytes with high d - glucose for 4 h , the administration of onoo subsequently increased intracellular calcium levels . in the presence of a membrane - permeable trpc blocker , 2-apb , the onoo - induced calcium increase was significantly attenuated ( 3.99  0.05 vs. 4.56  0.04 ; each n = 15 ; p < 0.01 compared with onoo alone ; fig . our present data show that high d - glucose  induced oxidative stress causes increased trp expression and calcium influx in human monocytes . an increased calcium influx through trp channels may be responsible for an increased activation of monocytes and enhanced atherosclerosis in patients with diabetes . the presence of high glucose levels is a characteristic feature of diabetes and is considered a major pathogenic factor for increased atherosclerosis and consecutive diseases including cardiovascular disease ( 19 ) . both an increased activation of monocytes and an increased generation of reactive oxygen species in monocytes have been described in patients with diabetes ( 6,20 ) . the production of mitochondrial superoxide has been implicated as a major underlying mechanism linking high glucose and cellular dysfunction ( 21,22 ) . in the present study , we confirmed that high glucose increases reactive oxygen species in monocytes . the high glucose  induced increase of reactive oxygen species was blocked by the superoxide dismutase mimetic tmp . activated monocytes in patients with diabetes are characterized by increased calcium concentrations ( 911 ) . according to our present results , increased trpc channel expression in monocytes may be responsible for an increased calcium influx after high glucose we observed that high d - glucose  induced oxidative stress increased the expression of both trpc3 and trpc6 mrna and trpc3 and trpc6 channel proteins in monocytes . the stimulating effect of high glucose could be blocked by concurrent administration of tmp , supporting the relevance of increased oxidative stress after high d - glucose . recently , shanmugam et al . ( 23 ) showed that exposure of monocytes to high glucose increased the expression of several inflammatory cytokines , including tnf- in an oxidant stress our present results support these findings , indicating that high glucose  induced upregulation of trp expression was associated with enhanced mrna levels of the inflammatory cytokine tnf-. we observed that high glucose elevates the expression of tnf- even higher than onoo . the increased tnf- response after high glucose compared with onoo indicates that irrespective of oxidative stress , other pathways ( e.g. , the polyol pathway ) contribute to increased inflammatory response in diabetes ( 24 ) . induced oxidative stress activates monocytes by enhancing inflammatory cytokines and trp channel expression promoting transmembrane calcium influx . ( 25 ) suggested that trpc channels are the molecular basis of oxidant - activated cation channels in endothelial cells . poteser et al . ( 26 ) reported that redox modulation of trpc channels is responsible for oxidative stress the present study extends their findings , showing that oxidative stress increased the expression of both trpc3 and trpc6 mrna and proteins . induced oxidative stress in patients with diabetes may activate monocytes both by interaction with trpc channels and by enhanced trpc expression . the present study indicates that induction of oxidative stress by several pathways may affect trp expression . induction of oxidative stress using lipopolysaccharide- or tnf-induced activation of nadph oxidase significantly increased trpc3 mrna expression , whereas the reduction of superoxide radicals using diphenylene iodonium significantly reduced trpc3 mrna expression . in the present study , we also confirmed that high d - glucose  induced oxidative stress increases trpm6 and trpm7 mrna expression in agreement with data from yamamoto et al . ( 27 ) , who showed the regulation of trpm expression by oxidative stress . to evaluate whether increased trpc mrna is a feature of diabetes , we also compared trpc mrna in monocytes from patients with type 2 diabetes and control subjects . we observed increased trpc6 mrna in monocytes from patients with type 2 diabetes . increased trpc6 protein expression has already been reported in platelets from patients with diabetes compared with control subjects ( 28 ) , supporting the view that increased trpc6 expression is a characteristic feature of peripheral blood cells from patients with diabetes . furthermore , hu et al . ( 29 ) showed increased trpc6 expression in the adrenal medulla from ossabaw miniature pigs with pre - diabetic metabolic syndrome . however , differences might be explained in part by use of native blood cells and cultured cells . in our study , however , several pathways including phospholipase c , protein kinase c , or phosphatidylinositol 3-kinase may modulate trp channel protein expression and function , as suggested by recent literature ( 3133 ) . in conclusion , the present study shows that high glucose induced oxidative stress increases trpc3 and trpc6 channel expression and calcium influx in human monocytes . these data point to a novel pathway for an increased activation of monocytes and hence atherosclerosis in patients with diabetes .