Radiation-induced endothelial dysfunction and fibrosis in rat lung: modification by the angiotensin converting enzyme inhibitor CL242817

The purpose of this study was to evaluate the angiotensin converting enzyme (ACE) inhibitor CL242817 as a modifier of radiation-induced pulmonary endothelial dysfunction and pulmonary fibrosis in rats sacrificed 2 months after a single dose of 60Co gamma rays (0-30 Gy) to the right hemithorax. CL242817 was administered in the feed continuously after irradiation at a regimen of 60 mg/kg/day. Pulmonary endothelial function was monitored by lung ACE activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Pulmonary fibrosis was evaluated by lung hydroxyproline (HP) content. Lung ACE and PLA activities decreased with increasing radiation dose, and cotreatment with CL242817 significantly ameliorated both responses. CL242817 dose-reduction factors (DRF) were 1.3-1.5 for ACE and PLA activity. Lung PGI2 and TXA2 production increased with increasing radiation dose, and CL242817 almost completely prevented both radiation responses. The slope of the radiation dose-response curves in the CL242817-treated rats was essentially zero, precluding calculation of DRF values for PGI2 and TXA2 production. Lung HP content also increased with increasing radiation dose, and CL242817 significantly attenuated this response (DRF = 1.5). These data suggest that the ability of ACE inhibitors to ameliorate radiation-induced pulmonary endothelial dysfunction is not unique to captopril [Ward et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 135-140 (1988)], rather it is a therapeutic action shared by other members of this class of compounds. These data also provide the first evidence that ACE inhibitors exhibit antifibrotic activity in irradiated rat lung.     

Pentoxifylline does not spare acute radiation reactions in rat lung and skin.

Male rats were exposed to single doses (0-30 Gy) of 60Co gamma rays to the right hemithorax. Half of each dose group consumed only control powdered chow after irradiation, and half consumed feed containing 0.10% (w/w) pentoxifylline (50 mg/kg/day). The severity of epilation and desquamation in the field of the radiation port was scored weekly. Two months after irradiation the animals were killed, and pulmonary endothelial function was monitored by the activity of lung angiotensin converting enzyme (ACE) and plasminogen activator (PLA), and by production of prostacyclin (PGI2) and thromboxane (TXA2). The amount of hydroxyproline (HP) in the lung served as an index of pulmonary fibrosis. Radiation produced a dose-dependent decrease in ACE and PLA activity in the right lung and an increase in the production of PGI2 and TXA2. This endothelial dysfunction was accompanied by an increase in wet weight and in protein and HP content in the irradiated lung. Pentoxifylline spared only the increase in lung wet weight and protein content, and actually elevated the radiation-induced hyperproduction of PGI2 and TXA2. The severity of the epilation and desquamation reactions increased with increasing radiation dose and time but was independent of diet. These data indicate that pentoxifylline, despite some promising pharmacological actions, has no beneficial effect on acute radiation reactions in rat lung and skin.     

Pulmonary endothelial dysfunction induced by unilateral as compared to bilateral thoracic irradiation in rats

Rats were sacrificed 2 months after a single dose of 10-30 Gy of 60Co gamma rays delivered to either a right unilateral or a bilateral thoracic port. Four indices of lung endothelial function were measured: the activities of angiotensin-converting enzyme (ACE) and plasminogen activator (PLA) and the production of prostacyclin (PGI2) and thromboxane (TXA2). The number of macrophages recovered by bronchoalveolar lavage (BAL) and the degree of right ventricular hypertrophy (an index of pulmonary hypertension) also were determined. Right lung ACE and PLA activity decreased linearly, and PGI2 and TXA2 production increased linearly with increasing radiation dose. The response curves for right unilateral and bilateral thoracic irradiation were not significantly different. In contrast, bilateral irradiation was more toxic than unilateral, since rats exposed to the former exhibited decreased body weight, an increased incidence of pleural effusions, an increase in the number of macrophages recovered by BAL, and right ventricular hypertrophy. These data demonstrate that pulmonary endothelial dysfunction induced by hemithorax irradiation represents a direct response of the endothelium to radiation injury and is not secondary to other phenomena such as shunting of function to the shielded lung.     

Monocrotaline pneumotoxicity in mice

Lung injury induced in rats by the pyrrolizidine alkaloid monocrotaline is a well-documented model of pulmonary hypertension. To our knowledge, however, monocrotaline-induced cardiopulmonary injury has rarely been described and has never been quantitated in mice. In the present study, adult male mice received 2.4, 4.8, or 24.0 mg monocrotaline/kg body weight/day in the drinking water continuously for 6 weeks. These doses represent 1, 2, and 10 times the severely pneumotoxic regimen in rats. Pulmonary endothelial function was monitored by right lung angiotensin converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Light and electron microscopy were performed on the left lungs. Cardiac right ventricular hypertrophy was evaluated by the right ventricle to left ventricle plus septum weight ratio (RV/LV + S). Monocrotaline-treated mice exhibited a dose-dependent decrease in lung ACE and PLA activities and an increase in PGI2 and TXA2 production, indicative of endothelial dysfunction. However, these responses were significant only after the highest monocrotaline dose. Light and electron microscopy revealed dose-dependent pulmonary inflammatory and exudative reactions. Unlike previous studies in rats, however, monocrotaline-treated mice developed relatively little lung fibrosis, cardiomegaly, or right ventricular hypertrophy, and no occlusive medial thickening of the pulmonary arteries, even at the highest dose level. These and previous data indicate that there are quantitative biochemical and qualitative morphological differences between mice and rats with respect to monocrotaline pneumotoxicity. Furthermore, in monocrotaline-treated mice (but not in rats) there appears to be a dissociation between lung endothelial dysfunction and inflammation on the one hand, and pulmonary hypertension and fibrosis on the other.     

Radiation injury in rat lung. IV. Modification by D-penicillamine

To determine whether D-penicillamine, known to reduce fibrosis in irradiated rat lung (W. F. Ward, A. Shih - Hoellwarth , and R. D. Tuttle , Radiology 146, 533-537, 1983), also ameliorates radiation injury in the pulmonary endothelium, we measured angiotensin-converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) production in the lungs of penicillamine-treated (10 mg/day, po, continuous after irradiation) and untreated rats from 2 weeks to 6 months after a single dose of 25 Gy of 60Co gamma rays to the right hemithorax. Both ACE and PLA activity in the irradiated right lung of untreated rats decreased dramatically between the 1st and 2nd months after exposure, then reached a plateau through 6 months at approximately 25 and 50% of the normal level, respectively. For the first 2 months after irradiation, penicillamine-treated animals exhibited significantly (P less than 0.05) higher activities of both ACE and PLA than did untreated rats. From 3 to 6 months after irradiation, however, the only significant drug effect on these enzymes was a 25% increase in PLA activity at 6 months. PGI2 production by the irradiated lung of untreated rats increased continuously, and at 6 months was approximately 10 times higher than normal. Penicillamine significantly (P less than 0.05) reduced this hypersecretion, and at 6 months after irradiation, PGI2 production by the lungs of drug-treated rats was only half that of untreated animals. In contrast, the drug had no significant effect on enzyme activities in the lungs of sham-irradiated rats. Thus the antifibrotic agent D-penicillamine delays the onset of radiation-induced enzyme dysfunction in the pulmonary endothelium. In addition at 6 months after irradiation, the lungs of penicillamine-treated rats exhibit 25% more PLA activity and only half as severe a hypersecretion of PGI2 as do the lungs of untreated animals. The drug is most effective in ameliorating endothelial damage during the first 2 months after irradiation, preceding the development of interstitial fibrosis. However, the effect of this penicillamine regimen on pulmonary endothelial function is not as large as its effect on collagen accumulation in irradiated rat lung.     

Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized Ascaris suum antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A2, prostaglandin (PG) D2 and PGI2 from the PG endoperoxide intermediate, PGH2, was assayed over a range of substrate concentrations from 3-200 microM. Synthesis of PGI2 by trachealis microsomes was approximately 5-fold greater than that of TXA2. PGI2 and TXA2 production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA2 production predominated over PGI2 by 9-fold, preparations from Ascaris-exposed animals synthesized 50% more TXA2 than controls at PGH2 concentrations of 25 microM and above, whereas synthesis of PGI2 and PGD2 were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA2 and PGI2 in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA2 synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.     

Radiation-induced pulmonary endothelial dysfunction and hydroxyproline accumulation in four strains of mice

C57BL mice exposed to 14 Gy of whole-thorax irradiation develop significant histologic lung fibrosis within 52 weeks, whereas CBA and C3H mice do not exhibit substantial fibrosis during this time. The purpose of the present study was to determine whether this strain-dependent difference in radiation histopathology is associated with genetic differences in pulmonary endothelial metabolic activity or in endothelial radioresponsiveness. C57BL/6J, C57BL/10J, CBA/J, and C3H/HeJ mice were sacrificed 12 weeks after exposure to 0 or 14 Gy of 300-kV X rays to the whole thorax. Lung angiotensin converting enzyme (ACE) activity and plasminogen activator (PLA) activity were measured as indices of pulmonary endothelial function; and lung hydroxyproline (HP) content served as an index of pulmonary fibrosis. Lung ACE and PLA activities in sham-irradiated C57BL/6J and CB57BL/10J mice were only half as high as those in sham-irradiated CBA/J and C3H/HeJ mice. Exposure to 14 Gy of X rays produced a slight but nonsignificant reduction in lung ACE and PLA activity in the C57BL strains, and a significant reduction in the CBA/J and C3H/HeJ mice. Even after 14 Gy, however, lung ACE and PLA activities in CBA/J and C3H/HeJ mice were higher than those in sham-irradiated C57BL/6J and C57BL/10J mice. Lung HP content in all four strains increased significantly after irradiation, but this increase was accompanied by an increase in lung wet weight. As a result, HP concentration (per milligram wet weight) remained constant or increased slightly in both C57BL strains and actually decreased in the CBA/J and C3H/HeJ mice. These data demonstrate significant genetic differences in both intrinsic pulmonary endothelial enzyme activity and endothelial radioresponsiveness among the four strains of mice. Specifically, strains prone to radiation-induced pulmonary fibrosis (C57BL/6J, C57BL/10J) exhibit only half as much lung ACE and PLA activity as do strains resistant to fibrosis (CBA and C3H).     

Plasminogen activator activity in lung and alveolar macrophages of rats exposed to graded single doses of gamma rays to the right hemithorax

Male rats were sacrificed 2 or 6 months after a single dose of 0-30 Gy of 60Co gamma rays to the right hemithorax. At autopsy, macrophages were lavaged from the right lung, counted, and frozen. The right (irradiated) and the left (shielded) lungs were frozen, then assayed for plasminogen activator (PLA) activity by the fibrin plate lysis method. Freeze-thawed macrophages were assayed for both PLA activity (125I-fibrin clot lysis method) and fibrinolytic inhibitor activity (inhibition of urokinase-induced fibrin lysis). There was a linear, dose-dependent decrease in right lung PLA activity over the dose range of 10-30 Gy at 2 and 6 months postirradiation, reductions of 3.1 and 2.6% per Gy, respectively. PLA activity at all radiation doses was 10-15% higher at 6 months than at 2 months (P less than 0.05), indicative of a partial recovery of this endothelial function in the irradiated lung. There were no significant changes in PLA activity in the shielded left lung at any dose or time. There also was a linear, dose-dependent increase in the number of macrophages lavaged from the right lung at both 2 and 6 months postirradiation, with larger numbers recovered after all doses at 2 months. PLA activity per 10(6) macrophages decreased with increasing radiation dose at both autopsy times, closely paralleling lung PLA activity. This radiation-induced decrease in macrophage PLA activity was not due to increased fibrinolytic inhibitor activity in the irradiated macrophages. These data quantitate the dose response and time course of radiation-induced fibrinolytic defects in rat lung and suggest that information obtained from a minimally invasive procedure such as bronchoalveolar lavage may serve as an index of the degree of pulmonary fibrinolytic dysfunction after irradiation.     

Effects of nitrogen dioxide exposure on prostacyclin synthesis in lung and thromboxane A2 synthesis in platelets in rats

Effects of nitrogen dioxide (NO2) exposure on prostacyclin (PGI2) synthesis in the rat lung and thromboxane A2 (TXA2) synthesis in the platelets were studied. Male Wistar rats were exposed to 10 ppm NO2 for 1, 3, 5, 7 and 14 days. PGI2 synthesizing activity of homogenized lung decreased. The damage of PGI2 synthesizing activity reaches its maximum at 3 days. At 14 days, PGI2 synthesizing activity returned to the normal level. The activity of PGI2 synthetase decreased significantly. The formation of lipid peroxides due to NO2 exposure may cause the depression of PGI2 synthesizing activity of lung. On the other hand, platelet TXA2 synthesizing activity increased. This increased TXA2 synthesizing activity lasted at least till 3 days. Then, it returned to the normal level. The counts of platelet were decreased significantly by 1, 3, 5 and 7 days NO2 exposure. Then the decreased counts of platelet returned to the normal level at 14 days NO2 exposure. These results indicate that the depression of PGI2 synthesizing activity of lung by NO2 exposure cause an increase in TXA2 synthesizing activity of platelets. It may contribute to induce platelet aggregation and to the observed decrease in the number of platelets during NO2 exposure.     

Chronic cigarette smoke exposure adversely alters 14C-arachidonic acid metabolism in rat lungs, aortas and platelets

Male rats were exposed to freshly generated cigarette smoke once daily, 5 times a week for 10 weeks. Inhalation of smoke was verified by elevated carboxyhemoglobin in blood sampled immediately after smoke exposure and by increased lung aryl hydrocarbon hydroxylase activity 24 hours after the last smoke exposure. Aortic rings isolated from smoke-exposed rats synthesized less prostacyclin (PGI2) from 14C-arachidonic acid than rings from sham rats. Platelets from smoke-exposed rats synthesized more thromboxane (TXA2) from 14C-arachidonic acid than platelets from room controls but not those from sham rats. Lung microsomes from smoke-exposed rats synthesized more TXA2 and had a lower PGI2/TXA2 ratio than lung microsomes from room controls and shams. It is concluded that chronic cigarette smoke exposure alters arachidonic acid metabolism in aortas, platelets and lungs in a manner resulting in decreased PGI2 and increased TXA2, thereby creating a condition favoring platelet aggregation and a variety of cardiovascular diseases.     

Serum copper concentration as an index of lung injury in rats exposed to hemithorax irradiation

Serum copper concentration was evaluated as an index of lung injury (monitored by lung prostacyclin production) with respect to the effects of time, dose, dose fractionation, and penicillamine dose modification in rats irradiated to the right hemithorax. Both lung PGI2 production and serum Cu concentration increased with increasing 60Co gamma-ray dose in animals sacrificed 2 or 6 months postirradiation, and the highest values for both responses were observed at the latter autopsy time. At 2 months postirradiation, the elevations in lung PGI2 production and serum Cu concentration also were spared similarly when total radiation doses were delivered in five equal daily fractions as compared to single doses. Finally, the ability of D-penicillamine to ameliorate the radiation-induced hyperproduction of PGI2 by rat lung was accompanied by an attenuation of the dose-dependent increase in serum Cu concentration at 2 months postirradiation in the drug-treated rats. In contrast, serum iron concentration was independent of time, dose, and penicillamine. At 2 months after irradiation, there also was a dose-dependent increase in lung hydroxyproline (collagen) content, the magnitude of which correlated closely with serum copper concentration in individual animals. Thus serum copper concentration is an accurate and minimally invasive index of lung injury in rats irradiated to the hemithorax and can predict lung hydroxyproline (collagen) content in individual irradiated rats.     

Thromboxane and prostacyclin production in the perfused rabbit lung

We investigated whether prostacyclin formation by the isolated rabbit lung can serve as a measure of pulmonary distress. The basal TXA2 and PGI2 formation was very low, and depended on the preperfusion history of the lung (low or high flow, use of dextran or artificial perfusate). The basal prostanoid production remained unchanged over a time period of 2 h. Neither was it influenced by the serotonin uptake inhibitor chlorimipramine and by small changes in temperature (33 degrees C vs 39 degrees C). The PGI2 formation was almost independent of hemodynamic alterations such as embolism or vasoconstriction. An enhanced production was only seen after a dramatic increase in flow (from 1.7-5 ml/sec), and a transient 3-fold increase was observed after administration of 1 mM H2O2. A substantial (up to 40-fold) but transient increase in TXA2 production was measured after 1 mM of H2O2, and the TXA2 production was positively correlated to the increase in pulmonary arterial pressure. However, thromboxane production was also dramatically augmented by hemodynamic alterations such as embolism, increased flow and--to a lesser extent--vasoconstriction. We conclude that the determination of the prostanoid production (and particularly the TXA2 formation) by the rabbit lung cannot be used as a direct measure of endothelial distress. To this end it is excessively biased by hemodynamic alterations such as recruitment and shear stress.     

Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta

Confluent monolayers of bovine aortic endothelial cells were examined 2-72 h after exposure to 0.5-5.0 Gy of 60Co gamma-rays. Accumulation of prostacyclin [PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity. Platelet adherence to irradiated and control monolayers also was studied. There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium. These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy. Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM). However, by 72 h this increase was less significant. The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment. There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment. However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets.     

Effects of split-dose irradiation on survival and oncogenic transformation induced by 31 MeV protons in C3H10T1/2 cells

Survival and oncogenic transformation were studied in C3H10T1/2 cells exposed to 31 MeV protons. Total doses of 0.5, 1 and 7 Gy were delivered as single and two equal fractions with various time intervals up to 10 h between doses. With split doses as compared with single doses to a total dose of 7 Gy, survival increased by a factor of 2.5 +/- 0.2, whereas the frequency of transformation per surviving cell declined by a factor of 3.1 +/- 0.5. Maximal split-dose recovery occurred within the first 5 h for both endpoints. Further, the transformation frequency decreased by factors of 3.1 +/- 0.6 and 1.5 +/- 0.3 respectively for total doses of 0.5 and 1.0 Gy split into two equal fractions. The data for 1 and 7 Gy are compatible with data in the literature for other low LET radiations.     

Effects of reactive oxygen species on eicosanoid metabolism in human endothelial cells.

The influence of reactive oxygen species (H2O2 was used as model substance) on the formation and release of PGI2 and TXA2 by cultured human endothelial cells was analyzed. In the presence of H2O2 concentrations which did not induce a general cell damage (analyzed by estimation of the cellular concentration of energy rich phosphates and extent of lipid peroxidation), the formation of both eicosanoids exhibited a sigmoidal shape with respect to time. Increasing H2O2 concentration shortened the half time of PGI2 and TXA2 production. The maximum rates of PGI2 and TXA2 formation were separated by a delay of the TXA2 production. The ratio of PGI2 and TXA2 formation was 100 to 1 at the time of maximum PGI2 formation and 1-2 to 1 at the time of maximum TXA2 formation. This effect of reactive oxygen species could contribute to the reduction of the protective function of the endothelium in hemostasis and vascular tone. Using antioxidants, the modulating function of reactive oxygen species on the eicosanoid metabolism in endothelial cells was verified.     

Up-regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.     

Monocrotaline pneumotoxicity in mice

Lung injury induced in rats by the pyrrolizidine alkaloid monocrotaline is a well-documented model of pulmonary hypertension. To our knowledge, however, monocrotaline-induced cardiopulmonary injury has rarely been described and has never been quantitated in mice. In the present study, adult male mice received 2.4, 4.8, or 24.0 mg monocrotaline/kg body weight/day in the drinking water continuously for 6 weeks. These doses represent 1, 2, and 10 times the severely pneumotoxic regimen in rats. Pulmonary endothelial function was monitored by right lung angiotensin converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI₂) and thromboxane (TXA₂) production. Light and electron microscopy were performed on the left lungs. Cardiac right ventricular hypertrophy was evaluated by the right ventricle to left ventricle plus septum weight ratio (RV/LV + S). Monocrotalinetreated mice exhibited a dose-dependent decrease in lung ACE and PLA activities and an increase in PGI₂ and TXA₂ production, indicative of endothelial dysfunction. However, these responses were significant only after the highest monocrotaline dose. Light and electron microscopy revealed dosedependent pulmonary inflammatory and exudative reactions. Unlike previous studies in rats, however, monocrotaline-treated mice developed relatively little lung fibrosis, cardiomegaly, or right ventricular hypertrophy, and no occlusive medial thickening of the pulmonary arteries, even at the highest dose level. These and previous data indicate that there are quantitative biochemical and qualitative morphological differences between mice and rats with respect to monocrotaline pneumotoxicity. Furthermore, in monocrotaline-treated mice (but not in rats) there appears to be a dissociation between lung endothelial dysfunction and inflammation on the one hand, and pulmonary hypertension and fibrosis on the other.     

Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact-inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time-dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.     

A selective inhibitor of thromboxane synthetase activity of rabbit heart tissue: a pyridazinic derivative

The effects of 2-(2 dimethylaminoethyl) 5-benzylidene 6-methyl (2H,4H)-3-pyridazinone (III) were studied on the biosynthesis of TXA2 and PGI2 in vitro the TXA2 and PGI2 synthetase activity of heart tissue. Biosyntheses of TXA2 and PGI2 were carried out using arachidonic acid as a substrate and horse platelet and aorta microsomes as sources of TXA2 and PGI2 synthetases respectively. TXB2 and 6-keto PGF1 alpha were determined by RIA. III--did not significantly modify either the biosynthesis of PGI2 in vitro or the PGI2 synthetase activity of heart tissue. did not significantly inhibit TXA2 biosynthesis in vitro but markedly reduced the TXA2 synthetase activity of heart tissue: for a microsomal fraction concentration of 100 micrograms protein, the ID50 was 6.37 X 10(-5) M +/- 1.29 X 10(-8) M. Thus III behaves as a specific inhibitor of the TXA2 synthetase activity of heart tissue and could have a beneficial use in therapeutics.