Table 3.

Effects of d-glucose and PMA or PKC activity in the cytosol and membrane fractions from human umbilical vein endothelial cells

	5 mm d-glucose		25 mm d-glucose
Conditions	Cytosol	Membrane	Cytosol	Membrane
Control	123 ± 13	27 ± 13*	55 ± 12*	147 ± 20‡
PMA (100 nm)	61 ± 5*	189 ± 11‡	49 ± 12*	149 ± 12‡
Calphostin C(100 nm)	107 ± 23	17 ± 6	131 ± 25§	43 ± 16§
PMA + calphostin C	146 ± 29†	17 ± 12†	150 ± 32§	55 ± 11§
4α-PDD (100 nm)	117 ± 22†	32 ± 12†	39 ± 19*	169 ± 27‡
l-NAME (100 μm)	145 ± 25†	19 ± 7*	27 ± 2*	187 ± 25‡
l-NAME + PMA	24 ± 9*	170 ± 22‡	35 ± 31*	145 ± 10‡
SNAP (100 μm)	107 ± 38	27 ± 12*	47 ± 14*	127 ± 12‡
SNAP + PMA	55 ± 15*	191 ± 27‡	36 ± 9*	173 ± 13‡
SNAP + l-NAME	143 ± 8*	16 ± 34*	41 ± 12*	182 ± 10‡
PD-98059 (10 μm)	116 ± 8†	17 ± 14†	15 ± 12*	147 ± 13‡
PD-98059 + PMA	17 ± 32*	197 ± 17‡	37 ± 12*	269 ± 64‡
PD-98059 + l-NAME	117 ± 27†	32 ± 11†	59 ± 13*	178 ± 41‡
PD-98059 + l-NAME+SNAP	129 ± 17†	22 ± 10†	39 ± 14*	161 ± 32‡

Cytosolic and membrane fractions of HUVECs were prepared from endothelial cell monolyaers cultured for 24 h in M199 containing 5 or 25 mm d-glucose (as described in Methods). The activity of PKC was estimated as the difference between ³²P information from [γ⁻³²P]ATP into a synthetic glycogen synthase PKC substrate peptide in the presence of CACl₂-phosphatidylserine-diolein vs. EGTA (Castro et al. 1998). Experiments were performed in endothelial cells exposed to M199 (Control) or M199 contaning different molecules for 30 min at the indicated concentrations. PMA, phorbol 12-myristate 13-acetate; 4α-PDD, 4α-phorbol 12, 13-didecanoate; l-NAME, N^G-nitro-L-arginine methyl ester; SNAP, S-nitroso-N-acetyl-l,d-penicillamine. Values are in pmol (mg protein)⁻¹ min⁻¹; means +/- s.e.m., n = 3-4 different cell cultures.

^*P < 0.05 vs. control values for cytosol fraction in 5 mm d-glucose

^†P < 0.04 vs. values in PMA-treated cells

^‡P < 0.05 vs. control values for membrane fraction in 5 mm d-glucose

^§P < 0.05 vs. corresponding control values in 25 mM D-glucose.