Lindsay Queen

Activation of nitric oxide synthase by β2‐adrenoceptors in human umbilical vein endothelium in vitro

Some animal studies suggest that β‐adrenoceptor‐mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of β2‐adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) inhibits this response. The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial β‐adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine‐3′,5′‐monophosphate (cyclic AMP). Vasorelaxant responses were determined in umbilical vein rings to the nonselective β‐adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2α. NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline, and adenylyl cyclase activity by the conversion of [α‐32P]‐ATP to [32P]‐cyclic AMP. Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by β2‐ (but not β1‐) adrenergic blockage, and by endothelium removal or 1 mM L‐NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L‐NMMA, with a reduction in Emax from 90.0±9.3% to 50.5±9.9% (P<0.05). Isoprenaline 1 μM increased NOS activity in HUVEC (34.0±5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration‐dependent manner; this response was inhibited by β2 (but not β1‐) adrenergic blockade. Forskolin 1 μM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 μM. The increase in L‐arginine to L‐citrulline conversion observed with each agent was abolished by co‐incubation with NOS inhibitors. These results indicate that endothelial β2‐adrenergic stimulation and cyclic AMP elevation activate the L‐arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.

Nitric oxide-dependent β2-adrenergic dilatation of rat aorta is mediated through activation of both protein kinase A and Akt

Vasorelaxation to β2‐adrenoceptor stimulation occurs through both endothelium‐dependent and endothelium‐independent mechanisms, and the former is mediated through Ca2+‐independent activation of endothelial‐type nitric oxide synthase (NOS‐3). Since Ca2+‐independent NOS‐3 activation may occur through its serine phosphorylation via protein kinase A (PKA) or Akt, we determined the PKA and Akt dependency of β2‐adrenergic relaxation of rat aorta. Rat aortic rings were pre‐incubated with the PKA inhibitor H‐89 (10−7 M), the phosphatidylinositol 3‐kinase (PI3K) inhibitor wortmannin (5 × 10−7 M), Akt inhibitor (10−5 M), or vehicle, in the absence or presence of the NOS inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 10−4 M). Rings were then contracted with phenylephrine (10−7 M), and concentration–relaxation responses determined to the β2‐adrenoceptor agonist albuterol. Rings exhibited a concentration‐dependent relaxation to albuterol: pEC50 6.9±0.2, Emax 88.2±4.0%. L‐NAME attenuated Emax to 60.2±3.5% (P<0.001). In the presence of L‐NAME, wortmannin or Akt inhibitor did not influence albuterol responses, whereas H‐89 reduced Emax further, to 27.5±2.2% (P<0.001). In the absence of L‐NAME, Emax to albuterol was reduced by H‐89, wortmannin or Akt inhibitor, to 56.2±2.2, 56.0±1.6 and 55.4±1.8%, respectively (P<0.001 for each); the combinations H‐89 plus wortmannin or H‐89 plus Akt inhibitor reduced Emax further still. Western blotting of NOS‐3 immunoprecipitates from rat aortas confirmed that albuterol increased serine phosphorylation of NOS‐3, and this increase was attenuated by H‐89 or Akt inhibitor. Our results indicate that β2‐adrenoceptor stimulation relaxes rat aorta through both NO‐dependent and independent mechanisms. The latter is predominantly PKA‐mediated, whereas the former occurs through both PKA and PI3K/Akt activation.

β2-Adrenoceptors Activate Nitric Oxide Synthase in Human Platelets

Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet beta-adrenoceptors (betaARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, after betaAR stimulation or cAMP elevation. Basal NOS activity was 0.11+/-0.03 pmol L-citrulline/10(8) platelets. The betaAR agonist isoproterenol 1 micromol/L and the adenylyl cyclase activator forskolin 1 micromol/L each increased NOS activity, to 0.26+/-0.04 and 0.23+/-0.03 pmol L-citrulline/10(8) platelets, respectively (P<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 micromol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca(2+). In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3+/-0.2% versus 2.6+/-0.2% respectively; P<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through beta(2)ARs, as determined by coincubation with betaAR subtype-selective antagonists. We conclude that beta(2)ARs activate platelet NOS by increasing cAMP, and that this activation is Ca(2+)-independent. beta(2)ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the L-arginine/NO system mediates the effects of beta(2)ARs on adhesion but not aggregation.

Mechanisms underlying β2‐adrenoceptor‐mediated nitric oxide generation by human umbilical vein endothelial cells

Endothelial β2‐adrenoceptor (β2AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l‐arginine transport and of phosphorylation of NO synthase 3 (NOS‐3) in β2AR‐mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l‐arginine uptake, NOS activity (from l‐arginine to l‐citrulline conversion), membrane potential (using [3H]tetraphenylphosphonium), as well as serine phosphorylation of NOS‐3 (by Western blotting and mass spectrometry), in HUVEC treated with βAR agonists or cyclic AMP‐elevating agents. β2AR stimulation increased l‐arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N‐ethylmaleimide. Blockade of l‐arginine uptake by l‐lysine inhibited NOS activity and, conversely, blockade of NOS using Nω‐nitro‐l‐arginine methyl ester (l‐NAME) inhibited l‐arginine transport. β2AR stimulation also caused a membrane hyperpolarization inhibitable by l‐NAME, suggesting that the increase in l‐arginine uptake occurred in response to NO‐mediated hyperpolarization. β2AR activation also increased NOS activity and phosphorylation of NOS‐3 on serine‐1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3‐kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that β2AR‐mediated NOS‐3 activation in HUVEC is mediated through phosphorylation of NOS‐3 on serine‐1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l‐arginine uptake resulting from NO‐mediated membrane hyperpolarization.

Amlodipine, but not verapamil or nifedipine, dilates rabbit femoral artery largely through a nitric oxide- and kinin-dependent mechanism

We investigated the nitric oxide (NO) dependence of vasorelaxation in response to different calcium channel blockers (CCB), in rabbit femoral artery in vivo. Anaesthetized rabbits underwent femoral artery ligation, and blood from the proximal artery was returned distal to the ligature through a constant infusion pump. The effects of local injection of CCB on perfusion pressure and plasma nitrite+nitrate (NOx, which reflects local NO biosynthesis) concentration in this system were determined. Intra‐arterial verapamil, nifedipine or amlodipine 10 μmol kg−1 each reduced perfusion pressure. Pre‐treatment with intra‐arterial NG‐nitro‐L‐arginine methyl ester (L‐NAME, a NO synthase inhibitor) 1 μmol kg−1 did not affect responses to verapamil or nifedipine, but attenuated the reduction in perfusion pressure to amlodipine, from 33.2±2.1% to 22.5±1.6% (P=0.002). Intra‐arterial amlodipine  –  unlike verapamil or nifedipine  –  increased femoral venous NOx, from 9.1±0.4 μM to 14.1±0.5 μM (P=0.005). The bradykinin B2 receptor antagonist HOE 140, 30 mg kg−1, attenuated the reduction in perfusion pressure and abolished the rise in venous NOx concentration, following intra‐arterial amlodipine. Amlodipine potently inhibited serum angiotensin converting‐enzyme (ACE) activity in vitro, as effectively as enalapril at similar concentrations. These results suggest that the vasorelaxant effects of nifedipine and verapamil are NO‐independent, whereas those of amlodipine are partly NO‐dependent, in rabbit femoral artery in vivo. This effect of amlodipine occurs through B2 receptor activation, and may be related to an increase in local bradykinin through inhibition of ACE.

β-Adrenergic receptors and nitric oxide generation in the cardiovascular system

Abstract.Nitric oxide plays a crucial role in cardiovascular homeostasis, with important vasodilatory, anti-thrombotic and anti-atherogenic properties. β-Adrenergic receptors (βARs), present on a wide variety of cardiovascular cells, including vascular endothelial cells, platelets, cardiac myocytes and leukocytes, have long been established as key players in maintaining cardiovascular homeostatic control. During the last few years a wealth of evidence has emerged which directly links stimulation of these cardiovascular βARs to nitric oxide (NO) generation, suggesting a new and important mechanism of adrenergic control of cardiovascular function. This review explores the cardiovascular cell systems in which this coupling of βARs and NO occurs, the intracellular signalling and regulatory mechanisms involved and the abnormalities in βAR-NO oxide coupling found in cardiovascular disease states.

β-Actin regulates platelet nitric oxide synthase 3 activity through interaction with heat shock protein 90

Cytoskeletal proteins are crucial in maintaining cellular structure and, in certain cell types, also play an essential role in motility and shape change. Nitric oxide (NO) is an important paracrine mediator of vascular and platelet function and is produced in the vasculature by the enzyme NO synthase type 3 (NOS-3). Here, we demonstrate in human platelets that the polymerization state of β-actin crucially regulates the activation state of NOS-3, and hence NO formation, through altering its binding of heat shock protein 90 (Hsp90). We found that NOS-3 binds to the globular, but not the filamentous, form of β-actin, and the affinity of NOS-3 for globular β-actin is, in turn, increased by Hsp90. Formation of this ternary complex among NOS-3, globular β-actin, and Hsp90, in turn, results in an increase in both NOS activity and cyclic guanosine-3′,5′-monophosphate, an index of bioactive NO, as well as an increased rate of Hsp90 degradation, thus limiting the duration for which NOS-3 remains activated. These observations suggest that β-actin plays a critical role in regulating NO formation and signaling in platelets.

Aspirin acetylates nitric oxide synthase type 3 in platelets thereby increasing its activity

AIMS Acute administration of aspirin increases nitric oxide (NO) synthesis by platelets, an effect not shared by other non-steroidal anti-inflammatory drugs. The aim of the present study was to determine the mechanism by which aspirin acutely increases the activity of NO synthase type 3 (NOS-3), the predominant NOS isoform expressed by platelets, and specifically whether this occurs through an increase in its acetylation. METHODS AND RESULTS Platelets isolated from the blood of healthy human subjects were exposed in vitro to vehicle or aspirin at different concentrations (5 micromol/L-4 mmol/L). Changes in intraplatelet Ca(2+) concentration were determined from fura-2 fluorescence. Following immunoprecipitation of NOS-3 from platelet lysates, its activity was determined from l-[(3)H]arginine to l-[(3)H]citrulline conversion, and its serine phosphorylation quantified by western blotting. Acetylation of NOS-3 in platelets was assessed by the incorporation of radioactivity into the immunoprecipitated enzyme from [acetyl-(14)C]aspirin. Following transfection of HeLa cells with NOS-3, NO biosynthesis in response to aspirin was determined from cyclic GMP measurement, and sites of NOS-3 acetylation were ascertained by liquid chromatography-tandem mass spectrometry. At all concentrations tested, aspirin increased the activity of NOS-3 from platelets. This was not associated with any measurable change in intraplatelet Ca(2+) concentration. Serine phosphorylation of NOS-3 in platelets was decreased, and this was especially marked for serine-1177 phosphorylation, whereas acetylation of NOS-3 was increased, by aspirin incubation. HeLa cells transfected with NOS-3 exhibited an increase in NO biosynthesis following aspirin exposure, and this was associated with acetylation of the enzyme on both serine-765 and serine-771. CONCLUSION Aspirin acetylates NOS-3 acutely in platelets, and this causes an increase in its activity as well as a decrease in its phosphorylation. It is also possible that aspirin indirectly affects NOS-3 activity by acetylating other substrates within the platelet, but this remains to be determined.

Aspirin modifies nitric oxide synthase activity in platelets: effects of acute versus chronic aspirin treatment

OBJECTIVE We examined the effects of aspirin on basal and beta-adrenoceptor (beta-AR)-mediated nitric oxide synthase (NOS) activity in normal platelets. METHODS NOS activity was determined from the conversion of L-[3H]arginine to L-[3H]citrulline, both basally and following beta-AR stimulation, in platelets from healthy human subjects following both short- and long-term aspirin administration. RESULTS Basal L-[3H]citrulline increased following aspirin 800 mg administered intravenously in vivo, from 0.31+/-0.12 to 0.76+/-0.14 pmol/10(8) platelets (P<0.01). Isoproterenol at 1 micromol/l increased platelet NOS activity before but not following intravenous aspirin. After short-term in vitro treatment with aspirin 10 micromol/l, 400 micromol/l or 4 mmol/l, basal platelet L-[3H]citrulline increased similarly, an effect not seen with indomethacin 100 micromol/l or ibuprofen 10 micromol/l. Platelet NOS activity was not increased by albuterol 1 micromol/l, in the presence of indomethacin, ibuprofen or aspirin in vitro. By contrast, oral aspirin 75 mg daily for 14 days did not affect basal platelet NOS activity, but abolished beta-adrenergic NOS activation. CONCLUSIONS Aspirin activates basal platelet NOS acutely, but not chronically, through a mechanism independent of cyclooxygenase (COX) inhibition. By contrast, both short- and long-term aspirin treatment inhibit platelet beta-adrenergic NOS activation by a COX-dependent mechanism. This indicates that aspirin exerts divergent effects on basal and beta-AR-stimulated platelet NOS activity, which are likely to be of clinical relevance.

Increased platelet aggregation in vivo in the Zucker Diabetic Fatty rat: differences from the streptozotocin diabetic rat

Diabetes mellitus, especially type 2, is associated with increased arterial thrombosis. Our aims were (i) to characterize and compare platelet aggregation in vivo and in vitro in a type 2 diabetes model; and (ii) to determine whether these results differ from those in a type 1 diabetes model.