Vasken Ohanian

Identification of Protein Kinase C Isoforms in Rat Mesenteric Small Arteries and Their Possible Role in Agonist-Induced Contraction

We have identified immunologically the protein kinase C (PKC) isoforms present in rat mesenteric small arteries, defined their distribution between particulate and soluble fractions, and studied their involvement in phorbol ester-induced contraction. Our analysis revealed the presence of the CA(2+)-dependent PKCs (alpha and gamma), Ca(2+)-independent PKCs (delta and epsilon), and the atypical isoform (zeta). PKCbeta could not be detected, whereas PKCgamma is likely to be of neural origin. All isoforms exhibited different distributions. PKCalpha, PKCepsilon, and PKCzeta were found in both particulate and soluble fractions. In contrast, PKCdelta was mainly in the particulate fraction, and PKCgamma was in the soluble fraction. Phorbol esters, which activate PKC and cause smooth muscle contraction, downregulated only the alpha and delta isoforms. This was associated with a parallel loss of contractile response to phorbol ester. The force developed to submaximal concentrations of noradrenaline was decreased after phorbol dibutyrate pretreatment, although the sensitivity and maximal response were unchanged. Phorbol ester pretreatment did not affect the contractile response to vasopressin. The sensitivity to non-receptor-mediated contraction, caused by k+ in the presence of prazosin, was slightly reduced by 4 alpha- and 4 beta-phorbol ester pretreatment. Maximal tension in response to this agonist was not affected. We conclude that PKCalpha and/or PKCdelta is necessary for phorbol ester-mediated contraction but is not essential for noradrenaline-, vasopressin-, or k(+)-induced contraction, demonstrating differences in the mechanisms involved in the contractile response between these agents.

Receptor Tyrosine Kinase Axl Modulates the Osteogenic Differentiation of Pericytes

Abstract— Vascular pericytes undergo osteogenic differentiation in vivo and in vitro and may, therefore, be involved in diseases involving ectopic calcification and osteogenesis. The purpose of this study was to identify factors that inhibit the entry of pericytes into this differentiation pathway. RNA was prepared from pericytes at confluence and after their osteogenic differentiation (mineralized nodules). Subtractive hybridization was conducted on polyA PCR-amplified RNA to isolate genes expressed by confluent pericytes that were downregulated in the mineralized nodules. The subtraction product was used to screen a pericyte cDNA library and one of the positive genes identified was Axl, the receptor tyrosine kinase. Northern and Western blotting confirmed that Axl was expressed by confluent cells and was downregulated in mineralized nodules. Western blot analysis demonstrated that confluent pericytes also secrete the Axl ligand, Gas6. Immunoprecipitation of confluent cell lysates with an anti-phosphotyrosine antibody followed by Western blotting using an anti-Axl antibody, demonstrated that Axl was active in confluent pericytes and that its activity could not be further enhanced by incubating the cells with recombinant Gas6. The addition of recombinant Axl-extracellular domain (ECD) to pericyte cultures inhibited the phosphorylation of Axl by endogenous Gas6 and enhanced the rate of nodule mineralization. These effects were inhibited by coincubation of pericytes with Axl-ECD and recombinant Gas6. Together these results demonstrate that activation of Axl inhibits the osteogenic differentiation of vascular pericytes.

αV Integrins Are Necessary for Eutrophic Inward Remodeling of Small Arteries in Hypertension

Human essential hypertension is characterized by eutrophic remodeling of small arteries, with little evidence of hypertrophy. Likewise, vessels of young hypertensive TGR(mRen2)27 animals have undergone similar structural alterations. The role of integrins in resistance arteries of TGR(mRen2)27 during the eutrophic-remodeling process was examined as blood pressure rose. Initially, 8 &agr; and 3 &bgr; integrins were identified and levels of expression investigated using RT-PCR. As pressure increased and remodeling advanced, integrin expression profiles revealed that only &agr;V was significantly raised. In conjunction, we confirmed elevated integrin &agr;V protein levels in TGR(mRen2)27 rat arteries and localization to the media using immunofluorescence. &bgr;1 and &bgr;3, but not &bgr;5 integrin subunits were coprecipitated with integrin &agr;V and are implicated in the eutrophic remodeling process. Administration of a peptide antagonist of &agr;V&bgr;3 abolished remodeling but enhanced growth, indicating that hypertrophy supervened as a response to hypertension-induced increases in wall stress. We have established that the only upregulated integrin, the &agr;V subunit of integrin &agr;V&bgr;3, has a crucial role in the hypertensive remodeling process of TGR(mRen2)27 rat resistance arteries. During hypertensive remodeling, functions of specific &agr;V&bgr;3-extracellular matrix interactions are likely to allow vascular smooth muscle cell–length autoregulation, which includes a migratory process, to maintain a narrowed lumen after a prolonged constricted state.

Involvement of tyrosine phosphorylation in endothelin-1-induced calcium-sensitization in rat small mesenteric arteries

1 We have studied the effect of endothelin‐1 stimulation on protein tyrosine phosphorylation levels in intact small mesenteric arteries of the rat and investigated the effects of tyrosine kinase inhibition on the contractile response to this agonist. 2 Endothelin‐1 stimulated a rapid (20 s), sustained (up to 20 min) and concentration‐dependent (1‐100 nM) increase in protein tyrosine phosphorylation levels which coincided temporally with the contractile response in intact and α‐toxin permeabilized small artery preparations. Tyrosine phosphorylation was increased in four main clusters of proteins of apparent molecular mass 28–33, 56–61, 75–85 and 105–115 kDa. Endothelin‐1‐induced protein tyrosine phosphorylation was independent of extracellular calcium, antagonized by the tyrosine kinase inhibitor tyrphostin A23 but not by the inactive tyrphostin Al. 3 In intact small arteries tyrphostin A23 inhibited the force developed to endothelin‐1 at all concentrations studied; at higher concentrations (10 and 100 nM) the profile of contraction was altered from a sustained to a transient response. Tyrphostin Al inhibited the contractile response to endothelin‐1 at all concentrations except 100 nM; the profile of the response was not altered. Neither tyrphostin affected the transient phasic contraction induced by endothelin‐1 (100 nM) in the absence of extracellular calcium. 4 In rat α‐toxin permeabilized mesenteric arteries endothelin‐1 caused a concentration‐dependent increase in force in the presence of 10 μM GTP and low (pCa 6.7) constant calcium, demonstrating increased sensitivity of the contractile apparatus to calcium. Tyrphostin A23 inhibited this response by approximately 50%, tyrphostin Al did not affect endothelin‐1‐induced calcium sensitization of force. 5 We conclude that increased tyrosine phosphorylation is important in the contractile response induced by endothelin‐1 in intact small mesenteric arteries. Furthermore our data implicate activation of this signalling pathway in the tonic phase of contraction possibly through modulation of the sensitivity of the contractile apparatus to calcium.

Role of the Actin Cytoskeleton in G-Protein–Coupled Receptor Activation of PYK2 and Paxillin in Vascular Smooth Muscle

Dynamic remodeling of the actin cytoskeleton occurs during agonist-induced smooth muscle contraction. Tyrosine phosphorylation of the adaptor protein paxillin has been implicated in regulation of actin filament formation and force development. We have investigated the role of the actin cytoskeleton in noradrenaline (NA)-induced and endothelin (ET)-induced activation of the calcium-dependent nonreceptor tyrosine kinase PYK2 and subsequent phosphorylation of paxillin in rat small mesenteric arteries. NA and ET induced a rapid and prolonged activation of PYK2, as shown by increased phosphorylation at Y402 and Y881, and a concomitant association of the kinase with a Triton X-100 insoluble membrane (cytoskeleton) compartment. Both agonists also increased phosphorylation of paxillin at Y31 and Y118 with a similar time course as PYK2 phosphorylation, and induced its association with the same membrane compartment as PYK2. Treatment of arteries with cytochalasin D disrupted stress fibers and inhibited NA-induced and ET-induced force in a myosin light chain 20 phosphorylation independent and reversible manner. However, cytochalasin D treatment had no effect on NA-induced and ET-induced phosphorylation of either PYK2 or paxillin but did prevent their association with the TritonX-100 insoluble membrane compartment. These results show that in mesenteric arteries an intact cytoskeleton and force development are not prerequisites for G-protein–coupled receptor–induced activation of PYK2 and paxillin, by tyrosine phosphorylation, in vascular tissue, but are necessary for the translocation of PYK2 and paxillin to the membrane.

Noradrenaline-induced paxillin phosphorylation, ERK activation and MEK-regulated contraction in intact rat mesenteric arteries.

In rat mesenteric arteries, noradrenaline (NA) induces a time-dependent increase in tyrosine phosphorylation of a number of proteins, one of which was identified as paxillin. NA-induced protein tyrosine phosphorylation was ablated by tyrosine kinase inhibition, virtually unaffected by protein kinase C (PKC) inhibition or PKC downregulation and was mimicked by KCl. NA also caused a time-dependent activation of the extracellular signal-regulated kinases (ERK)1 and ERK2. These responses were blocked by the ERK-activating kinase (MEK) inhibitor PD98059 and by tyrosine kinase inhibition but only modestly attenuated by PKC downregulation or inhibition. Pretreatment of cannulated mesenteric arteries (50 mm Hg internal pressure) with PD98059 significantly reduced the contractile responsiveness of the vessels to NA (1.56 ± 0.14 µM, EC50 control; 3.32 ± 0.49 µM, EC50 + PD98059, p < 0.01). Thus, NA induces time-dependent increases in protein-tyrosine phosphorylation and ERK activation in rat mesenteric arteries that could suggest a role for Ca2+-dependent non-receptor tyrosine kinases and ERKs in the response of small arteries to NA. In addition, the modulation of NA-induced mesenteric artery contraction by inhibition of the MEK/ERK pathway further implicates ERK in the regulation of, though perhaps not the mediation of NA-induced small artery contraction.

Phospholipase C-δ1 modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1

Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-delta(1), a PLC isoform implicated in alpha(1)-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced PIP(2) hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with (33)Pi or myo-[(3)H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-delta(1) was localized by immunoblot analysis and neutralized by delivery of PLC-delta(1) antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-delta(1) was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-delta(1) levels in these domains. PLC-delta(1) is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [(33)P]PIP(2) hydrolysis and [(3)H]inositol phosphate formation but with no effect on ET-1-induced [(33)P]PIP(2) hydrolysis. Neutralization of PLC-delta(1) by PLC-delta(1) antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-delta(1) antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-delta(1) in NA-induced PIP(2) hydrolysis and sustained contraction in intact vascular tissue.

MNAR functionally interacts with both NH2- and COOH-terminal GR domains to modulate transactivation

Glucocorticoids are potent anti-inflammatory agents, acting through the glucocorticoid receptor (GR) to regulate target gene transcription. However, GR may also exert acute effects, including activation of signaling kinases such as c-Src and protein kinase B, possibly via the scaffold protein, modulator of nongenomic action of the estrogen receptor (MNAR). MNAR inhibited GR transactivation in A549 cells, but in HEK293 cells there was a ligand concentration-dependent biphasic effect. Transactivation driven by low ligand concentrations was inhibited by MNAR expression, whereas higher ligand concentrations were potentiating. Further analysis revealed that MNAR inhibited transactivation by the ligand-independent activation function (AF)1 but potentiated the COOH-terminal AF2 domain. The effect of MNAR was independent of c-Src activity, demonstrated by inhibitors and c-Src knockdown studies. In support of the role of MNAR in modulating GR transactivation, coimmunoprecipitation studies showed interaction between MNAR and GR in the nucleus but not the cytoplasm. Furthermore, MNAR and c-Src were also found to physically interact in the nucleus. Immunofluorescence studies showed MNAR to be predominantly a nuclear protein, with significant colocalization with GR. Deletion studies revealed that MNAR 884-1130 was coimmunoprecipitated with GR, and furthermore this fragment inhibited GR transactivation function when overexpressed. In addition, MNAR 1-400, which contains multiple LxxLL motifs, also inhibited GR transactivation. Taken together, MNAR interacts with GR in the nucleus but not cytoplasm and regulates GR transactivation in a complex manner depending on cell type. MNAR is capable of regulating both AF1 and AF2 functions of the GR independently. MNAR expression is likely to mediate important cell variation in glucocorticoid responsiveness, in a c-Src-independent mechanism.

Age-related remodeling of small arteries is accompanied by increased sphingomyelinase activity and accumulation of long-chain ceramides.

The structure and function of large arteries alters with age leading to increased risk of cardiovascular disease. Age‐related large artery remodeling and arteriosclerosis is associated with increased collagen deposition, inflammation, and endothelial dysfunction. Bioactive sphingolipids are known to regulate these processes, and are also involved in aging and cellular senescence. However, less is known about age‐associated alterations in small artery morphology and function or whether changes in arterial sphingolipids occur in aging. We show that mesenteric small arteries from old sheep have increased lumen diameter and media thickness without a change in media to lumen ratio, indicative of outward hypertrophic remodeling. This remodeling occurred without overt changes in blood pressure or pulse pressure indicating it was a consequence of aging per se. There was no age‐associated change in mechanical properties of the arteries despite an increase in total collagen content and deposition of collagen in a thickened intima layer in arteries from old animals. Analysis of the sphingolipid profile showed an increase in long‐chain ceramide (C14–C20), but no change in the levels of sphingosine or sphingosine‐1‐phosphate in arteries from old compared to young animals. This was accompanied by a parallel increase in acid and neutral sphingomyelinase activity in old arteries compared to young. This study demonstrates remodeling of small arteries during aging that is accompanied by accumulation of long‐chain ceramides. This suggests that sphingolipids may be important mediators of vascular aging.

Endothelin-1 stimulates hydrolysis of phosphatidylcholine by phospholipases C and D in intact rat mesenteric arteries.

A characteristic of endothelin-1 (ET-1)-induced contraction is the prolonged duration of the response. Phosphatidylcholine (PC) hydrolysis has been implicated in sustained agonist-induced effects through the formation of the second messengers phosphatidic acid (PA) and diacylglycerol (DAG). Therefore, we have investigated the activation of PC-phospholipase D (PLD) and PC-phospholipase C (PLC) in intact rat mesenteric small arteries stimulated with ET-1 and determined whether PC-derived DAG is necessary for ET-1-induced contraction. PLD activity, measured as phosphatidylethanol (PEt) production in vessels labelled with [3H] or [14C]myristate, was increased in a concentration- and time-dependent manner following ET-1 stimulation, as were [3H]PA levels, peaking at 10 min (ET-1 100 nM) and remaining above control levels for up to 20 min. Inclusion of 0.5% ethanol during ET-1 stimulation reduced [3H]PA levels, but did not alter the time course of formation. In addition, [14C]choline release was increased, confirming PLD-mediated PC hydrolysis. In contrast, DAG levels, measured by [3H]myristate labelling and mass assay, increased transiently at 5 min of ET-1 stimulation only. Analysis of the subclasses of DAG demonstrated an increase in all types of DAG without any enrichment with arachidonate-containing species, indicating PC, not inositol lipid hydrolysis was the source of DAG. The PC-PLC inhibitor D609, 2.5 µg/ml, completely abolished the ET-1-induced increase in [14C]DAG without affecting the increase in [14C]PA, PLD activity or the contractile response. In [14C]choline-labelled vessels, [14C]phosphocholine levels were increased by ET-1 with a similar time course to DAG production. Removal of extracellular Ca2+ and addition of 0.1 or 2 mM EGTA completely inhibited ET-1-stimulated PLD activity. The tyrosine kinase inhibitor tyrphostin A23 (100 µM) abolished ET-1-induced [3H]PA, [3H]PEt and [14C]DAG increases, whilst the negative analogue A1 was without effect. These data suggest that ET-1 couples via a calcium-dependent tyrosine kinase mechanism to PLD and PC-PLC in vascular smooth muscle. PC-derived DAG did not appear to be necessary for the contractile response, whereas sustained formation of PA, generated by PLD activity, implies that this lipid second messenger could be involved in the prolonged contractile response to this peptide.