Ming C. Gong

The effects of the Rho‐kinase inhibitor Y‐27632 on arachidonic acid‐, GTPγS‐, and phorbol ester‐induced Ca2+‐sensitization of smooth muscle

The effects of the Rho‐kinase inhibitor, Y‐27632 [1] on Ca2+‐sensitization of force induced by arachidonic acid (AA), phorbol 12,13‐dibutyrate (PDBu), GTPγS, and by the stable thromboxane analog, 9,11‐dideoxy‐9α,11α‐methanoepoxy‐PGF2α (U‐46619), were determined in α‐toxin‐permeabilized smooth muscles. Y‐27632 relaxed (up to 99%) Ca2+‐sensitization by GTPγS (10 μM) and U‐46619 (1 μM), but not by PDBu (20 μM), and reduced GTPγS‐induced myosin light chain (MLC20) phosphorylation from 28% to 17% (P=0.002). GTPγS‐induced force sensitization was inhibited by Y‐27632 more potently when the inhibitor was added during the plateau of force than prior to stimulation. In α‐toxin‐permeabilized smooth muscle, Y‐27632 inhibited AA (50 μM)‐induced Ca2+‐sensitization of force (by 66±1.3%) and reduced MLC20 phosphorylation. In contrast, Y‐27632 did not relax force Ca2+‐sensitized by AA in smooth muscle permeabilized with Triton X‐100. We conclude that (i) AA induces Ca2+‐sensitization through dual mechanisms, one mediated by Rho‐kinase (or a related kinase), and (ii) Rho‐kinase is not required for phorbol ester‐induced Ca2+‐sensitization.

Longitudinal analysis of arterial blood pressure and heart rate response to acute behavioral stress in rats with type 1 diabetes mellitus and in age-matched controls

We recorded via telemetry the arterial blood pressure (BP) and heart rate (HR) response to classical conditioning following the spontaneous onset of autoimmune diabetes in BBDP/Wor rats vs. age-matched, diabetes-resistant control (BBDR/Wor) rats. Our purpose was to evaluate the autonomic regulatory responses to an acute stress in a diabetic state of up to 12 months duration. The stress was a 15-s pulsed tone (CS+) followed by a 0.5-s tail shock. The initial, transient increase in BP (i.e., the “first component,” or C1), known to be derived from an orienting response and produced by a sympathetic increase in peripheral resistance, was similar in diabetic and control rats through ∼9 months of diabetes; it was smaller in diabetic rats 10 months after diabetes onset. Weakening of the C1 BP increase in rats that were diabetic for >10 months is consistent with the effects of sympathetic neuropathy. A longer-latency, smaller, but sustained “second component” (C2) conditional increase in BP, that is acquired as a rat learns the association between CS+ and the shock, and which results from an increase in cardiac output, was smaller in the diabetic vs. control rats starting from the first month of diabetes. A concomitant HR slowing was also smaller in diabetic rats. The difference in the C2 BP increase, as observed already during the first month of diabetes, is probably secondary to the effects of hyperglycemia upon myocardial metabolism and contractile function, but it may also result from effects on cognition. The small HR slowing concomitant with the C2 pressor event is probably secondary to differences in baroreflex activation or function, though parasympathetic dysfunction may contribute later in the duration of diabetes. The nearly immediate deficit after disease onset in the C2 response indicates that diabetes alters BP and HR responses to external challenges prior to the development of structural changes in the vasculature or autonomic nerves.

Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle.

1. Diacylglycerol (DAG; 10 microM), an activator of conventional and novel protein kinases C (cPKCs and nPKCs), induced Ca2+ sensitization of force in isolated intact and alpha‐toxin‐permeabilized femoral artery (FA) and portal vein (PV), and increased the phosphorylation of myosin light chain (MLC20) at the same peptides phosphorylated by myosin light chain kinase. 2. Ca2+ sensitization by DAG was specifically inhibited by a pseudosubstrate peptide inhibitor of cPKCs (PKC alpha(22‐30) peptide; 50 microM). Similarly, GF 109203X (600 nM), an inhibitor of cPKCs and nPKCs, completely abolished Ca2+ sensitization by phorbol 12,13‐dibutyrate (PDBu; 1 microM). In contrast, Ca2+ sensitization induced by the alpha1‐adrenergic agonist phenylephrine (100 microM) was not inhibited by these inhibitors of cPKCs and nPKCs. 3. A pseudosubstrate peptide inhibitor of the atypical PKCs (aPKCs) PKC zeta(116‐124) (50 microM) significantly (about 50%) inhibited the Ca2+ sensitization of force and MLC20 phosphorylation induced by 100 microM phenylephrine and by 300 microM arachidonic acid, but not that by DAG (10 microM) or PDBu (1 microM). 4. A phospholipase A2 (PLA2) inhibitor, ONO‐RS‐082 (10 microM), abolished the release of arachidonic acid and partially (by 40%) inhibited the Ca2+ sensitization induced by phenylephrine in FA smooth muscle. This effect was not additive to the inhibition observed with the aPKC inhibitor peptide, suggesting that arachidonic acid and aPKCs exert their effects via the same pathway, probably through activation of aPKC(s) by arachidonic acid. 5. Western blot analysis with antibodies to aPKCs revealed aPKCs zeta, lambda (or iota) and an unidentified 64 kDa protein. The distribution (cytosolic and particulate) of these proteins was not affected by PDBu (1 microM). 6. Our results are consistent with a significant role for atypical (or related) PKCs through a PLA2‐arachidonic acid‐aPKC pathway in agonist‐induced Ca2+ sensitization, in parallel with a similar, but minor role of the DAG‐cPKC cascade. The inability of the combination of the two (aPKC and cPKC) inhibitors to completely eliminate Ca2+ sensitization also suggests the presence of a third, still unidentified, pathway of this mechanism.

ACE2 is expressed in mouse adipocytes and regulated by a high-fat diet

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low- (LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 +/- 253; HF: 6,829 +/- 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.

Hypertension and Disrupted Blood Pressure Circadian Rhythm in Type 2 Diabetic db/db Mice

Human Type 2 diabetes is associated with increased incidence of hypertension and disrupted blood pressure (BP) circadian rhythm. Db/db mice have been used extensively as a model of Type 2 diabetes, but their BP is not well characterized. In this study, we used radiotelemetry to define BP and the circadian rhythm in db/db mice. We found that the systolic, diastolic, and mean arterial pressures were each significantly increased by 11, 8, and 9 mmHg in db/db mice compared with controls. In contrast, no difference was observed in pulse pressure or heart rate. Interestingly, both the length of time db/db mice were active (locomotor) and the intensity of locomotor activity were significantly decreased in db/db mice. In contrast to controls, the 12-h light period average BP in db/db mice did not dip significantly from the 12-h dark period. A partial Fourier analysis of the continuous 72-h BP data revealed that the power and the amplitude of the 24-h period length rhythm were significantly decreased in db/db mice compared with the controls. The acrophase was centered at 0141 in control mice, but became scattered from 1805 to 0236 in db/db mice. In addition to BP, the circadian rhythms of heart rate and locomotor activity were also disrupted in db/db mice. The mean arterial pressure during the light period correlates with plasma glucose, insulin, and body weight. Moreover, the oscillations of the clock genes DBP and Bmal1 but not Per1 were significantly dampened in db/db mouse aorta compared with controls. In summary, our data show that db/db mice are hypertensive with a disrupted BP, heart rate, and locomotor circadian rhythm. Such changes are associated with dampened oscillations of clock genes DBP and Bmal1 in vasculature.

Role of calcium-independent phospholipase A2β in high glucose-induced activation of RhoA, Rho-kinase, and CPI-17 in cultured vascular smooth muscle cells and vascular smooth muscle hypercontractility in diabetic animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A2β (iPLA2β) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA2β mRNA, protein, and iPLA2 activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA2β protein up-regulation. Inhibiting iPLA2β activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA2β in iPLA2β-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA2β inhibition. Moreover, increases in iPLA2 activity and iPLA2β protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA2β, but not iPLA2γ, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA2β up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

Smooth-muscle BMAL1 participates in blood pressure circadian rhythm regulation.

As the central pacemaker, the suprachiasmatic nucleus (SCN) has long been considered the primary regulator of blood pressure circadian rhythm; however, this dogma has been challenged by the discovery that each of the clock genes present in the SCN is also expressed and functions in peripheral tissues. The involvement and contribution of these peripheral clock genes in the circadian rhythm of blood pressure remains uncertain. Here, we demonstrate that selective deletion of the circadian clock transcriptional activator aryl hydrocarbon receptor nuclear translocator-like (Bmal1) from smooth muscle, but not from cardiomyocytes, compromised blood pressure circadian rhythm and decreased blood pressure without affecting SCN-controlled locomotor activity in murine models. In mesenteric arteries, BMAL1 bound to the promoter of and activated the transcription of Rho-kinase 2 (Rock2), and Bmal1 deletion abolished the time-of-day variations in response to agonist-induced vasoconstriction, myosin phosphorylation, and ROCK2 activation. Together, these data indicate that peripheral inputs contribute to the daily control of vasoconstriction and blood pressure and suggest that clock gene expression outside of the SCN should be further evaluated to elucidate pathogenic mechanisms of diseases involving blood pressure circadian rhythm disruption.

Group VIA Phospholipase A2 (iPLA2β) Participates in Angiotensin II-induced Transcriptional Up-regulation of Regulator of G-protein Signaling-2 in Vascular Smooth Muscle Cells

Rgs2 (regulator of G-protein signaling-2)-deficient mice exhibit severe hypertension, and genetic variations of RGS2 occur in hypertensive patients. RGS2 mRNA up-regulation by angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) is a potentially important negative feedback mechanism in blood pressure homeostasis, but how it occurs is unknown. Here we demonstrate that group VIA phospholipase A2 (iPLA2β) plays a pivotal role in Ang II-induced RGS2 mRNA up-regulation in VSMC by three independent approaches, including pharmacologic inhibition with a bromoenol lactone suicide substrate, suppression of iPLA2β expression with antisense oligonucleotides, and genetic deletion in iPLA2β-null mice. Selective inhibition of iPLA2β by each of these approaches abolishes Ang II-induced RGS2 mRNA up-regulation. Furthermore, using adenovirus-mediated gene transfer, we demonstrate that restoration of iPLA2β-expression in iPLA2β-null VSMC reconstitutes the ability of Ang II to up-regulate RGS2 mRNA expression. In contrast, Ang II-induced vasodilator-stimulated phosphoprotein phosphorylation and Ang II receptor expression are unaffected. Moreover, in wild-type but not iPLA2β-null VSMC, Ang II stimulates iPLA2 enzymatic activity significantly. Both arachidonic acid and lysophosphatidylcholine, products of iPLA2β action, induce RGS2 mRNA up-regulation. Inhibition of lipoxygenases, particularly 15-lipoxygenase, and cyclooxygenases, but not cytochrome P450-dependent epoxygenases inhibits Ang II- or AA-induced RGS2 mRNA expression. Moreover, RGS2 protein expression is also up-regulated by Ang II, and this is attenuated by bromoenol lactone. Disruption of the Ang II/iPLA2β/RGS2 feedback pathway in iPLA2β-null cells potentiates Ang II-induced vasodilator-stimulated phosphoprotein and Akt phosphorylation in a time-dependent manner. Collectively, our results demonstrate that iPLA2β participates in Ang II-induced transcriptional up-regulation of RGS2 in VSMC.

Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle.

Excitatory agonists can induce significant smooth muscle contraction under constant free Ca2+ through a mechanism called Ca2+ sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca2+-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca2+-independent phospholipase A2 (iPLA2) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA2 activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca2+ sensitization of contraction from 49 ± 3.6 to 12 ± 1.0% (p< 0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca2+, phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA2 in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA2 is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca2+-sensitization of contraction in vascular smooth muscle.

Endothelin-2 induces oviductal contraction via endothelin receptor subtype A in rats

Proper function of the oviduct is critical to reproductive success with regulated contraction and relaxation facilitating transportation of the germ cells to the site of fertilization. Endothelin-2 (EDN2) is a potent vasoconstrictor produced by granulosa cells of the preovulatory follicle at the time of ovulation; however, whether this gonadotropin surge-induced peptide played a role in facilitating germ cell transportation by inducing oviductal contraction was unknown. The objectives of these experiments were (1) to determine whether the endothelin receptor system was present in the oviduct, (2) to test the hypothesis that EDN2 induces oviductal contraction via a specific endothelin receptor subtype, (3) to determine, as a possible alternate source of the ligand, whether mRNA for EDN2 was expressed in cumulus-oocyte complexes (COCs) within the oviduct, and (4) to determine whether EDN2 could overcome prostaglandin E(2) (PGE(2))-induced oviductal relaxation. Microarray and real-time PCR analysis indicated that mRNA for both the endothelin receptor subtypes (ET(A) and ET(B)) was present in the oviduct, whereas immunohistochemical examination revealed that ET(A) protein was the dominant isoform, present in the luminal epithelial cells of the oviduct. Real-time PCR analysis demonstrated that mRNA for EDN2 was expressed in COCs after ovulation. Isometric tension analysis indicated that EDN2 was a potent oviductal constrictor and that the contractile effect of EDN2 was mediated by the ET(A) and not the ET(B) receptor subtype. The oviductal contraction induced by EDN2 also reversed oviductal relaxation induced by PGE(2). In summary, ET(A) receptor-specific EDN2-induced contraction as a facilitator of oviductal function suggests a novel pathway involved in germ cell transport and hence mammalian fertility.