Inactivation of viral and prion pathogens by gamma-irradiation under conditions that maintain the integrity of human albumin.

BACKGROUND AND OBJECTIVES: The administration of therapeutic plasma protein concentrates has been associated with the real risk of transmitting viral diseases and the theoretical risks of prion transmission. Our objective was to determine if gamma-irradiation can inactivate viral or prion infectivity without damaging a protein biotherapeutically. MATERIALS AND METHODS: Human albumin 25% solution, spiked with four model viruses (including porcine parvovirus) or with brain homogenate from scrapie-infected hamsters, was gamma-irradiated at constant low-dose rates and assayed for viral and prion infectivity or for albumin integrity. RESULTS: At a radiation dose of 50 kGy, viruses were inactivated by >/= 3.2 to >/= 6.4 log10 and scrapie by an estimated 1.5 log10, whereas albumin was only moderately aggregated and fragmented. CONCLUSIONS: gamma-Irradiation can preferentially inactivate viral and prion pathogens without excessive damage to albumin structure.

Regulation of endothelial cell prostaglandin synthesis by glutathione.

Prostaglandin synthesis in in vitro systems is dependent on glutathione and peroxide concentrations. We tested the effects of glutathione depletion and H2O2 exposure on prostaglandin synthesis in cultured porcine aortic endothelial cells. Depletion of glutathione using buthionine sulfoximine (BSO), diethylmaleate, and 2,4-chlorodinitrobenzene increased prostaglandin synthetic capacity. Production of prostacyclin, but not prostaglandin E2, from exogenous arachidonic acid was significantly greater than in controls. Glutathione depletion also resulted in enhanced production of prostacyclin from exogenous prostaglandin H2. These responses were not due to direct effects of glutathione-depleting agents on prostaglandin synthetic enzymes. Exposure to H2O2 also altered prostaglandin synthetic capacity in endothelial cells. While 5 microM H2O2 stimulated prostaglandin production from exogenous arachidonate, 25 and 50 microM were found to be inhibitory. Prostaglandin synthetic capacity was greater in BSO-treated cells which were exposed to 5 and 10 microM H2O2 than in cells exposed to H2O2 alone. However, prostaglandin synthetic capacity was greatly reduced in BSO-treated cells exposed to 50 microM H2O2. Thus, normal levels of cellular glutathione exert an inhibitory influence on prostaglandin synthesis. However, glutathione depletion increases the sensitivity of prostaglandin synthesis to inhibition by 50 microM H2O2.

Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells.

We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was shown to have no direct effect on conversion of arachidonic acid to prostaglandins. In intact cells menadione caused only a 40% inhibition of the conversion of PGH2 to prostacyclin. Enzymes involved in the incorporation and the release of arachidonic acid were not affected by menadione (20 microM, 15 min). Menadione undergoes oxidation/reduction reactions in intact cells leading to partial reduction of oxygen-forming, reactive oxygen species. In our cells menadione was found to increase KCN-resistant oxygen consumption. Further, an increased accumulation of H2O2 was observed with a time course consistent with menadione-induced inhibition of prostaglandin synthesis. We conclude that menadione at sublethal doses caused inhibition of prostaglandin synthesis. The mechanism involves inactivation of PGH2 synthase by a reactive species resulting from metabolism of menadione by endothelial cells.

Recovery of porcine aortic endothelial cell prostaglandin synthesis following inhibition by sublethal concentrations of hydrogen peroxide.

Confluent monolayers of porcine aortic endothelial cells exposed for 10 min to 100 microM H2O2 lose their capacity to produce prostaglandins in response to addition of saturating exogenous arachidonic acid. Significant recovery of prostaglandin I2 and E2 synthesis occurred within 3 h and full enzymatic capacity returned by 6 h. Reducing the injury by exposure to half the amount of H2O2 allowed prostaglandin I2 production to recover to a greater extent in 3 h, while cells exposed for 60 min to either 0.5 or 1.0 mM H2O2 demonstrated no recovery. Pre-treatment with either actinomycin D or cycloheximide also prevented recovery following exposure to 100 microM peroxide. Injured cells did not recover when incubated with balanced salts after removal of peroxide, while incubation with medium 199 allowed for the complete return of synthetic capacity. Addition of 1% fetal calf serum in medium 199 did not facilitate recovery. Production of prostaglandins from endogenous arachidonic acid, released by either bradykinin or the ionophore A23187, was also inhibited by H2O2 exposure, however, full recovery of this stimulated synthesis occurred within 3 h. Cycloheximide pre-treatment completely inhibited recovery of bradykinin-induced prostaglandin I2 synthesis. These data demonstrate that sublethal concentrations of H2O2 irreversibly inactivate fatty acid cyclooxygenase and that synthesis of new enzyme is required for recovery. This return of activity occurs more rapidly for production of prostaglandins from endogenous arachidonic acid compared with production following addition of exogenous substrate.

Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells.

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.

Effect of hydrogen peroxide on prostaglandin production and cellular integrity in cultured porcine aortic endothelial cells.

Oxidative damage to the vascular endothelium may play an important role in the pathogenesis of atherosclerosis and aging, and may account in part for reduced vascular prostacyclin (PGI2) synthesis associated with both conditions. Using H2O2 to induce injury, we investigated the effects of oxidative damage on PGI2 synthesis in cultured endothelial cells (EC). Preincubation of EC with H2O2 produced a dose-dependent inhibition (inhibitory concentration [IC50] = 35 microM) of PGI2 formation from arachidonate. The maximum dose-related effect occurred within 1 min after exposure although appreciable H2O2 remained after 30 min (30% of original). In addition, H2O2 produced both a time- and dose-dependent injury leading to cell disruption, lactate dehydrogenase release, and 51Cr release from prelabeled cells. However, in dramatic contrast to H2O2 effects on PGI2 synthesis, loss of cellular integrity required doses in excess of 0.5 mM and incubation times in excess of 1 h. The superoxide-generating system, xanthine plus xanthine oxidase, produced a similar inhibition of PGI2 formation. Such inhibition was dependent on the generation of H2O2 but not superoxide in that catalase was completely protective whereas superoxide dismutase was not. H2O2 (50 microM) also effectively inhibited basal and ionophore A23187 (0.5 microM)-stimulated PGI2 formation. However, H2O2 had no effect on phospholipase A2 activity, because ionophore A23187-induced arachidonate release was unimpaired. To determine the effects on cyclooxygenase and PGI2 synthase, prostaglandin products from cells prelabeled with [3H]arachidonate and stimulated with ionophore A23187, or products formed from exogenous arachidonate were examined. Inhibition of cyclooxygenase but not PGI2 synthase was observed. Incubation of H2O2-treated cells with prostaglandin cyclic endoperoxide indicated no inhibition of PGI2 synthase. Thus, in EC low doses of H2O2 potently inhibit cyclooxygenase after brief exposure whereas larger doses and prolonged exposure are required for classical cytolytic effects. Surprisingly, PGI2 synthase, which is known to be extremely sensitive to a variety of lipid peroxides, is not inhibited by H2O2. Lipid solubility, enzyme location within the EC membrane, or the local availability of reducing factors may explain these results, and may be important determinants of the response of EC to oxidative stress.

Arachidonic acid metabolism in cultured aortic endothelial cells. Effect of cAMP and 3-isobutyl-1-methylxanthine.

To investigate the hypothesis that cyclic AMP (cAMP) regulates arachidonic acid metabolism in vascular tissue, we have studied the effects of forskolin (FSK), an activator of adenylate cyclase, and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, on hormone-stimulated prostacyclin (PGI2) synthesis in porcine aortic endothelial cells grown in culture. In these experiments, bradykinin (1 microgram/ml) and A23187 (0.2 microM) potently stimulated PGI2 biosynthesis (9- and 10-fold respectively). However, prostaglandin synthesis in response to either of these agents was not affected by FSK even though FSK elevated intracellular levels of cAMP 10-fold. IBMX failed to elevate basal cAMP levels when incubated with unstimulated cells. Stimulation of IBMX-treated (0.1 but not 1.0 or 4.0 mM) cells with bradykinin, however, did result in increased cAMP levels, presumably due to PGI2 formation and subsequent activation of adenylate cyclase. In addition to phosphodiesterase inhibition, IBMX inhibited PGI2 formation (72% at 1 mM) in a dose-dependent manner so that, at higher doses of IBMX, cAMP levels returned to baseline. Thus, prostacyclin synthesis inhibition by IBMX could not be attributed to elevated cAMP. In other experiments, IBMX (1 mM) was found to directly inhibit arachidonic acid release (32%) and arachidonic acid metabolism (65%) in endothelial cells and to inhibit arachidonic acid conversion to PGE2 by sheep seminal vesicle microsomes (65%). These data suggest that IBMX directly inhibits both phospholipase and cyclooxygenase activities. These experiments do not support the contention that cAMP regulates these enzymes in cultured aortic endothelial cells.

The role of calcium in the regulation of prostacyclin synthesis by porcine aortic endothelial cells.

Both bradykinin (EC50 = 8 ng/ml) and the ionophore A23187 (EC50 = 3 X 10(-7) M) potently stimulated arachidonate release and prostaglandin synthesis in porcine aortic endothelial cells. The response to each was completely dependent on extracellular Ca2+ (EC50 = 3 X 10(-7) M); no role for intracellular Ca2+ was noted. The rapid Ca2+ influx prompted by either activator was consistent with the time course for arachidonate release. Whereas the arachidonate released in response to bradykinin was transient, that released in response to A23187 was more prolonged, and paralleled a continued influx of Ca2+. Ca2+ entry elicited by bradykinin was mediated by channels which could not be blocked by verapamil. When Mn2+ was substituted for Ca2+, no stimulation of prostacyclin synthesis was seen in response to A23187; however, the bradykinin response was unaffected. The mechanism of these effects was studied using doses of bradykinin or A23187 which resulted in increases in Ca2+ influx and prostacyclin synthesis of similar magnitude for each agonist. Under these conditions, trifluoperazine blocked elevated prostacyclin synthesis (ID50 = 5-6 X 10(-6) M for each agonist). Trifluoperazine sulfoxide, however, was much less active. Pimozide inhibited bradykinin-stimulated prostacyclin synthesis at low doses (ID50 = 3 X 10(-6) M). Trifluoperazine was much less effective against high doses of A23187 (4 X 10(-6) M). These data suggest that arachidonate release and prostacyclin synthesis are dependent on influx of extracellular calcium and subsequent activation of a Ca2+-dependent phospholipase by a calmodulin-mediated mechanism.

Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta.

To address the hypothesis that metabolites of arachidonic acid are important regulators of prostaglandin (PG) synthesis in intact vascular tissue, we studied arachidonate metabolism in rabbit aortas in response to a continuous infusion of arachidonic acid, 10 micrograms/ml. Prostacyclin (PGI2; measured as 6-keto-PGF1 alpha) production rate accelerated during the first 2 min, reached peak velocity at 2 min, and then progressively decelerated. The velocity profile of PGI2 production was similar to that previously reported for cyclooxygenase holoenzyme assayed in vitro, and was consistent with progressive inactivation of the enzymes leading to PGI2 synthesis. We determined the specific inhibition of cyclooxygenase and prostacyclin synthetase by measuring PGI2 and PGE2 production rates and by infusing cyclic endoperoxides. Our results indicate preferential inactivation of cyclooxygenase during arachidonate metabolism, most likely due to cyclooxygenase-derived oxidative intermediates. This was a dose-dependent response and resulted in a progressive decrease in the 6-keto-PGF1 alpha/PGE2 ratio. Exogenously added 15-hydroperoxy eicosatetraenoic acid, on the other hand, actually stimulated cyclooxygenase activity at low doses, while markedly inhibiting prostacyclin synthetase. This finding, along with the accelerating nature of arachidonate metabolism, is consistent with the concept of "peroxide tone" as a mediator of cyclooxygenase activity in this system. These results demonstrate that arachidonate metabolites regulate PG synthesis in intact blood vessels. The progressive enzymatic inhibition intrinsic to arachidonate metabolism may be a model for similar changes occurring in states of enhanced lipid peroxidation. These metabolic alterations might greatly influence the numerous vascular functions known to involve arachidonic acid metabolism.

Mechanism of bradykinin-stimulated prostacyclin synthesis in porcine aortic endothelial cells.

Porcine aortic endothelial cells studied at confluence were found to synthesize both prostacyclin and prostaglandin E2. Addition of arachidonic acid, bradykinin, the calcium ionophore A23187 or thrombin stimulated prostaglandin formation, whereas addition of angiotensin II did not. Bradykinin was found to stimulate very potently arachidonic acid release from cells prelabelled with [3H]arachidonate, the response being dose-dependent and half-maximal at 8 ng/ml. The rate of release of label (primarily arachidonate) from cells was increased by bradykinin (100 ng/ml) approximately 8-fold, with a return to control levels by 10 min. The calcium ionophore, A23187, similarly released [3H]arachidonic acid from prelabelled cells; the rate of release was approximately linear for 15 min. Both bradykinin and ionophore A23187 stimulated [3H]arachidonate release from endothelial cell phospholipids, an effect which was abolished in a dose-dependent manner by mepacrine. Release in response to bradykinin was prevented by incubation in Ca2+-free medium. Trifluoperazine, a compound which can inhibit calmodulin-mediated events, blocked the release of label stimulated by bradykinin. These data indicate that the likely mechanism of bradykinin-stimulated prostaglandin production in endothelial cells involves the activation of a phospholipase via a Ca2+-calmodulin-dependent pathway.

The ability of vascular tissue to produce prostacyclin decreases with age.

The ability of aortae from young and mature swine to produce prostacyclin (PGI2) has been determined. PGI2 was measured as its hydration product, 6-keto-PGF1 alpha and assayed by stable isotope dilution GCMS. There was no significant difference in 6-keto-PGF1 alpha production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PFG1 alpha as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary difference an unlikely explanation for the age-related decrease in PGI2 synthesis. Both young and mature vascular tissues produced essentially only PGI2; insignificant amounts of PGE2 and PGF2 alpha were found.

Comparison of left ventricular free wall and septal diastolic compliance in the dog.

Septal to free wall dimensions are frequently employed for the analysis of diastolic compliance. However, the diastolic properties of these anatomically distinct regions of left ventricle are not well characterized. Regional compliance was studied in eight open-chest anesthetized dogs. Pairs of 2-mm-diameter piezoelectric crystals were implanted in the left ventricular free wall or septum 1.38 +/- 0.06 cm apart at a midwall location 58% +/- 1.9 of the left ventricular endocardial-epicardial or left ventricular endocardial-right ventricular endocardial distance. Left ventricular end-diastolic pressure was increased from an average of 8.1-21.0 mmHg, with a resulting average maximum end-diastolic strain of 11% (end-diastolic (ED) segment length/control ED length). Regional stiffness was assessed at all sites based on the relationship between left ventricular end-diastolic pressure and regional strain. Neither strain nor calculated stiffness coefficients differed significantly among the three sites. Septal transmural pressure (left ventricular end-diastolic pressure--right ventricular end-diastolic pressure) was nearly constant as left ventricular end-diastolic pressure increased during volume infusion and thus did not account for the observed septal strain.

Regional differences in myocardial performance in the left ventricle of the dog.

To determine whether significant regional differences in shortening exist in the canine left ventricle, the shortening characteristics of small segments of the circumferentially oriented hoop axis fibers and the more longitudinally oriented fibers near the epicardium were examined using pairs of ultrasound crystals placed at three levels of the left ventricular free wall in the open-chest dog. Mean control shortening of the hoop axis fibers near the apex of the left ventricle averaged 20% of the end-diastolic length, significantly greater than shortening at the midventricular (13%) or basal (14%) levels. During transient periods of aortic constriction, end-diastolic length increased significantly and the extent of shortening was maintained for the hoop axis fibers at the apical and midventricular levels; end-diastolic length did not change and shortening decreased at the basal level. The epicardial fibers shortened an average of 5.6% of their end-diastolic length during control conditions at all three sites and showed small, parallel changes in shortening and end-diastolic length during aortic constriction. We conclude that significantly greater hoop axis shortening occurs near the apex of the left ventricle and that at this level a uniformly contracting model is inappropriate. In addition, the response of the hoop axis fibers to increased aortic impedance is not homogeneous, with a significant reduction in shortening occurring only at the base of the left ventricle where end-diastolic length does not increase.