Vascular Calcium Sensitivity Research Articles

Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling

Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30 mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30 mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1α. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.

Bradykinin induces vascular contraction after hemorrhagic shock in rats

BackgroundBradykinin (BK) has many biological effects in inflammation, allergy, and septic shock. Studies have shown that low doses of BK can induce vascular relaxation and high doses can induce vascular contraction in many pathophysiological conditions, but the role and mechanisms that high doses of BK have on vascular contraction in hemorrhagic shock are not clear. MethodsWith hemorrhagic-shock rats and hypoxia-treated superior mesenteric artery (SMA), we investigated the role and mechanisms of high doses of BK-induced vascular contraction in hemorrhagic shock. ResultsHigh doses of BK (500–50,000 ng/kg in vivo or 10−10 to 10−5 mol/L in vitro) dose dependently induced vascular contraction of SMA and increased the vascular calcium sensitivity in normal and hemorrhagic-shock rats. Less than 10−10 mol/L of BK induced vascular dilation BK-induced increase of vascular contractile response and calcium sensitivity was reduced by denudation of the endothelium, 18α-glycyrrhetic acid (an inhibitor of myoendothelial gap junction) and connexin 43 antisense oligodeoxynucleotide. Further studies found that high concentrations of BK-induced vascular contraction in hemorrhagic shock was closely related to the activation of Rho A–Rho kinase pathway and Protein Kinase C (PKC) α and ε. ConclusionsHigh doses of BK can induce vascular contraction in hemorrhagic shock condition, which is endothelium and myoendothelial gap junction dependent. Cx43-mediated activation of Rho A–Rho kinase and Protein Kinase C (PKC) pathway plays a very important role in this process. This finding provided a new angle of view to the biological role of BK in other pathophysiological conditions such as hemorrhagic shock or hypoxia.

Abstract 373: Mineralocorticoid Receptor and Rhoa/rho Kinase Contribute to Vascular Dysfunction in Obese Db/db Mice

Increasing evidence indicates that adipose tissue modulates vascular function. Our previous studies demonstrated that adipocytes secrete aldosterone. We hypothesized that adipocyte-releasing factors induce vascular dysfunction through mineralocorticoid receptor (MR)-dependent mechanisms. This study explored the small G protein RhoA/Rho kinase as a pathway linking aldosterone/MR and the paracrine effects of adipose tissue in the vasculature, since the activation of this pathway regulates vascular contraction. Diabetic obese mice (db/db) and their control counterparts (db/+) were treated with MR antagonist (MRA) (K canrenoate, 30mg/Kg/day, S.C., 4 weeks) or RhoA/Rho kinase inhibitor, fasudil (30 mg/Kg/day, S.C., 3 weeks). Blood pressure levels were similar between groups. Fasudil, but not MRA, reduced plasma glucose levels in db/db mice (db/+:12.3±6; db/db: 30.1±3; MRA-treated db/db: 32.5±2; fasudil treated db/db: 17.1±2; mmoL/mL). Arteries from db/db mice displayed reduced relaxation to 10 - 5 M acetycholine (Ach; db/+:79.4±3.9% vs db/db: 14.3±3.1%). Arteries from MRA-treated db/db mice exposed to fat conditioned medium (FCM) had improved responses to Ach (MRA-treated db/db: 28.3±1.9% vs MRA-treated db/db +FCM: 42.8±4.3%). Increased norepinephrine (NE)-induced contraction was observed in db/db mice in endothelium-denuded arteries (10 -5 M NE; db/+: 1.5 ± 0.1 vs db/db: 2.2 ± 0.1; mN/mm). NE responses were reduced in MRA-treated db/db mice (1.7±0.1 mN/mm) or fasudil (1.6±0.2 mN/mm). Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Fasudil treatment reduced calcium-induced contraction in both groups with greater effect in db/db mice (5 mmol/L CaCl 2 ; db/+: 73 % vs db/db: 57 %). MR blockade did not affect vascular RhoA/Rho kinase activity (RhoA membrane to cytosol translocation) in db/db mice. Our data suggest that in db/db mice: MR and RhoA/Rho kinase signaling contribute to adipose modulation of vascular/contraction. While MR plays a role in vascular relaxation, RhoA/Rho kinase signalling is important in contractile dysfunction, adipocyte secretion, and glucose metabolism - processes that may contribute to cardiovascular injury in obesity-associated diabetes.

Role of protein kinase G on the post-shock mesenteric lymph blockage ameliorating vascular calcium sensitivity

To investigate the role of protein kinase G (PKG) in blocking post-shock mesenteric lymph (PSML) return ameliorating the calcium sensitivity in hemorrhagic shock rats. Male Wistar rats were randomly divided into sham, shock, shock+ligation (shock plus mesenteric lymph duct ligation (MLDL)), shock+drainage (shock plus PSML drainage) groups. After shock (hypotension 40 mm Hg) for three hours or corresponding times, the superior mesenteric artery (SMA) was taken out for detecting the PKG and phospho PKG (p-PKG) contents, and the vascular rings of SMA were prepared for assaying the calcium sensitivity using an isolated organ perfusion system. The PKG and p-PKG contents of SMA in shock group were significantly increased than that of sham group, and MLDL or PSML drainage reducing the levels of PKG and p-PKG. Meanwhile, the vascular calcium sensitivity in shock group was significantly lower than that of sham group, MLDL or PSML drainage enhanced the calcium sensitivity. After incubating with PKG regulators in shock+ligation and shock+drainage groups, the PKG agonist 8Br-cGMP reduced the contractility of vascular rings to gradient calcium ions and Emax and the PKG inhibitor agonist KT5823 elevated the calcium sensitivity significantly. Protein kinase G plays an important role in post-shock mesenteric lymph blockage improving vascular calcium sensitivity.

Effects of Interleukin-1β on Vascular Reactivity After Lipopolysaccharide-induced Endotoxic Shock in Rabbits and Its Relationship With PKC and Rho Kinase

Calcium desensitization plays a critical role in the occurrence of vascular hyporeactivity after shock. Interleukin (IL)-1β participates in the regulation of vascular reactivity via both nitric oxide (NO)-dependent and NO-independent mechanisms. However, the specific NO-independent pathway remains to be established. The issue of whether IL-1β modulates vascular reactivity via regulation of calcium sensitivity in the NO-independent mechanism is unclear. In the current study, effects of IL-1β on vascular calcium sensitivity and its relationship with PKC and Rho kinase were investigated in vivo and in vitro using a rabbit model of lipopolysaccharide (LPS)-induced endotoxic shock and superior mesenteric arteries (SMAs), respectively. The calcium sensitivity profile of SMAs displayed a biphasic change after LPS-induced endotoxic shock (significant increase at 0.5 hour and 1 hour after LPS administration and marked decrease after 2 hours) and was negatively related to changes in serum IL-1β. The IL-1 receptor antagonist, IL-1ra (4 μg/mL), partly reversed LPS-induced calcium desensitization. In vitro incubation with IL-1β (50-200 ng/mL) reduced the calcium sensitivity of SMAs and suppressed the activities of Rho kinase and PKC and the phosphorylation of 20-kDa myosin light chain. These effects of IL-1β were shown to be regulated by the PKC agonist, phorbol 12-myristate 13-acetate, and Rho kinase agonist and antagonist, angiotensin II, and Y-27632, respectively. Our results collectively suggest that IL-1β participates in vascular hyporeactivity after endotoxic shock via regulation of vascular calcium sensitivity. Moreover, this regulatory effect of IL-1β seems closely related to downregulation of the activities of PKC and Rho kinase.

Protein Kinase C Isoforms Responsible for the Regulation of Vascular Calcium Sensitivity and Their Relationship to Integrin-Linked Kinase Pathway After Hemorrhagic Shock

The aim of this study was to investigate the protein kinase C (PKC) isoforms involved in the regulation of vascular calcium sensitivity after hemorrhagic shock, the related mechanism, and the role of integrin-linked kinase (ILK). Using superior mesenteric artery from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells, the effects of PKC isoforms agonists and antagonists on vascular calcium sensitivity, their relationship with myosin light chain phosphatase (MLCP), myosin light chain (MLC20) phosphorylation, and ILK were observed. The results indicated that PKCα and ε agonists, thymelea toxin and carbachol, restored shock-induced decrease of vascular calcium sensitivity; PKCα antagonist, Gö-6976 and PKCε pseudosubstrate inhibition peptide, aggravated shock-induced calcium desensitization, whereas the agonists and antagonists of PKCδ and ζ had no effects on shock-induced calcium desensitization. PKCα and ε agonists reversed the increased MLCP activity and the decreased MLC20 phosphorylation induced by shock or hypoxia. ILK inhibitor abolished the effects of PKCα and ε agonists on vascular calcium sensitivity, MLCP activity, and MLC20 phosphorylation. These findings suggested that PKCα and ε may be the main isoforms responsible for the regulation of vascular calcium sensitivity after hemorrhagic shock, which enforce their regulation through MLCP-MLC20 pathway, and ILK may be a downstream molecule of PKCα and ε.

INVOLVEMENT OF CPI-17 AND ZIPPER-INTERACTING PROTEIN KINASE IN THE REGULATION OF PROTEIN KINASE C-α, PROTEIN KINASE C-ε ON VASCULAR CALCIUM SENSITIVITY AFTER HEMORRHAGIC SHOCK

Our previous studies have found that protein kinase C (PKC) alpha and epsilon take part in the regulation of calcium sensitization and vascular reactivity after hemorrhagic shock. However, the regulatory mechanism is still unclear. The aim of this study was to investigate the role of PKC-potentiated inhibitory protein for protein phosphatase 1 of 17 kDa (CPI-17) and zipper-interacting protein kinase (ZIPK) in the regulation of PKC-alpha and PKC-epsilon on vascular calcium sensitivity after hemorrhagic shock. Using superior mesenteric artery from hemorrhagic shock rats (at 40 mmHg for 2 h), the effects of PKC-alpha and PKC-epsilon agonists on calcium sensitivity and the phosphorylation of 20-kDa myosin light chain (MLC20), and the modulating effects of CPI-17 and ZIPK in this process were observed. In hypoxia-treated vascular smooth muscle cells (VSMCs), the effects of PKC-alpha and PKC-epsilon agonists on protein expression and activity of CPI-17 and ZIPK and the modulating effects of CPI-17 and ZIPK on the regulation of PKC-alpha and PKC-epsilon on the activity of myosin light chain phosphatase (MLCP) were observed. The results indicated that PKC-alpha and PKC-epsilon agonists thymelea toxin and carbachol seemed to increase the calcium sensitivity and MLC20 phosphorylation of superior mesenteric artery after hemorrhagic shock and antagonize the increase of MLCP activity in VSMC after hypoxia. The effects of PKC-alpha and PKC-epsilon agonists were abolished by CPI-17 and ZIPK neutralizing antibody. The protein expression and activity of CPI-17 and ZIPK in VSMC were suppressed after hypoxia but increased by PKC-alpha and PKC-epsilon agonists. Immunoprecipitation experiment showed that PKC-alpha and PKC-epsilon, especially PKC-epsilon, may directly bind with ZIPK but not CPI-17 in VSMC, and ZIPK may directly bind with CPI-17. These results suggest that CPI-17 and ZIPK participate in the regulation of PKC-alpha and PKC-epsilon on vascular calcium sensitivity after hemorrhagic shock. Protein kinase C alpha and epsilon regulation of MLCP-MLC20 phosphorylation pathway in the process of VSMC contraction is mainly through ZIPK; CPI-17 may play an indirect modulating role.

REGULATORY EFFECTS OF MYOENDOTHELIAL GAP JUNCTION ON VASCULAR REACTIVITY AFTER HEMORRHAGIC SHOCK IN RATS

Myoendothelial gap junction (MEGJ), one kind of gap junction between vascular endothelial cell and vascular smooth muscle cell, can transmit electrical and chemical signals to keep the electric and machinery activity synchronism of vasculature. After severe trauma or shock, vascular reactivity to vasoconstrictors or vasodilators is greatly reduced. However, whether MEGJ participates in the regulation of vascular reactivity after hemorrhagic shock, what type of MEGJ is involved, and what is the possible mechanism are unknown. With the hemorrhagic shock Sprague-Dawley rats and their superior mesenteric arteries (SMAs), the effects of 18alpha-glycyrrhetic acid, a lipophilic aglycone that disrupts gap junction plaques, on vascular contractile response to norepinephrine (endothelium-independent vascular constrictor), myricetin (endothelium-dependent vasoconstrictor) and relaxation reactivity to sodium nitroprusside (endothelium-independent vasodilator), and acetylcholine (Ach; endothelium-dependent vasodilator) were observed. Meanwhile, the relationship of the mRNA/protein expression of connexins 37, 40, and 43(Cx40 and Cx43) to the changes of vascular reactivity after hemorrhagic shock and the effect of antisense oligodeoxynucleotide of Cx40 or Cx43 on vascular calcium sensitivity and vascular reactivity were investigated. The results indicated that 18alpha-glycyrrhetic acid antagonized myricetin and Ach-induced SMA reactivity, but had no effect on norepinephrine- and sodium nitroprusside-induced vascular response. The mRNA and protein expression of Cx37 and Cx40 of SMA were negatively associated with the vascular reactivity, whereas Cx43 seemed to be a positive relationship to vascular reactivity. Antisense oligodeoxynucleotide of Cx40 significantly increased the calcium sensitivity, myricetin-induced vasoconstriction, and Ach-induced vasodilation, whereas antisense oligodeoxynucleotide of Cx43 depressed them. It was suggested that MEGJ plays an important role in the regulation of endothelium-dependent vascular reactivity after hemorrhagic shock. The involved types were mainly Cx40 and Cx43. The possible mechanism that Cx40/Cx43 regulates the endothelium-dependent vasoconstrictor reactivity may be related to their regulating effects on the calcium sensitivity of vascular smooth muscle cell.