Expression of RhoA, Rho kinase activity and muscle contraction in diabetes and hyperglycemia.

<p>Smooth muscle of the fundus of wild type (WT) and ob/ob mice and smooth muscle from WT mice treated with 5.5 mM (NG) or 30 mM glucose (HG) for 48 h were used to measure the expression of RhoA, Rho kinase activity, MYPT1 phosphorylation and muscle contraction. (A, B, F, G) Expression of RhoA was measured by qRT-PCR and western blot and results with mRNA was expressed as fold change. The molecular weight of RhoA is 24 kDa. Values are means±SE of 4–6 experiments. (C and H) Rho kinase activity was measured in response to acetylcholine (ACh, 1μM) by immunokinase assay and the results are expressed as cpm/mg protein. Values are means±SE of 4–6 experiments. (D and I) MYPT1 phosphorylation was measured by western blot using phospho-specific antibody. The molecular weight of MYPT1 is 140 kDa. (E and J) Muscle contraction was measured as an increase in sustained contraction in response to acetylcholine (10 μM) in organ bath experiments and the results are expressed as mN/100 mg of tissue. Values are means±SE of 4–6 experiments.</p

Expression of microRNA-133a in diabetes and hyperglycemia.

<p>(A) Expression of microRNA-133a (miR-133a) was measured by qRT-PCR in RNA isolated from the smooth muscle of WT or ob/ob mice with or without intraperitoneal injections of pre- miR-133a (5 mg/kg of body weight over 3 days). (B) Expression of miR-133a was measured by qRT-PCR in RNA isolated from the smooth muscle cells treated with NG or HG for 48 h in the presence or absence of transfection with pre-miR-133a. Values are means±SE of 3–5 experiments.</p

Regulation of RhoA expression and Rho kinase activity by miR-133a in diabetes and hyperglycemia.

<p>Expression of RhoA and Rho kinase activity was measured in the smooth muscle of WT or ob/ob mice with or without intraperitoneal injections pre-miR-133a or antagomiR-miR133a (5 mg/kg of body weight over 3 days) (A, B, E and F) and in smooth muscle cells treated with NG or HG for 48 h in the presence or absence of transfection with pre-miR-133a or antagomiR-133a (C and G). RhoA mRNA expression was measured by qRT-PCR and the results are expressed as fold increase from control. RhoA protein expression was measured by western blot and the results are expressed as densitometric values. The molecular weights of RhoA and β-Actin are 24 and 42 kDa, respectively. (D) Rho kinase activity was measured by immunokinase assay and the results are expressed as cpm/mg protein. Values are means±SE of 3–5 experiments.</p

Effect of N-acetylcysteine (NAC) on the expression of miR-133 and RhoA, and muscle contraction in diabetes and hyperglycemia.

<p>(A and E) Expression of miR-133a was measured by qRT-PCR in RNA isolated from the smooth muscle of WT or ob/ob mice with or without intraperitoneal (IP) injections of NAC (300 mg/kg) and in RNA isolated from smooth muscle cells treated with NG or HG for 48 h in the presence or absence of NAC (1 mM). Results are expressed as fold change from control. Values are means±SE of 4–6 experiments. (B, C, F and G) Expression of RhoA was measured by qRT-PCR and western blot in smooth muscle of WT or ob/ob mice with or without IP injections of NAC (300 mg/kg) and in smooth muscle cells treated with NG or HG for 48 h in the presence or absence of NAC. Results are expressed as fold change from control for mRNA levels. The molecular weights of RhoA and β-actin are 24 and 42 kDa, respectively. Values are means±SE of 4–6 experiments. (H) Rho kinase activity was measured in response to acetylcholine (ACh, 1μM) by immunokinase assay and the results are expressed as cpm/mg protein. Values are means±SE of 4–5 experiments. (D and I) Muscle contraction was measured as increase in sustained contraction in response to acetylcholine (10 μM) in organ bath experiments in smooth muscle of WT or ob/ob mice with or without IP injections NAC (300 mg/kg) and in smooth muscle treated with NG or HG for 48 h in the presence or absence of NAC (1mM). Results are expressed as mN/100 mg of tissue. Values are means±SE of 4–6 experiments.</p

Oxidative stress in diabetes and hyperglycemia.

<p><b>S</b>mooth muscle of the fundus of WT and ob/ob mice and smooth muscle from WT mice treated with NG or HG for 48 h were used to measure expression of the RAGE, SOD-1 and level of H₂O₂ and ROS. (A, B, G and H) Expression of RAGE was measured by qRT-PCR and western blot and results with mRNA was expressed as fold change. Values are means±SE of 4–6 experiments. (C and I) Expression of SOD-1 was measured by qRT-PCR and the results are expressed as fold increase from control. Values are means±SE of 4–6 experiments. (D and J) H₂O₂ levels were measured by calorimetry and the results are expressed as μM/100 mg. Values are means±SE of 4–6 experiments. (E and K) ROS levels were measured by fluorometry and the results are expressed as percent increase from control. Values are means±SE of 4–6 experiments. (F) MPO levels were measured by fluorometry and the results are expressed as percent increase from control. Values are means±SE of 4–5 experiments.</p

Expression of NOX-4 and microRNA-25 in diabetes and hyperglycemia.

<p>Smooth muscle of the fundus from WT and ob/ob mice, and smooth muscle of the fundus from WT mice treated with NG or HG for 48 h were used to measure the expression of NOX-4 and microRNA-25 (miR-25). (A and C) Analysis of NOX-4 expression by western blot. The molecular weights of NOX4 and β-actin are 67 and 42 kDa, respectively. (B and D) mRNA expression of NOX-4 was measured by qRT-PCR and the results are expressed as fold change. Values are means±SE of 4–6 experiments. (E and F) Expression of miR-25 was measured by qRT-PCR and results are expressed as fold change. Values are means±SE of 4–6 experiments.</p

Feedback inhibition of PAR2-AP-stimulated Rho kinase activity by PKA derived from NF-kB pathway.

<p>(<b>A</b>) Gastric muscle cells were incubated with myristoylated PKI for 10 min or with the RhoA inhibitor <i>Clostridium botulinum</i> C3 exoenzyme (2 µg/ml) for 2 h, and then treated with PAR2-AP for 10 min. Cells were homogenized in lysis buffer and proteins were separated on SDS-PAGE. Phosphorylation (i.e., activation) of IKK2 was measured by western blot phospho-specific (Ser^177/181) antibody and phosphorylation of p65 subunit (activation of NF-kB) was measured using phospho-specific (Ser⁵³⁶) antibody. Degradation of IkBa was measured using antibody to IkBa. Western blot of the b-actin protein is shown for a control loading. (<b>B</b>) Freshly dispersed gastric smooth muscle cells were treated with PAR2-AP (1 µM) for 10 min in the presence or absence of IKK IV (10 µM), MG-132 (10 µM), or myristoylated PKI (1 µM). Results are expressed as cpm/mg protein above basal levels (3125±502 cpm/mg protein). Treatment of cells with IKK IV (3021±486 cpm/mg protein), MG-132 (2859±602 cpm/mg protein), or myristoylated PKI (3254±574 cpm/mg protein) alone had no effect on basal Rho kinase activity. (<b>C</b>) Cultured smooth muscle cells expressing dominant negative mutants of IKK2 (K44A) or IkBa (S32A/S36A) or the PKA phosphorylation site-deficient RhoA (S188A) were treated with PAR2-AP (1 µM). Rho kinase activity was measured using [³²P]ATP by immunokinase assay. Results are expressed as cpm/mg protein. Values are means ± S.E. of four experiments. ## Significant increase from control response to PAR2-AP (P<0.01). (<b>D</b>) Phosphorylation of RhoA was measured in cells labelled with [³²P]Pi. Control cells or cells expressing phosphorylation-site deficient RhoA(S188A) were treated with PAR2-AP (1 µM) and phosphorylation of RhoA was analysed by SDS-PAGE and autoradiography. (<b>E</b>) Dissociation of PKA catalytic subunit from NF-kB. Control cells, cells transfected with PAR2 siRNA or RhoA(S188A) were treated with PAR2-AP for 10 min. NF-kB immunoprecipitates were separated by SDS/PAGE and membranes probed with antibodies to the catalytic subunit of PKA (PKACS, 1∶2000) or NF-kB (1:1000).</p

Delayed gastric emptying in diabetes.

<p>WT and ob/ob mice were treated with IP injections of NAC (300 mg/kg) and fasted for 24 h and then fed normal chow ad libitum for 4 h. Gastric emptying was monitored immediately after 3 h of feeding. Results are expressed as the amount of leftover food in the stomach as milligrams per gram of body weight. Values are means±SE of 4 experiments.</p

Schematic diagram of the mechanism involved in the upregulation of RhoA/Rho kinase pathway and hypercontraction in diabetes.

<p>In smooth muscle, activation of RhoA by contractile agonist causes stimulation of Rho kinase activity and Rho kinase-dependent phosphorylation of MYPT1 resulting in inhibition of MLCP activity and increase in MLC₂₀ phosphorylation and muscle contraction. In diabetic smooth muscle, oxidative stress causes upregulation of RhoA/Rho kinase pathway and hypercontraction. The decrease in miR-133a expression in response to oxidative stress leads to an increase in RhoA expression, Rho kinase activity, MYPT1 phosphorylation and muscle contraction.</p

Figure 1

<p>(<b>A</b>) Expression of PAR2 in rabbit gastric smooth muscle cells. Western blot was performed on homogenates prepared from cultured gastric smooth muscle cells from rabbit. Lysates were prepared from control cells and cells transfected with PAR2-specific siRNA and proteins were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and probed with specific antibodies to PAR2 (1∶1000). Immunoreactive bands for PAR2 was detected by enhanced chemiluminescence. Suppression of PAR2 expression in cells transfected with PAR2 siRNA suggests that selectivity of antibodies and validity of siRNA transfection. (<b>B</b>) Activation of G proteins PAR2-AP in gastric smooth muscle cells. Membranes isolated from freshly dispersed smooth muscle cells were incubated with ³⁵S-labeled guanosine 5-O-(3-thiotriphosphate) ([³⁵S]GTPgS) in the presence or absence of PAR2-activating peptide (PAR2-AP) (1 µM) for 20 min. Aliquots were added to wells coated with Ga_q, Ga_i1, Ga_i2, Ga_i3, Ga_s, Ga₁₂ or Ga₁₃ for 2 h and bound radioactivity was measured. Results are expressed as cpm/mg protein. PAR2-AP caused a significant increase in the binding of [³⁵S]-GTPgS-Ga complexes to wells coated with Ga_i1 and Ga_i2, Ga_q, and Ga₁₃. PAR2-AP did not cause significant increase in the binding to Ga_i3, Ga₁₂, and Ga_s (7±10% to 11±8% increase above basal levels). Values are means ± S.E. of four experiments. ** Significant increase in G protein activation (P<0.001).</p