A pharmacological study on the mechanism of the endothelin-induced contraction of the bovine cerebral artery]

Using helical strips of the bovine middle cerebral arteries, changes in vascular tension were measured during isometric contractions induced by endothelin. 1) Both Ca(++)-free media and Ca(++)-antagonists depressed the endothelin-induced contractions only by 40% of the control, suggesting the involvement of both Ca(++)-entry from outside the muscle cell and intracellular Ca(++)-release from the sarcoplasmic reticulum. 2) Endothelin-induced contractions were significantly depressed by 1 microgram/ml tetrodotoxin (TTX). Relative size of depression by TTX was practically the same as that observed in Na(+)-free media without TTX. These results indicated a partial involvement of Na(+)-entry through TTX-sensitive Na(+)-channels. 3) Endothelin-induced contractions were effectively depressed by NCDC, an inhibitor of phospholipase C, suggesting the involvement of PI-turnover in the contraction. 4) Protein kinase inhibitors such as H-7 and H-8 effectively depressed endothelin-induced contractions. This result suggested the phosphorylation of a certain protein by protein kinase C as a cause of long lasting contractions. 5) A phospholipase A2 (PL A2) inhibitor, quinacrine, significantly depressed the endothelin-induced contractions, suggesting a possible involvement of PL A2. However, neither the cyclooxygenase inhibitor nor the lipoxygenase inhibitor depressed endothelin-induced contractions.     

Endothelin-induced Ca-independent contraction of the porcine coronary artery

Front surface fluorometry and porcine coronary arterial strips loaded with fura-2 were used to investigate the effect of endothelin on cytosolic Ca concentrations, [Ca]i, and on contractile force, the objective being to elucidate the mechanism of action. Both in the presence and absence of extracellular Ca, endothelin induced rapid and dose-dependent increases in [Ca]i and in contraction. When caffeine-sensitive and histamine-sensitive intracellular Ca stores were depleted, in Ca-free medium, a transient contraction but no increase in [Ca]i followed the subsequent application of endothelin. This Ca-independent component was largely inhibited by the relative protein kinase C inhibitor, H-7, but not inhibited by W-7, calmodulin antagonist. This component is probably linked to activation of protein kinase C.     

ETB-mediated contraction differs between left descending coronary artery and its next branch

Pig left descending coronary artery (main artery) and its next branch (branch arteries) differ in many properties. Here we report on the receptor types and the Ca²⁺ pools utilized for endothelin (ET) contraction in 3 mm long de-endothelialized rings of the main (weight 7.38 ± 0.38 mg) and the branch (1.07 ± 0.03 mg) arteries. KCl (60 mM) contracted the main and the branch arteries with force of 41.8 ± 3.1 and 16.9 ± 1.0 mN (millinewton), respectively. Force of contraction for all the other agents was normalized taking the KCl value as 100%. We determined the total ET-induced responses using ET-1 and those mediated by ET_B using IRL1620. In Ca²⁺-containing solutions, ET-1 contracted the main arteries with pEC_B = 8.2 ± 0.1 and a maximum force of 98 ± 5%. The branch arteries also gave similar values of pEC₅₀ (8.4 ± 0.1) and maximum force (99 ± 14%). IRL1620 contracted the main and the branch arteries with pEC₅₀ = 7.9 ± 0.1 but the maximum force was significantly higher in the branch arteries (44 ± 3%) than in the main (15 ± 2%). In Ca²⁺-free solutions, the pEC₅₀ values for ET-1 or IRL-1620 did not change but the maximum force of contraction was diminished considerably in both main and branch arteries. Thus, the left coronary artery and its next branch differ in that the role of ET_B receptors is greater in the latter.     

Electron microscopy of histamine-induced contraction of the in vitro swine coronary artery.

Dose-dependent contractions of the in vitro swine coronary artery were induced by application of histamine and acetylcholine, but not of angiotensin II, ergonovine, noradrenaline, prostaglandin F2 alpha and serotonin. Ultrastructural changes especially of the tunica intima during the contractions were observed at 2, 5 and 30 min after application of histamine and acetylcholine. The intimal gutter spirally running along the longitudinal axis of the vessel was obscured, and the intimal surface became extensively indented. Exclusively in the histamine-treated samples, the increase in number and size of the intracellular vacuoles and the dilation of the intercellular clefts to the extent of the intercellular vacuoles were observed in the endothelium. Moreover, the enhancement of the endothelial permeability was indicated by the marker experiments using horseradish peroxidase. Such endothelial cell damages and the enhancement of the endothelial permeability may amplify the coronary artery contraction.     

Bradykinin facilitates noradrenaline spillover during contraction in the canine gracilis muscle.

Noradrenaline spillover from skeletal muscle vascular areas increases during exercise but the underlying mechanisms are not well understood. Muscle contraction itself causes changes in many factors that could affect noradrenaline spillover. For instance, it has been reported that bradykinin is synthesized in skeletal muscle areas during contraction. Because the B2 bradykinin receptor facilitates noradrenaline spillover, it may be involved in the increase associated with contraction. In this experiment, we studied the effect of bradykinin on noradrenaline spillover in the in situ canine gracilis muscle, using the specific B2 antagonist HOE 140. The drug did not modify noradrenaline spillover at rest, but did cause a significant decrease during muscle contraction, from 558 to 181 pg min(-1). As reported previously in the literature, fractional extraction of noradrenaline decreased during muscle contraction. This effect was independent of HOE 140 treatment. In light of our results, it seems that bradykinin formation during muscle contraction may play an important part in the observed increase in noradrenaline spillover but does not affect fractional extraction.     

Endothelin-1 inhibits and enhances contraction of porcine coronary arterial strips with an intact endothelium.

Using front-surface fluorometry and fura-2-loaded porcine coronary arterial strips with an intact endothelium, changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension of smooth muscle were simultaneously monitored in an attempt to determine the vasoactive properties of endothelin-1 (ET-1). ET-1 in low concentrations (0.1-1nM) caused a significant transient decrease in [Ca2+]i and tension of the strips precontracted with 10(-7) M U-46619. The maximal decreases in [Ca2+]i and tension were obtained with 0.6nM ET-1. In higher concentrations (1nM-100nM), there was no reduction in [Ca2+]i or tension; the contraction induced by U-46619 was potentiated. The decreases in [Ca2+]i and tension induced by ET-1 were inhibited by the mechanical removal of the endothelium or by pretreatment with NG-nitro-L-arginine and were slightly attenuated by indomethacin. Thus, ET-1 in low concentrations can induce endothelium-dependent transient relaxations accompanied by transient reductions of [Ca2+]i in isolated porcine coronary arteries. This effect is mainly mediated by the release of endothelium-derived relaxing factor.     

Phosphorylation in coronary artery cold-induced contraction in the newborn lamb

Myocardial dysfunction after hypothermic protection has been linked to various mechanisms. Coronary vasospasm in particular may be responsible for ischemic injury during reperfusion. Herein we hypothesized that coronary arteries (CA) sustain a cold-induced contraction during hypothermia mediated by a protein tyrosine kinase (PTK)-/protein tyrosine phosphatase (PTP)-dependent pathway. Isolated newborn lamb CA rings were studied in a tissue bath for isometric contraction during 2-h profound (17 degrees C) or ultra-profound (7 degrees C) hypothermia. In parallel, protein tyrosine phosphorylation was evaluated by use of the Western blot technique. Na-orthovanadate (SOV) and genestein (GEN) were used separately and in combination to evaluate the effect of PTK/PTP activation on CA contraction and tyrosine phosphorylation during cooling (17 or 7 degrees C) vs 37 degrees C. Cooling from 37 to 7 degrees C induced transient contraction at approximately 17 degrees C (29% KCl response), which was more prominent during rewarming to 37 degrees C (36% KCl). Cooling to 17 degrees C resulted in sustained contraction (7-10% KCl), which was reversible upon rewarming. Cold-induced contraction was significantly enhanced by SOV (7- to 10-fold at 17 degrees C; 2-fold at 7 degrees C) and abolished by GEN. Concurrently, tyrosine phosphorylation of 33-, 45-, and 104-kDa proteins increased during cooling (35-100% at 17 degrees C; 46-66% at 7 degrees C). Tyrosine phosphorylation was similarly enhanced by SOV (1.7- to 2.3-fold at 17 degrees C; 2.9- to 3.9-fold at 7 degrees C) and abolished by GEN in the presence or absence of SOV. These results support a prominent role for the PTK/PTP signal transduction pathway in the coronary artery cold-induced contraction. This information provides one possible biomolecular mechanism linked to ischemia/reperfusion pathophysiology of CA in neonatal hearts exposed to hypothermic myocardial protection.     

Bradykinin protects cardiac c-kit positive cells from high-glucose-induced senescence through B2 receptor signaling pathway

Cardiac c-kit positive cells are cardiac-derived cells that exist within the heart and have a great many protective effects. The senescence of cardiac c-kit positive cells probably leads to cell dysfunction. Bradykinin plays a key role in cell protection. However, whether bradykinin prevents cardiac c-kit positive cells from high-glucose-induced senescence is unknown. Here, we found that glucose treatment causes the premature senescence of cardiac c-kit positive cells. Bradykinin B2 receptor (B2R) expression was declined by glucose-induced senescence. Bradykinin treatment inhibited senescence and reduced intracellular oxygen radicals according to senescence-associated β-galactosidase staining and 2′,7′-dichlorodihydrofluorescein diacetate staining. Moreover, the mitochondrial membrane potential was damaged, as measured by JC-1 staining. The mitochondrial membrane potential was preserved under bradykinin treatment. The concentration of superoxide was decreased, and the concentration of intracellular adenosine triphosphate was increased after bradykinin treatment. Western blot showed that bradykinin leads to AKT and mammalian target of rapamycin (mTOR) phosphorylation and decreased levels of P53 and P16 when compared with glucose treatment alone. Antagonists of B2R, phosphoinositide 3-kinase (PI3K), mTOR, and B2R small interfering RNA prevented the protective effect of bradykinin. P53 antagonist also inhibited the glucose-induced senescence of cardiac c-kit positive cells. In conclusion, bradykinin prevents the glucose-induced premature senescence of cardiac c-kit positive cells through the B2R/PI3K/AKT/mTOR/P53 signal pathways.     

Vascular endothelium (review). I. General morphology. 2B: phylogenesis of the vascular endothelium

The phylogenetic descent of vascular endothelium from mesenchyme--derived precursors is described related to the development of a vessel--bound microcirculation. Endothelial precursors in primitive animals may have migrated into tissue clefts gradually forming vascular tubes. True microcirculatory vessels at first appear in the nemertines, a closed vascular system is present in some annelids whereas in arthropods an open lacunar system predominates. The first appearance of true endotheliocytes is under discussion; the author gives some evidence that it is present already in some annelids. Precursor of the endothelial wall of vessels may be the so called "Leydig's membrane", covered with amoebocytes and other mesenchymal cells. The molluscs exhibit many variants of endothelium. In the fishes, the vascular system begins to split into a blood and a lymphatic system. Obviously the specialization of endothelium correlates with the level of evolution. Despite the complicated course, the evolution of endothelium may be regarded as monophyletic.     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

Bradykinin inhibits high glucose- and growth factor-induced collagen synthesis in mesangial cells through the B2-kinin receptor

Mesangial matrix expansion is an early lesion leading to glomeruloclerosis and chronic renal diseases. A beneficial effect is achieved with angiotensin I-converting enzyme inhibitors (ACEI), which also favor bradykinin (BK) B2 receptor (B2R) activation. To define the underlying mechanism, we hypothesized that B2R activation could be a negative regulator of collagen synthesis in mesangial cells (MC). We investigated the effect of BK on collagen synthesis and signaling in MC. Inflammation was evaluated by intercellular adhesion molecule-1 (ICAM-1) expression. BK inhibited collagen I and IV synthesis stimulated by high glucose, epithelial growth factor (EGF), and transforming growth factor-β (TGF-β) but did not alter ICAM-1. Inhibition of collagen synthesis was B2R but not B1R mediated. PKC or phosphatidylinositol 3-kinase (PI3K) inhibitors mimicked the BK effect. B2R activation inhibited TGF-β- and EGF-induced Erk1/2, Smad2/3, Akt S473, and EGFR phosphorylation. A phosphatase inhibitor prevented BK effects. The in vivo impact of B2R on mesangial matrix expansion was assessed in streptozotocin-diabetic rodents. Deletion of B2R increased mesangial matrix expansion and albuminuria in diabetic mice. In diabetic rats, matrix expansion and albuminuria were prevented by ACEI but not by ACEI and B2R antagonist cotreatment. Consistently, the lowered BK content of diabetic glomeruli was restored by ACEI. In conclusion, deficient B2R activation aggravated mesangial matrix expansion in diabetic rodents whereas B2R activation reduced MC collagen synthesis by a mechanism targeting Erk1/2 and Akt, common pathways activated by EGF and TGF-β. Taken together, the data support the hypothesis of an antifibrosing effect of B2R activation.     

A novel loop diuretic, torasemide, inhibits thromboxane A2-induced contraction in the isolated canine coronary artery.

We examined the effect of a novel antihypertensive diuretic, torasemide, on the vasoconstriction induced by TXA2 in the isolated canine coronary artery. Carbocyclic thromboxane A2 (CTA2), a stable analogue of the potent coronary vasoconstrictor thromboxane A2, exhibited a slow onset and progressive contraction of isolated canine coronary arteries at 2 x 10(-8) M. Torasemide (10(-7) approximately 10(-4) M) elicited a dose-dependent vasodilating action in the isolated canine coronary arteries contracted by CTA2, whereas indapamide or furosemide had little effect on this preparation. The maximum vasodilating response to torasemide was 45 +/- 12% of vasodilating effect induced by 10(-4) M papaverine. These results suggest that torasemide is a promising antihypertensive agent with a coronary protective effect.     

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.     

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.     

Involvement of COX-1 in A3 adenosine receptor-mediated contraction through endothelium in mice aorta

We investigated whether A(3) adenosine receptor (A(3)AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A(3) knockout (A(3)KO) mice aorta. A(3)AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC(50): 2.9 +/- 0.2 x 10(-9) M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A(3)KO +E aorta. At 10(-7) M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A(3)KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (-E), in both WT and A(3)KO aorta. A(3)AR gene and protein expression were reduced by 74 and 72% (P < 0.05), respectively, in WT -E compared with WT +E aorta, while being undetected in A(3)KO +E/-E aorta. Indomethacin (nonspecific COXs blocker, 10(-5) M), SC-560 (specific COX-1 blocker, 10(-8) M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10(-6) M), and furegrelate (thromboxane synthase inhibitor, 10(-5) M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B(2) production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A(3)KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A(3)KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A(3)AR antagonist in WT +E aorta. Expression of both A(3)AR and COX-1 was located mostly on endothelium of WT and A(3)KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A(3)AR-mediated contraction via endothelium.     

Mechanisms of metabisulfite-induced bronchoconstriction: evidence for bradykinin B2-receptor stimulation.

Sodium metabisulfite (MBS) is a food preservative that can trigger bronchoconstriction in asthmatic subjects. Previous studies designed to identify the mechanisms involved in this response have yielded conflicting results. We noted certain similarities between the pharmacology of MBS-induced airway responses and those elicited by bradykinin (BK), another provocating agent in asthmatic subjects. Therefore we used allergic sheep to determine whether MBS-induced bronchoconstriction 1) had a pharmacology similar to that previously seen with BK in this model, including protection by a BK B2-receptor antagonist, NPC-567, and 2) was associated with increased concentrations of immunoreactive kinins in bronchoalveolar lavage. We measured specific lung resistance before and immediately after inhaled buffer and increasing concentrations of MBS (30 breaths of 25, 50, and 100 mg/ml) and calculated the concentration producing 100% increase in specific lung resistance over baseline (PC100). In seven sheep, geometric mean control PC100 was 33.1 mg/ml. Pretreatment with either the anticholinergic agent ipratropium bromide (180 micrograms; PC100 87.1 mg/ml) or the antiasthma drug nedocromil sodium (1 mg/kg aerosol; PC100 97.7 mg/ml) blocked the MBS-induced bronchoconstriction (P less than 0.05), whereas the histamine H1-receptor antagonist chlorpheniramine (2 mg/kg iv) was ineffective. Furthermore the MBS-induced bronchoconstriction was not affected by the neutral endopeptidase inhibitor thiorphan (40 breaths of a 1 mg/ml solution) or the angiotensin-converting enzyme inhibitor enalaprilat (2.5 mg aerosol). In six sheep the MBS-induced bronchoconstriction was completely blocked by NPC-567 (20 breaths, 5 mg/ml aerosol): after treatment with NPC-567 mean PC100 was 100 mg/ml compared with 57.5 mg/ml in the control trial (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory functions of the coronary endothelium

In this brief review three functions of the coronary endothelium are surveyed: (a) its barrier and exchange function, (b) the prevention of coagulation and platelet aggregation, and (c) its role in vasoregulation. Impairment of these functions can occur in ischemia, hypertension, arteriosclerosis and inflammation. (Mol Cell Biochem116: 163–169, 1992)     

HSP90 and Akt modulate Ang-1-induced angiogenesis via NO in coronary artery endothelium.

This study examines the notion that heat shock protein (HSP) 90 binding to nitric oxide (NO), endothelial NO synthase (eNOS), and PI3K-Akt regulate angiopoietin (Ang)-1-induced angiogenesis in porcine coronary artery endothelial cells (PCAEC). Exposure to Ang-1 (250 ng/ml) for periods up to 2 h resulted in a time-dependent increase in eNOS phosphorylation at Ser 1177 that occurred by 5 min and peaked at 60 min. This was accompanied by a gradual increase in NO release. Ang-1 also led to stimulation of HSP90 binding to eNOS and a significant increase in Akt phosphorylation. Thirty minutes of pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of HSP90) or 500 nM wortmannin [a specific phosphatidylinositol 3 (PI3)-kinase (PI3K) inhibitor] significantly attenuated Ang-1-stimulated eNOS phosphorylation and NO production. Exposure to Ang-1 caused an increase in endothelial cell migration, tube formation, and sprouting from PCAEC spheroids, and pharmacological blockage of HSP90 function or inhibition of PI3K-Akt pathway completely abolished these effects. Inhibition of nitric oxide synthase by NG-nitro-l-arginine methyl ester (2.5 mM) also resulted in a significant decrease in Ang-1-induced angiogenesis. We conclude that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to Ang-1-induced eNOS phosphorylation, NO production, and angiogenesis in PCAEC.