Ultraviolet radiation-induced phospholipase A2 activation occurs in mammalian cell membrane preparations.

Ultraviolet erythema in human skin is mediated in part by membrane derivatives of arachidonic acid (AA). UVA (320-400 nm) and UVB (290-320 nm) have been shown to induce release of AA from intact mammalian cells in culture. In order to investigate the mechanism of this release we examined the effect of UVA and UVB on release of [3H] AA from membrane preparations of murine fibroblasts. C3H 10T1/2 cells were prelabelled for 24 h with [3H] AA. The membrane fractions of the cells were separated after lysis by differential centrifugation. The membranes were irradiated in suspension and the [3H] AA released from the membranes was determined by scintillation spectroscopy of supernatants 3-4 h after irradiation. Both UVA and UVB induced release of AA from the membrane preparations. The response to UVB was small but significant, reaching levels approximately 150% of control release at doses of 1,200-4,000 J/m2. The response to UVA was larger; doses of 2.5-5.0 J/cm2 induced release equal to twice control (200%) levels, while doses of 10-20 J/cm2 induced maximal release at levels approximately 400% of control. Time course studies with UVB and UVA showed maximal release at 4 h after irradiation. When the membrane preparations were incubated with a polyclonal anti-phospholipase A2 antibody the UV induced release of [3H] AA was completely inhibited in both UVB (1200 J/m2) and UVA (10 J/cm2) treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission.

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated (60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission 'in vitro'. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A(2) (PLA(2)) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27 degrees C, CTX (14 microg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37 degrees C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 microg/ml) was inneffective on the neuromuscular transmission either at 27 or 37 degrees C. The PLA(2) enzymatic activity of CTX at 37 degrees C was 84 and that at 27 degrees C was 27 micromol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 micromol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA(2) enzymatic activity.

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes.

The activities of enzymes involved in lipid metabolism--phospholipase A2 (PLA2) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)--were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.

Synthesis of photoreactive phosphatidylethanolamine and its interaction with phospholipase A2.

A photoreactive derivative of phosphatidylethanolamine, N-(4-azidobenzoyl)phosphatidylethanolamine (AB-PE), was synthesized by acylation of phosphatidylethanolamine with an N-hydroxysuccinimide ester of 4-azidobenzoic acid. The substantial photosensitivity exhibited by AB-PE correlated with a marked decrease in the absorption spectra of the compound. The compound proved sensitive to lipase and phospholipase A2 hydrolysis but resistant to phospholipase C and D activities. Photolysis of a sonicated dispersion of AB-PE containing phospholipase A2 resulted in irreversible inhibition of the enzyme. Addition of natural phosphatidylethanolamine provided protection against photoinactivation.

Presence of a light-independent phospholipase A2 in bovine retina but not in rod outer segments.

Rod outer segments (ROS) are responsible for the visual transduction process. Rhodopsin, which constitutes 85-90% of ROS proteins, absorbs light photons, changes its conformation, and then binds to a heterotrimeric G-protein called transducin. As a consequence, transducin dissociates into Talpha and Tbetagamma subunits. The presence in ROS of a phospholipase A2 (PLA2) stimulated by light and guanosine 5'-O-(3-thio)triphosphate was first demonstrated in 1987 (Jelsema, C. L.(1987) J. Biol. Chem. 262, 163-168). This led that author to conclude that ROS PLA2 could be involved in the phototransduction process, and raised the possibility of receptor-mediated activation of PLA2 via G-proteins in cell types other than rods. However, the biochemical characteristics and the role of this PLA2 have not been fully elucidated. We have tried to reproduce some of the results previously reported in order to further characterize this enzyme. We have found that, in our hands, there is neither light-dependent nor GTP-dependent PLA2 activity in intact purified ROS. We also failed to detect PLA1 activity in those ROS preparations. Nevertheless, we detected significant amounts of PLA2 activity in two subretinal fractions adjacent to ROS: RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Müller cells, and rod inner segments). The enzyme present both in RPE and P200 is light- and GTP-independent, Ca2+- and Mg2+-independent, and seems to be optimally active in the alkaline pH range. Our results suggest that there is, if any, vanishingly little PLA2 or PLA1 activity in intact purified ROS and that the activity levels previously reported in the literature could have been due to a contamination by either RPE or P200. This is supported by our observation that some contaminated ROS preparations were "PLA2 active."

Evaluation of the effect of gamma rays on the venom of Vipera lebetina by biochemical study.

Snake bites represent a serious public health problem in many areas of the world. In Algeria, two widespread snakes are Vipera lebetina and Cerastes cerastes. Vipera lebetina venom causes local hemorrhage and necrosis, and it may lead to permanent limb loss. The principal causes of mortality after snakebites are acute renal failure and hemorrhage, which occur not only locally, at the site of the bite, but also systemically, contributing to the cardiovascular shock characteristic of severe envenomation. Gamma radiation has been shown to be effective for attenuating venom toxicity. Vipera lebetina venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60Co source, and the venom's toxic, enzymatic, and structural properties were analyzed. Intraperitoneal injection of the irradiated venoms (100-500 microg/20 g mouse body mass) revealed a significant decrease of the toxicity. Irradiated venoms with 1 and 2 kGy doses were four and nine times less toxic, respectively, than the native venom. A biochemical characterization of in vitro enzymatic activities was performed. Vipera lebetina displayed in vitro caseinolytic, amidolytic, esterasic, coagulant, and phospholipase A2 activities. Caseinolytic, amidolytic, esterasic, and coagulative activities were reduced for the irradiated venoms; only phospholipase A2 activity was abolished in the irradiated venom with a dose of 2 kGy. The native and irradiated venoms were separated by gel filtration and electrophoresis. Chromatographic and electrophoretic profiles were drastically changed as compared with the native venom. Vipera lebetina venom detoxified by gamma rays was used for active immunization, and the presence of antibody in the immune sera was detected by ELISA. The immunogenic properties were preserved and the antisera obtained with the irradiated venoms could cross-react. Antisera were able to neutralize the toxic effect of V. lebetina native venom. These results indicate that irradiation of V. lebetina venom with a dose of 2 kGy can promote a significant detoxification, keeping the immunological properties intact.

Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins.

Light stimulates phospholipase A2 activity in rod outer segments (ROS) of bovine retina as measured by the liberation of arachidonate from phosphatidylcholine, in in vitro assays of dark-adapted ROS. A role for GTP-binding proteins (G or N proteins) in the light activation of phospholipase A2 is suggested by the capacity for guanosine 5'-O-(thiotriphosphate) (GTP gamma S) to activate phospholipase A2 in dark-adapted ROS. In contrast, addition of GTP gamma S coincident with light exposure inhibited the light activation of phospholipase A2, suggesting that phospholipase A2 activity in the ROS is under dual regulation by G proteins. Transducin, the major G protein of the ROS, mediates the activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. Treatment of dark-adapted ROS with either toxin inhibits both basal and light-activated phospholipase A2, mimicking the action of the toxins on the light-induced cGMP phosphodiesterase activity of ROS. There is a loss of light-sensitive phospholipase A2 activity coincident with extraction of transducin from ROS membranes. In addition, the light-sensitive phospholipase A2 activity can be partially restored by the addition of purified transducin to the extracted ROS membranes. Light activation of phospholipase A2 in ROS membranes thus occurs by a transducin-dependent mechanism.