Injection Of Phospholipase A2 Research Articles

Ultrastructural analysis of early toxic effects produced by bee venom phospholipase A2 and melittin in Sertoli cells in rats

In this study, we aimed to investigate the testicular toxicity of two molecules derived from bee venom (BV): phospholipase A2 (PlA2) and melittin (Mlt). Ultrastructural effects of purified BV PlA2 and Mlt were assessed consecutive to repeated dose (30 days) and acute toxicity studies. For the subchronic treatment, PlA2 and Mlt were injected in daily doses equivalent to those released by a bee sting (105 μg PlA2/kg/day and 350 μg Mlt/kg/day), while in the acute treatment their doses corresponded to those released by 100 bee stings (9.3 mg PlA2/kg and 31 mg Mlt/kg). Both PlA2 and Mlt affected the Leydig cells and the cells in seminiferous tubules, the Sertoli cells first of all. PlA2 injection resulted in detachment of the Sertoli cells from the surrounding cells, and extracellular vacuolations, cytoplasmic vacuolations in their basal region and in branches as well, detachment of spermatids, residual bodies and sometimes even spermatocytes into the lumen, changes that had a higher magnitude after the acute treatment. Mlt injection induced similar ultrastructural alterations, but more severe, including degeneration of cellular organelles and cellular necrosis, resulting into rarefaction of the seminiferous epithelium; the ultrastructural changes had a higher magnitude after the 30 repeated dose treatment. We concluded that either of the two molecules tested here, PlA2 and Mlt, were Sertoli cells toxicants at the used doses, and they participated both in the BV testicular toxicity. We consider the observed changes as part of a preceding mechanism of the more severe alterations produced by the BV. It also remains possible that these early unspecific changes reported here could represent the response of the SCs not only to the components of bee venom, but to molecules of other venoms as well. The Sertoli cells were the primary target of PlA2 and Mlt in the spermatogenic epithelium, and their alteration led to further degenerative changes of the germ cells. Since the exposure to PlA2 and Mlt caused severe alteration, including cell death and detachment of immature germ cells into the lumen, we may also conclude that the bee venom molecules had a potential to interfere with normal progression of spermatogenesis. All the degenerative changes observed in the Sertoli cells were accompanied with changes of the Leydig cells.

Pancreatic Fat Necrosis and Hemorrhagic Necrosis as Separate Morphological and Functional Entities

Peculiarities of pathomorphogenesis of two destructive forms of acute pancreatitis were compared using three experimental models. We have shown that the direction of the pathological process is determined by specific combinations of pathogenic factors. Pathological processes caused by common bile duct ligation (ductal hypertension) alone and in combination with injection of phospholipase A2 are characterized by mixed pattern with strong predominance of fat necrosis symptoms (coagulative acinar necrosis, inflammatory demarcation, and sites of lipolysis) and hemorrhagic necrosis, respectively. Trypsin injection into the pancreatic tissue induces rapidly progressing hemorrhagic pancreatic necrosis with massive hemorrhages, extensive acinar necrosis, and weak cell reaction. Considerable differences in pathomorphogenesis and outcome of the pathological process allow us to consider pancreatic fat necrosis and hemorrhagic necrosis as separate morphological and functional entities, which is essential for the prognosis and treatment strategy.

Intracerebral injection of phospholipase A2 inhibits dopamine-mediated behavior in rats: possible implications for schizophrenia.

Phospholipase A2 (PLA2) is a key enzyme in the phospholipid metabolism. In the CNS intracellular PLA2 plays an essential role in signal transduction by affecting both dopamine (DA) release and DA-receptor sensitivity. In schizophrenia a disordered phospholipid metabolism and increased activity of PLA2 have been reported. In this study we investigated the effects of intracerebral PLA2 injections on dopaminergic neurotransmission in rats using Ungerstedt's model of rotational behavior. Circling behavior induced by the DA agonist apomorphine after unilateral PLA2 injections into the substantia nigra pars compacta was recorded. Seven and 21 days after intranigral PLA2 injection, apomorphine induced an ipsilateral rotation indicating a long-lasting inhibition of ipsilateral nigrostriatal dopaminergic pathway by PLA2 application. In schizophrenia a reduced dopaminergic activity in the frontal cortex has been hypothesized. Recent spectroscopy studies reported on an accelerated break-down of membrane phospholipids in the frontal cortex from schizophrenics. The present findings suggest that increased PLA2 activity in schizophrenia could accelerate the breakdown of membrane phospholipids and thus contribute to a hypodopaminergy in the frontal cortex of schizophrenic patients.

Phospholipase A2-induced electrophysiologic and histologic changes in rabbit dorsal lumbar spine tissues.

The present study was designed to characterize the effect of phospholipase A2 on the discharge of perispinal sensory nerves in the anesthetized New Zealand white rabbit. To examine the effects of phospholipase A2 on the neural response of somatosensory neurons innervating the lumbar facet joint and surrounding tissues. An irritating component of disc tissue may be phospholipase A2, which has been found at extraordinary high levels in herniated and painful discs. Phospholipase A2 has been shown to be inflammatory, but its effect on nerve response has never been shown. Surgically isolated facet joint capsules from rabbits were investigated by means of electrophysiologic and histologic techniques. Phospholipase A2 was injected into the characterized nerve receptive field, and responses were evaluated over time with varying doses. The injection of phospholipase A2 into the nerve receptive fields produced neurotoxicity with a 1500-U dose, sensitization of the nerves and recruitment of "silent units" with a 750-U dose, and no electrophysiologic effect with a 400-U dose. The tissues injected with phospholipase A2 and control solutions were examined histologically using a hematoxylin and eosin staining technique. In all three doses, the inflammatory changes were observed as soon as 2 hours after the injections. In control subjects, no changes were observed. After phospholipase A2 injection, the discharge rate of the units showed dose and time dependent patterns. Regardless of the different doses, histologic changes were observed as soon as 2 hours after the phospholipase A2 injections.

The role of phospholipase a2 in pancreatic acinar cell injury

The integrity of rat pancreatic acinar cells under the influence of human phospholipase A2 (PLA2) was studied. Isolated pancreatic acini showed no increased discharge of aspartylaminotransferase (ASAT) when incubated either in solutions containing human pancreatic PLA2 or the bile salt sodium deoxycholate (DEC), the latter in concentrations that augment PLA2 activity but have no destructive detergent effect. When human pancreatic PLA2 was injected into the rat pancreatic duct, uneven distribution was observed at 15 min and 3 h in immunohistochemical sections. Edema and a mild inflammatory reaction were the main changes in the pancreas. The necrotic areas seen by light and electron microscopy were quite small and located mostly at the periphery of lobules corresponding the spread of the injected material. Necrosis was of the coagulation type and showed equal extent after the injection of PLA2 with or without DEC. Internalized human pancreatic PLA2 was present already 15 min after the injection in the cytoplasm of some intact acinar cells, indicating a functioning protective mechanism. It was concluded that pancreatic acinar cells are quite resistant to PLA2-catalyzed hydrolysis of membrane phospholipids in vitro, but additional trauma, e.g., pressure caused by intraductal injection, and tissue related factors, such as the mediators of the inflammatory reaction, make acinar cells susceptible to the effect of PLA2.

Preliminary studies on phospholipase A2‐induced mouse paw edema as a model to evaluate antiinflammatory agents

Phospholipase A2 (PLA2) is a key component of the inflammatory process because of its role in the generation of eicosanoids and platelet-activating factor (PAF). Manipulation of PLA2 activity offers a novel therapeutic approach for the development of antiinflammatory agents; however, there is a need for a suitable in vivo model. Injection of 1 microgram of snake venom PLA2 (A. piscivorus piscivorus, D-49) into the mouse hind footpad produced a significant three- to four-fold rise in paw edema within 10 min, compared to the saline control. Edema formation depended on enzyme concentration and appeared specific for PLA2 since edema was negated by enzyme pretreatment with p-bromophenacyl bromide, a nonspecific PLA2 inhibitor. Moreover, injection of a protein such as bovine serum albumin did not result in significant edema. Coinjection of phenidone (lipoxygenase inhibitor, 50 micrograms), indomethacin (cyclooxygenase inhibitor, 50 micrograms), cyproheptadine (antihistamine/antiserotonin, 50 micrograms), aristolochic acid (putative PLA2 inhibitor, 100 micrograms), or kadsurenone (PAF antagonist, 50 micrograms) with PLA2 (1 microgram/paw) resulted in partial reduction (44.5, 34.2, 54.7, 64, and 50% inhibition, respectively) of edema formation. Oral administration of cyproheptadine (10 mg/kg), indomethacin (10 mg/kg), BW 755c (100 mg/kg), or dexamethasone (1 mg/kg) 1-3 h before challenge also decreased PLA2-induced edema (63.0, 30.1, 47.8, or 62.5% inhibition, respectively). The data suggest that mouse paw edema resulting from PLA2 injection is a multicomponent event, influenced by both autacoids and lipid mediators of inflammation.

Extracellular phospholipase A2 mediates inflammatory hyperaemia.

Inflammatory erythema is primarily the gross manifestation of enhanced regional blood flow or hyperaemia. Hyperaemia to areas of injury is thought to modulate the delivery of circulating inflammatory cells to the responding site1,2 and in experimental models, it was shown synergistically to enhance the extravasation of plasma proteins and mediators3,4. It has recently been proposed that blood flow to inflammatory sites may be modulated by a novel vasoactive moiety, distinct from prostaglandins and plasminogen activator5,6, which is released by macrophages in response to antigenic or mitogenic stimulation. Intradermal (i.d.) injection of this hyperaemia-inducing activity (HIA) from activated macrophages elicited a sustained hyperaemia in the test rabbit7. We have now investigated this vasoactive mediator further. Using a sensitive assay for phospholipase A2 (PLA2)8, we have identified this enzyme in vasoactive conditioned medium of glycogen-induced peritoneal exudate cells (PEC) of rabbits. In the vasoactive conditioned medium, PLA2 and HIA cofractionated on gel filtration chromatography, and both the enzyme activity and biological activity were abrogated by treatment with the active site-directed histidine reagent p-bromophenacyl bromide (pBPB). Intradermal injection of PLA2 purified from snake venom induced a hyperaemia with kinetics resembling those of HIA. We propose that antigen-induced release of PLA2 by activated macrophages may mediate the hyperaemia associated with sites of chronic inflammation.