Effects of maturation and acute hypoxia on receptor-IP(3) coupling in ovine common carotid arteries

Whereas previous studies have established that many mechanisms mediating pharmacomechanical coupling are subject to regulation, evidence of physiological regulation of the coupling efficiency between receptor activation and second-messenger production is scarce. The present studies address the hypothesis that acute hypoxia and maturation can influence the mass of second-messenger production for each activated agonist-bound receptor ("receptor gain"). For this assessment, receptor density and agonist affinity values were used to calculate 5-hydroxytryptamine (5-HT) concentrations that would produce standardized numbers of bound receptors (8.5 fmol/mg protein) in each experimental group and thus minimize effects of age or hypoxia on receptor density or agonist affinity. After 3 min of exposure to these 5-HT concentrations, normoxic magnitudes of contraction were similar (as %potassium maxima) in fetal (50 +/- 14%) and adult (40 +/- 9%) arteries, but hypoxia (PO(2) approximately 9--12 Torr for 30 min) depressed contractile tensions with a significantly different time course and magnitude in fetal (30 +/- 10%) and adult (17 +/- 11%) arteries (P < 0.05). Basal inositol 1,4,5-trisphosphate (IP(3)) values (in pmol/mg protein) were significantly greater in fetal (94 +/- 16) than in adult (44 +/- 6) arteries, and integrated areas above baseline for the IP(3) time courses (in nmol-s/mg protein) were significantly greater in fetal than in adult arteries both in normoxic (14.3 +/- 1.8 vs. 9.1 +/- 1.6) and hypoxic (15.0 +/- 2.1 vs. 8.6 +/- 1.2) conditions (P < 0.05). Hypoxia altered the IP(3) time courses both in the fetus and the adult but had no significant effect on IP(3 )mobilization or receptor gain. These data demonstrate that for the 5-HT(2a) receptor predominant in this preparation, receptor gain can be experimentally determined, is not influenced by acute hypoxia, but is greater in fetal than in adult ovine carotid arteries.     

Acute hypoxia modulates 5-HT receptor density and agonist affinity in fetal and adult ovine carotid arteries

In light of recent observations that receptor-ligand binding and coupling are physiologically regulated, the present study examined the hypothesis that the direct effects of hypoxia on vascular contractility involve modulation of pharmacomechanical coupling via changes in agonist affinity and/or receptor density. Because the direct effects of hypoxia on vascular smooth muscle contractility can vary with age, we carried out these experiments using both fetal and adult arteries. In common carotid arteries from near-term fetal and adult sheep, hypoxia (PO(2) = 9-12 Torr for 30 min) reduced the maximum responses to potassium by 17.8 +/- 3.5% (fetus) and 20.5 +/- 2.2% (adult), significantly reduced the pD(2) for 5-HT in the fetus (7.01 +/- 0.1 to 6.3 +/- 0.2) but not the adult (6.1 +/- 0.1 to 6.0 +/- 0.1), and significantly reduced 5-HT-induced maximum contractions (as % maximum response to 120 mM K(+)) not in the fetus (from 114 +/- 7 to 70 +/- 10%, not significant) but only in the adult (from 83 +/- 15 to 25 +/- 7%, P < 0.05) arteries. Hypoxia significantly attenuated 5-HT binding affinity (pK(A), determined by partial irreversible blockade with phenoxybenzamine) in both fetal (from 6.5 +/- 0.2 to 6.0 +/- 0.2) and adult arteries (from 6.2 +/- 0. 2 to 5.7 +/- 0.1) and also decreased receptor density (fmol/mg protein, determined by competitive binding with ketanserin and mesulergine) in adult (from 18.3 +/- 1.1 to 10.9 +/- 1.0) but not in fetal (21.0 +/- 1.0 to 23.2 +/- 1.4) arteries. These results suggest that acute hypoxia modulates receptor-ligand binding via age-dependent modulation of agonist affinity and receptor density. These effects may contribute to hypoxic vasodilatation and help explain why the effects of hypoxia on vascular contractility differ between fetuses and adults.     

Caldesmon phosphorylation is catalyzed by two kinases in permeabilized and intact vascular smooth muscle

Smooth muscle contraction is initiated by myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+) dependent MLC kinase. However, many aspects of smooth muscle contraction cannot be accounted for by MLC phosphorylation. One hypothesis that has received experimental support involves the thin filament protein caldesmon. Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon relieves this inhibitory effect. The primary candidates for catalysis of caldesmon phosphorylation are the p42/p44 ERK MAP kinases. However, we and others have shown that inhibition of the ERK MAP kinases has no effect on many smooth muscles. The goal of this study was to determine if evidence for a second endogenous caldesmon kinase may be obtained. We used Triton X-100 skinned and intact tissues of the swine carotid artery to address this goal. Caldesmon phosphorylation was evident in resting and Ca(2+) stimulated Triton X-100 skinned fibers. Ca(2+)-dependent caldesmon phosphorylation was partially sensitive to the ERK MAP kinase inhibitor PD98059, whereas all caldesmon phosphorylation was sensitive to the general kinase inhibitor, staurosporine. Histamine increased caldesmon phosphorylation levels in intact swine carotid artery, which was sensitive to both PD98059 and staurosporine. Histamine increased ERK MAP kinase activity, which was reversed by PD98059, staurosporine, and EGTA. Histamine-induced contractions were inhibited by staurosporine but not by PD98059. We interpret these results to suggest that although ERK MAP kinases catalyze caldesmon phosphorylation, a second staurosporine sensitive kinase is also important in caldesmon phosphorylation and it is this pathway that may be more important in contractile regulation.     

PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries

Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric tension and intracellular free calcium concentration ([Ca(2+)](i)) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 x 10(-6) M] in the absence or presence of ERK1/2 inhibition (U-0126, 10(-5) M). After PDBu +/- ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmon(Ser789), myosin light chain(20) (MLC(20)), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca(2+)](i). PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmon(Ser789) and MLC(20) levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 x 10(-7) M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC(20) phosphorylation in the thick filament pathway and increases Ca(2+) sensitivity. In addition, ERK1/2-dependent phosphorylation of caldesmon(Ser789) was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase.     

Evidence for modulation of smooth muscle force by the p38 MAP kinase/HSP27 pathway

Mitogen-activated protein (MAP) kinases signal to proteins that could modify smooth muscle contraction. Caldesmon is a substrate for extracellular signal-related kinases (ERK) and p38 MAP kinases in vitro and has been suggested to modulate actin-myosin interaction and contraction. Heat shock protein 27 (HSP27) is downstream of p38 MAP kinases presumably participating in the sustained phase of muscle contraction. We tested the role of caldesmon and HSP27 phosphorylation in the contractile response of vascular smooth muscle by using inhibitors of both MAP kinase pathways. In intact smooth muscle, PD-098059 abolished endothelin-1 (ET-1)-stimulated phosphorylation of ERK MAP kinases and caldesmon, but p38 MAP kinase activation and contractile response remained unaffected. SB-203580 reduced muscle contraction and inhibited p38 MAP kinase and HSP27 phosphorylation but had no effect on ERK MAP kinase and caldesmon phosphorylation. In permeabilized muscle fibers, SB-203580 and a polyclonal anti-HSP27 antibody attenuated ET-1-dependent contraction, whereas PD-098059 had no effect. These results suggest that ERK MAP kinases phosphorylate caldesmon in vivo but that activation of this pathway is unnecessary for force development. The generation of maximal force may be modulated by the p38 MAP kinase/HSP27 pathway.     

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.     

Postnatal maturation modulates relationships among cytosolic Ca2+, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries

The present study tests the hypothesis that age-related changes in patterns of agonist-induced myofilament Ca(2+) sensitization involve corresponding differences in the relative contributions of thick- and thin-filament regulation to overall myofilament Ca(2+) sensitivity. Posterior communicating cerebral arteries from term fetal and nonpregnant adult sheep were used in measurements of cytosolic Ca(2+), myosin light chain (MLC) phosphorylation, and contractile tensions induced by varying concentrations of K(+) or serotonin [5-hydroxytryptamine (5-HT)]. The results were used to assess the relative contributions of the relationships between cytosolic Ca(2+) and MLC phosphorylation (thick-filament reactivity), along with the relationships between MLC phosphorylation and contractile tension (thin-filament reactivity), to overall myofilament Ca(2+) sensitivity. For K(+)-induced contractions, both fetal and adult arteries exhibited similar basal myofilament Ca(2+) sensitivity. Despite this similarity, thick-filament reactivity was greater in fetal arteries, whereas thin-filament reactivity was greater in adult arteries. In contrast, 5-HT-induced contractions exhibited increased myofilament Ca(2+) sensitivity compared with K(+)-induced contractions for both fetal and adult cerebral arteries, and the magnitude of this effect was greater in fetal compared with adult arteries. When interpreted together with our previous studies of 5-HT-induced myofilament Ca(2+) sensitization, we attributed the present effects to agonist enhancement of thick-filament reactivity in fetal arteries mediated by G protein receptor activation of a PKC-independent but RhoA-dependent pathway. In adult arteries, agonist stimulation enhanced thin-filament reactivity was also probably mediated through G protein-coupled activation of RhoA-dependent and PKC-independent mechanisms. Overall, the present data demonstrate that agonist-enhanced myofilament Ca(2+) sensitivity can be partitioned into separate thick- and thin-filament effects, the magnitudes of which are different between fetal and adult cerebral arteries.     

Maturation enhances fluid shear-induced activation of eNOS in perfused ovine carotid arteries

The present study tests the hypothesis that age-dependent increases in endothelial vasodilator capacity are due to maturational increases in endothelial nitric oxide (NO) synthesis and release. Intact 4-cm carotid artery segments taken from term fetal lambs and nonpregnant adult sheep were perfused by using a closed system that enabled independent control of flow and inflow pressure and facilitated complete recovery of all NO released. Fluid shear stress induced a graded release of NO (in nmol NO x min x cm(-2) of luminal surface area) that was significantly greater in adult (890 +/- 140) than in fetal (300 +/- 40) carotid arteries at corresponding values of shear stress (5.9 +/- 0.3 dyn/cm2) but was independent of inflow pressure in both age groups. These age-related differences in NO release were not attributable to corresponding differences in endothelial NO synthase (eNOS) abundance, as eNOS protein levels (in ng of eNOS/cm2 of luminal surface area) were similar in adult (14 +/- 2) and fetal (12 +/- 1) arteries. Adult (80 +/- 15) and fetal (89 +/- 32) levels of eNOS mRNA (in 10(6) copies/cm2 of luminal surface area) were also similar. However, when NO release was normalized relative to the associated mass of eNOS protein to estimate eNOS-specific activity in situ, this value (in nmol NO x microg of eNOS(-1) x min(-1)) was significantly greater in adult (177 +/- 44) than in fetal (97 +/- 36) arteries when the endothelium was maximally activated by A-23187. Similarly, the slope of the relation between fluid shear stress and estimated eNOS-specific activity (in nmol NO x microg of eNOS(-1) x min(-1) per dyn/cm2) was also significantly greater in adult (6.8 +/- 0.1) than in fetal (2.9 +/- 0.1) arteries, which suggests that eNOS may be more sensitive to or more efficiently coupled to activating stimuli in adult compared with fetal arteries. We conclude that maturational increases in endothelial vasodilator capacity are attributable to age-dependent increases in NO release secondary to elevated eNOS-specific activity and involve more efficient coupling between endothelial activation and enhancement of eNOS activity in adult compared with fetal arteries.     

MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction

We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral alpha-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.     

Age-dependent modulation of endothelium-dependent vasodilatation by chronic hypoxia in ovine cranial arteries.

Although abundant evidence indicates that chronic hypoxia can induce pulmonary vascular remodeling, very little is known of the effects of chronic hypoxia on cerebrovascular structure and function, particularly in the fetus. Thus the present study explored the hypothesis that chronic hypoxemia also influences the size and shape of cerebrovascular smooth muscle and endothelial cells, with parallel changes in the reactivity of these cells to endothelium-dependent vasodilator stimuli. To test this hypothesis, measurements of endothelial and vascular smooth muscle cell size and density were made in silver-stained common carotid and middle cerebral arteries from term fetal and nonpregnant adult sheep maintained at an altitude of 3,820 m for 110 days. Chronic hypoxia induced an age-dependent remodeling that led to smooth muscle cells that were larger in fetal arteries but smaller in adult arteries. Chronic hypoxia also increased endothelial cell density in fetal arteries but reduced it in adult arteries. These combined effects resulted in an increased (adult carotid), decreased (adult middle cerebral), or unchanged (fetal arteries) per cell serosal volume of distribution for endothelial factors. Despite this heterogeneity, the magnitude of endothelium-dependent vasodilatation to A23187, measured in vitro, was largely preserved, although sensitivity to this relaxant was uniformly depressed. N(G)-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and endothelium denudation each independently blocked A23187-induced vasodilation without unmasking any residual vasoconstrictor effect. Indomethacin did not significantly attenuate A23187-induced relaxation except in the hypoxic adult middle cerebral, where a small contribution of prostanoids was evident. Vascular sensitivity to exogenous nitric oxide (NO) was uniformly increased by chronic hypoxia. From these results, we conclude that chronic hypoxia reduced endothelial NO release while also upregulating some component of the NO-cGMP-PKG vasodilator pathway. These offsetting effects appear to preserve endothelium-dependent vasodilation after adaptation to chronic hypoxia.     

Inhibition of cross-bridge formation has no effect on contraction-associated phosphorylation of p38 MAPK in mouse skeletal muscle

Mitogen-activated protein kinases (MAPKs), in particular p38 MAPK, are phosphorylated in response to contractile activity, yet the mechanism for this is not understood. We tested the hypothesis that the force of contraction is responsible for p38 MAPK phosphorylation in skeletal muscle. Extensor digitorum longus (EDL) muscles isolated from adult male Swiss Webster mice were stimulated at fixed length at 10 Hz for 15 min and then subjected to Western blot analysis for the phosphorylation of p38 MAPK and ERK1/2. Contralateral muscles were fixed at resting length and were not stimulated. Stimulated muscles showed a 2.5-fold increase in phosphorylated p38 MAPK relative to nonstimulated contralateral controls, and there was no change in the phosphorylation of ERK1/2. When contractile activity was inhibited with N-benzyl-p-toluene sulfonamide (BTS), a specific inhibitor of actomyosin ATPase, force production decreased in both a time- and concentration-dependent manner. Preincubation with 25, 75, and 150 µM BTS caused 78 ± 4%, 97 ± 0.2%, and 99 ± 0.2% inhibition in contractile force, respectively, and was stable after 30 min of treatment. Fluorescence measurements demonstrated that Ca²⁺ cycling was minimally affected by BTS treatment. Surprisingly, BTS did not suppress the level of p38 MAPK phosphorylation in stimulated muscles. These data do not support the view that force generation per se activates p38 MAPK and suggest that other events associated with contraction must be responsible. calcium; energetics; mechanical stress; metabolism     

Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38(MAPK) phosphorylation. Phosphorylation of ERK1/2 or p38(MAPK) was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38(MAPK) was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38(MAPK) inhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90(Rsk)), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90(Rsk) and MAPKAP-K2 most closely reflects ERK and p38(MAPK) stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38(MAPK). These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.     

Hypoxic regulation of the fetal cerebral circulation.

Fetal cerebrovascular responses to acute hypoxia are fundamentally different from those observed in the adult cerebral circulation. The magnitude of hypoxic vasodilatation in the fetal brain increases with postnatal age although fetal cerebrovascular responses to acute hypoxia can be complicated by age-dependent depressions of blood pressure and ventilation. Acute hypoxia promotes adenosine release, which depresses fetal cerebral oxygen consumption through action of adenosine on neuronal A1 receptors and vasodilatation through activation of A2 receptors on cerebral arteries. The vascular effect of adenosine can account for approximately half the vasodilatation observed in response to hypoxia. Hypoxia-induced release of nitric oxide and opioids can account for much of the adenosine-independent cerebral vasodilatation observed in response to hypoxia in the fetus. Direct effects of hypoxia on cerebral arteries account for the remaining fraction, although the vascular endothelium contributes relatively little to hypoxic vasodilatation in the immature cerebral circulation. In contrast to acute hypoxia, fetal cerebral blood flow tends to normalize during acclimatization to chronic hypoxia even though cardiac output is depressed. However, uncompensated chronic hypoxia in the fetus can produce significant changes in brain structure and function, alteration of respiratory drive and fluid balance, and increased incidence of intracranial hemorrhage and periventricular leukomalacia. At the level of the fetal cerebral arteries, chronic hypoxia increases protein content and depresses norepinephrine release, contractility, and receptor densities associated with contraction but also attenuates endothelial vasodilator capacity and decreases the ability of ATP-sensitive and calcium-sensitive potassium channels to promote vasorelaxation. Overall, fetal cerebrovascular adaptations to chronic hypoxia appear prioritized to conserve energy while preserving basic contractility. Many gaps remain in our understanding of how the effects of acute and chronic hypoxia are mediated in fetal cerebral arteries, but studies of adult cerebral arteries have produced many powerful pharmacological and molecular tools that are simply awaiting application in studies of fetal cerebral artery responses to hypoxia.     

Role of cAMP-dependent protein kinase A activity in endothelial cell cytoskeleton rearrangement

To examine signaling mechanisms relevant to cAMP/protein kinase A (PKA)-dependent endothelial cell barrier regulation, we investigated the impact of the cAMP/PKA inhibitors Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and PKA inhibitor (PKI) on bovine pulmonary artery and bovine lung microvascular endothelial cell cytoskeleton reorganization. Rp-cAMPS as well as PKI significantly increased the formation of actin stress fibers and intercellular gaps but did not alter myosin light chain (MLC) phosphorylation, suggesting that the Rp-cAMPS-induced contractile phenotype evolves in an MLC-independent fashion. We next examined the role of extracellular signal-regulated kinases (ERKs) in Rp-cAMPS- and PKI-induced actin rearrangement. The activities of both ERK1/2 and its upstream activator Raf-1 were transiently enhanced by Rp-cAMPS and linked to the phosphorylation of the well-known ERK cytoskeletal target caldesmon. Inhibition of the Raf-1 target ERK kinase (MEK) either attenuated or abolished Rp-cAMPS- and PKI-induced ERK activation, caldesmon phosphorylation, and stress fiber formation. In summary, our data elucidate the involvement of the p42/44 ERK pathway in cytoskeletal rearrangement evoked by reductions in PKA activity and suggest the involvement of significant cross talk between cAMP- and ERK-dependent signaling pathways in endothelial cell cytoskeletal organization and barrier regulation.     

MicroRNA-31 regulated by the extracellular regulated kinase is involved in vascular smooth muscle cell growth via large tumor suppressor homolog 2

Aberrant growth of vascular smooth muscle cells (VSMCs) is a major cellular event in the pathogenesis of many proliferative vascular diseases. Recently, microRNA-31 (miR-31) has been found to play a critical role in cancer cell proliferation. However, the biological role of miR-31 in VSMC growth and the mechanisms involved are currently unknown. In the present study, the expression of rat mature miR-31 (rno-miR-31) was determined in cultured VSMCs and in rat carotid arteries. We identified that rno-miR-31 is an abundant miRNA in VSMCs, and its expression was significantly increased in proliferative VSMCs and in vascular walls with neointimal growth. The up-regulation of rno-miR-31 in proliferative VSMCs was inhibited by the inhibitor of mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK). By both gain-of-function and loss-of-function approaches, we demonstrated that rno-miR-31 had a proproliferative effect on VSMCs. We further identified that LATS2 (large tumor suppressor homolog 2) is a downstream target gene product of rno-miR-31 that is involved in rno-miR-31-mediated effect on VSMC proliferation. The LATS2 as a target gene protein of rno-miR-31 is verified in vivo in balloon-injured rat carotid arteries. The results suggest that MAPK/ERK/miR-31/LATS2 may represent a novel signaling pathway in VSMC growth. miR-31 is able to enhance VSMC proliferation via its downstream target gene product, LATS2.     

Time-dependent potentiation of contractile response to norepinephrine in canine isolated cerebral arteries.

The time course of contractile responses to alpha-adrenoceptor agonists was investigated using various arteries isolated from dogs and monkeys. The contractile response to norepinephrine was increased during the time course of the experiment in canine basilar and internal carotid arteries, whereas the response of isolated canine external carotid arteries and monkey internal carotid arteries did not change significantly. Treatment with 10(-7) M propranolol, 5 x 10(-6) M cocaine plus 10(-5) M hydrocortisone, or 5 x 10(-5) M acetylsalicylic acid did not significantly affect the time-dependent potentiation of the norepinephrine-induced contraction in canine internal carotid arteries. The time-dependent enhancement in the response to norepinephrine was also observed in the arterial preparations from which the endothelial cells were removed. The contractile response of canine internal carotid arteries to phenylephrine did not alter significantly throughout the experiments. On the other hand, the responses to clonidine and xylazine were markedly enhanced with time. Significant potentiation of the norepinephrine-induced contraction was observed in canine internal carotid arteries treated with 10(-8) M prazosin, whereas 10(-8) M yohimbine attenuated the time-dependent potentiation. These results suggest that the contractile responses of isolated canine basilar and internal carotid arteries to norepinephrine are potentiated during the course of the experiment, which is likely to be related, in part, to an enhancement in alpha 2-adrenoceptor mediated contraction.     

Focal adhesion signaling is required for myometrial ERK activation and contractile phenotype switch before labor

In late pregnancy rapidly increasing fetal growth dramatically increases uterine wall tension. This process has been implicated in the activation of the myometrium for labor, but the mechanisms involved are unclear. Here, we tested, using a rat model, the hypothesis that gestation-dependent stretch, via activation of focal adhesion signaling, contributes to the published activation of myometrial ERK at the end of pregnancy. Consistent with this hypothesis, we show here that ERK is targeted to adhesion plaques during late pregnancy. Furthermore, myometrial stretch triggers a dramatic increase in myometrial contractility and ERK and caldesmon phosphorylation, confirming the presence of stretch sensitive myometrial signaling element. Screening by anti-phosphotyrosine immunoblotting for focal adhesion signaling in response to stretch reveals a significant increase in the tyrosine phosphorylated bands identified as focal adhesion kinase (FAK), A-Raf, paxillin, and Src. Pretreatment with PP2, a Src inhibitor, significantly suppresses the stretch-induced increases in FAK, paxillin, Src, ERK and caldesmon phosphorylation and myometrial contractility. Thus, focal adhesion-Src signaling contributes to ERK activation and promotes contraction in late pregnancy. These results point to focal adhesion signaling molecules as potential targets in the modulation of the myometrial contractility and the onset of labor.     

Fetal and adult cerebral artery K(ATP) and K(Ca) channel responses to long-term hypoxia.

High-altitude long-term hypoxia (LTH) alters cerebral vascular contractile and relaxation responses in both fetus and adult. We tested the hypotheses that LTH-mediated vascular responses were secondary to altered K+ channel function and that in the fetus these responses differ from those of the adult. In middle cerebral arteries (MCA) from both nonpregnant adult and fetal (approximately 140 days gestation) sheep, which were either acclimatized to high altitude (3,820 m) or sea-level controls, we measured norepinephrine (NE)-induced contractions and intracellular Ca2+ concentration ([Ca2+]i) simultaneously, in the presence or absence of different K+ channel openers or blockers. In adult MCA, LTH was associated with approximately 20% decrease in NE-induced tension and [Ca2+]i, with a significant increase in Ca2+ sensitivity. In contrast, in fetal MCA, LTH failed to affect significantly NE-induced contraction or [Ca2+]i but significantly decreased the ATP-sensitive K+ (K(ATP)) channel and Ca2+-activated K+ (K(Ca)) channel-mediated relaxation. The significant effect of K(ATP) and K(Ca) channel activators on the relaxation responses and the fact that K+ channels play a key role in myogenic tone support the hypotheses that K+ channels play an important role in hypoxia-mediated responses. These results also support the hypothesis of significant developmental differences with maturation from fetus to adult.