title: Significance of endothelial prostacyclin and nitric oxide in peripheral and pulmonary circulation.
abstract: BACKGROUND
  Vasoprotective function of endothelial cells is associated, among others, with biosynthesis and release of nitric oxide (NO), prostacyclin (PGI2), prostaglandin E2 (PGE2), carbon monoxide (CO) and plasminogen activator (t-PA). These endothelial mediators calm down activated platelets and leukocytes, prevent the occurrence of parietal thrombotic events, promote thrombolysis, maintain tissue perfusion and protect vascular wall against acute damage and against chronic remodeling. Endothelial dysfunction in patients suffering from atherosclerosis or diabetes type 2 is associated not only with suppression in release of the above mediators but also with deleterious discharge of prostaglandin endoperoxides (PGH2, PGG2), superoxide anion (O2-, peroxynitrite (ONOO-), and plasminogen activator inhibitor (PAI-1). We looked for mechanisms of protective endothelial function, with a special respect to the differences between peripheral and pulmonary circulation.
  
  
  METHODS
  Cultured endothelial cells of bovine aorta (BAEC) were used to study physiological and pharmacological mechanisms of increasing free cytoplasmic calcium [Ca2+]i. A porphyrinic sensor quantified the release of NO from BAEC. In cultured human umbilical vein endothelial cells (HUVEC) we looked for induction by bradykinin (Bk) of mRNAs for a number of enzymes. In blood perfused rat lungs we studied protective role of NO against injury inferred by lipopolysaccharide on pulmonary microcirculation that was accomplished by thromboxane A2 (TXA2), platelet activating factor (PAF), cysteinyl-leukotrienes (cyst-LTs) and the complement system. In vivo we analyzed the influence of Bk, perindopril and quinapril ('tissue type' angiotensin converting enzyme inhibitors, ACE-Is) on endothelial function in entire circulation of anaesthetized rats using a thrombolytic bioassay and EIA for 6-keto-PGF1 alpha and t-PA antigen.
  
  
  RESULTS
  In BAEC Bk via kinin B2 receptors raised in a concentration-dependent manner (1 pM-10 nM) free cytoplasmic calcium ions [Ca2+]i, that triggered the release of NO from BAEC. Calcium ionophore (A23187, 1-100 nM) as well as receptor agonists such as adenosine diphosphate (ADP, 10 nM-1 microM), adrenaline (Adr, 1-10 microM) or acetylcholine (Ach, 10-100 microM) produced a similar rise in endothelial [Ca2+]i as did Bk at a nanomolar concentration. 'Tissue type' ACE-Is, e.g. quinapril or perindopril acted through accumulation of endogenous Bk. However, the potency of ACE-I to change endothelial function is by several orders of magnitude lower than that for exogenous Bk. In vivo the major difference between thrombolytic actions by quinapril or perindopril on one hand, and by exogenous Bk on the other was longevity of thrombolysis by ACE I and a distinct hypotensive action of exogenous Bk. Still, the long-lasting isolated thrombolytic effect of ACE I was mediated entirely by endogenous BK as evidenced by the preventive action of icatibant, a kinin B2 receptor antagonist. Moreover, in vivo the immediate thrombolysis by ACE-I was mediated by PGI2 rather than by NO or t-PA, as shown by pharmacological analysis, and by direct blood assays of 6-keto-PGF1 alpha and t-PA antigen. Bradykinin as a mediator of pleiotropic endothelial action of several cardiovascular drugs (e.g. ACE-I) may complete its mission not only through B2 receptor and [Ca2+]i--mediated release of PGI2 or NO. Here, we describe a new route of the Bk action. Bk mediated induction of the [Ca2+]i-independent, so called 'inducible', endothelial isoenzymes required for generation of CO, PGI2 and PGE2. After 4 hours of incubation of HUVEC with Bk (10 nM) it induced mRNAs for haemooxygenase 1 (HO-1), cyclooxygenase 2 (COX-2), prostaglandin E synthase (PGE-S) whereas mRNA for nitric oxide synthase 2 (NOS-2) was weakly affected. We proved also that unlike in peripheral circulation, in pulmonary circulation only NO but not PGI2 would play a protective role. In the blood-perfused lung, endotoxaemia liberates lipids, such as TXA2, PAF and cyst-LTs. These toxic lipids along with the activated complement mediate pulmonary damage. Pulmonary endothelial nitric oxide is the only local protector against lung injury evoked by the phagocytised bacterial lipopolysaccharide.
  
  
  CONCLUSIONS
  Summing up, in peripheral circulation endogenous Bk is the most efficient activator of protective endothelial function. For instance, thrombolytic action of 'tissue type' ACE-I depends on the Bk-released PGI2. Acting as an agonist of endothelial B2 kinin receptors Bk rises [Ca2+]i with a subsequent activation of constitutive COX 1 and NOS-3. This is followed by an immediate release of PGI2 and NO. Moreover, acting as 'microcytokine' Bk induces mRNAs for HO-1, COX-2 and PGE S, the isoenzymes responsible for a delayed endothelial biosynthesis of CO, PGI2 and PGE2. (ABSTRACT TRUNCATED)
authors.affiliation: Jagiellonian University, 16 Grzegorzecka str., 31-531 Cracow, Poland. mfgrygle@kinga.cyf-kr.edu.pl
authors.forename: R J
authors.initials: RJ
authors.lastname: Gryglewski
authors.forename: S
authors.initials: S
authors.lastname: Chłopicki
authors.forename: W
authors.initials: W
authors.lastname: Uracz
authors.forename: E
authors.initials: E
authors.lastname: Marcinkiewicz
mesh_terms: D015121:6-Ketoprostaglandin F1 alpha / Q000097:blood; D000109:Acetylcholine / Q000494:pharmacology; D000125:Achilles Tendon / Q000098:blood supply; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic; D001920:Bradykinin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019446:Endotoxemia / Q000503:physiopathology; D004837:Epinephrine / Q000494:pharmacology; D011464:Epoprostenol / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics; D051547:Heme Oxygenase-1; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics; D007546:Isoquinolines / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000502:physiology* / Q000503:physiopathology; D008565:Membrane Proteins; D008833:Microcirculation / Q000187:drug effects / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020913:Perindopril / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics; D011652:Pulmonary Circulation / Q000187:drug effects / Q000502:physiology*; D000077583:Quinapril; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044005:Tetrahydroisoquinolines; D010959:Tissue Plasminogen Activator / Q000097:blood; D014158:Transcription, Genetic / Q000187:drug effects; D014471:Umbilical Veins
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D007527:Isoenzymes; D007546:Isoquinolines; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D012333:RNA, Messenger; D044005:Tetrahydroisoquinolines; D009569:Nitric Oxide; D000001:Calcimycin; D015121:6-Ketoprostaglandin F1 alpha; D000244:Adenosine Diphosphate; D011464:Epoprostenol; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C496513:Ptgs1 protein, rat; D010959:Tissue Plasminogen Activator; D016285:Iloprost; D000109:Acetylcholine; D000077583:Quinapril; D001920:Bradykinin; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester; D020913:Perindopril; D004837:Epinephrine