B. Salafsky

SCHISTOSOMA MANSONI: PRODUCTION OF CERCARIAL EICOSANOIDS AS CORRELATES OF PENETRATION AND TRANSFORMATION

A method was developed, using a 0.25% agar matrix, to incorporate varying concentrations of linoleate and correlate cercarial transformation and eicosanoid production in vitro. Schistosoma mansoni cercariae were stimulated to penetrate over a wide range of linoleate concentrations; however, the transformation process occurred over a narrow range. Approximately 25% of cercariae penetrated the agar matrix in controls (no linoleate) and 0.003 mM linoleate. Penetration rates rose gradually until, at linoleate concentrations of 0.3 mM or greater, penetration approached 100%. The transformation process did not begin until the linoleate concentration in agar reached 2.0 mM (3.8%), and achieved maximum (91%) at 3.0 mM. A concentration of 9.0 mM linoleate gave 100% penetration and transformation rates, but penetration was superficial and cercariae were not viable. Cercarial eicosanoid production was concentration-related. Various eicosanoid classes were associated with cercarial penetration and transformation. Penetration rates were correlated with increasing leukotriene (LT, R = 0.9541) and hydroxyeicosatetraenoic acid (HETE, R = 0.8363) levels, while transformation rates correlated with increasing prostaglandin levels (R = 0.9225). Correlating eicosanoid production with penetration and transformation rates strengthened the hypothesis that successful cercarial penetration and transformation are dependent on both skin essential fatty acid levels and resulting cercarial eicosanoid production.

Selective effects of ethanol on opiate receptor subtypes in brain

Large concentrations of ethanol in vitro decreased ligand binding to mu and delta opiate receptors in the frontal cortex of the C57BL mouse, but did not alter binding to kappa opiate receptors. Mu and delta receptors were equally sensitive to the inhibitory effect of ethanol. Since the effects of ethanol were significant only in large concentrations, ethanol may alter opiate binding through its membrane lipid-perturbing actions, and the selectivity of the effects of ethanol may reflect differences in the microenvironments of the opiate receptor subtypes in membranes. After chronic ingestion of ethanol by mice, in vivo, there was a selective decrease in the number of mu receptors in the frontal cortex. This change may result from indirect effects of ethanol on the opiate receptor and may contribute to specific central effects of ethanol.

THE ROLE OF ESSENTIAL FATTY ACIDS AND PROSTAGLANDINS IN CERCARIAL PENETRATION (SCHISTOSOMA MANSONI)

We used rat skin membranes to test the putative role of prostaglandins (PG) and essential fatty acids (EFA) in the penetration response of Schistosoma mansoni cercariae. To examine the effects of EFA on cercarial penetration an EFA-deficient rat model was used. Dams were fed an EFA-deficient diet during lactation and the pups were weaned to this diet. Cercarial penetration of EFA-deficient rat skin membranes was not reduced from control levels until 12 wk on the diet. At this time a decrease of 64.3% was observed. This decrease remained constant up to 16 wk, after which the study was terminated. Other normal rats were treated with 20 mg/kg ibuprofen, a PG inhibitor, to examine the role of PG in the penetration response. Treated rat skin contained a mean of 2 micrograms ibuprofen per 30 mm3 of skin (25-mm skin disc) at 1.5 hours post-injection. Skin from treated rats inhibited penetration by over 81%. These studies indicate that skin EFA and PG may have a critical role in the completeness of penetration by cercariae through the skin, although it is not clear whether cercarial or host PG are involved in the penetration response.

Prenatal and postnatal exposure to diazepam: effects on opioid receptor binding in rat brain cortex.

The effects of chronic perinatal or postnatal administrations of diazepam on opiate receptor development in cerebral cortex and striatum were studied at two different post-treatment ages, using tritiated ethylketocyclazocine, the prototypic kappa-opiate, for measurements of opiate binding site ontogeny. Prenatal plus postnatal exposure to diazepam markedly decreased the total number of binding sites for [3H]ethylketocyclazocine in the rat cerebral cortex and striatum at 14 days of age. Postnatal diazepam treatment alone did not alter [3H]ethylketocyclazocine binding site development in cortex or striatum. These results suggest that development of opiate receptors in rat brain can be altered by prenatal exposure to diazepam.

The role of prostaglandins in cercarial (Schistosoma mansoni) response to free fatty acids.

In examining the structure-activity relationship of a diverse group of chemicals reported to prevent cercarial penetration after topical application, we noticed a moiety that was common to free fatty acids and prostaglandins. Because unsaturated fatty acids have been reported to stimulate cercarial penetration, we hypothesized that cercarial stimulation by skin and fatty acids may invoke prostaglandin mechanisms in cercariae, skin, or both. Thus we compared the stimulation of cercariae by a series of essential and nonessential fatty acids and demonstrated an inhibition of this response by ibuprofen and aspirin, known cyclo-oxygenase inhibitors, and by 13-azaprostanoic acid, a potent antagonist of the thromboxane/endoperoxide receptor. These data led us to postulate a major role for prostaglandins in the cercarial penetration response.

Effects of ethanol on ouabain inhibition of mouse brain (Na+,K+)ATPase activity

Plots of ouabain inhibition of mouse cerebral cortical (Na+,K+)ATPase activity fitted a two-site model significantly better than a one-site model, consistent with the presence of two forms of the enzyme with different affinities for ouabain. The fraction of enzyme activity with high affinity for ouabain (HAO: Ki = 500 nM), suggested to be localized neuronally, constituted the major portion (60-70%) of activity. Ouabain inhibition of both components of enzyme activity was reduced as KCl concentrations were increased. In vitro, only high concentrations of ethanol affected (Na+,K+)ATPase activity and ouabain inhibition of activity. Ethanol (500 mM) selectively reduced the activity, and increased the sensitivity to ouabain inhibition, of the HAO component, with no significant effect on the low-affinity (LAO) component. On the other hand, following chronic treatment of mice with ethanol in vivo, in a paradigm that produced tolerance and physical dependence, the sensitivity to ouabain of the HAO form of the enzyme was selectively increased. The relative proportions, and the activities of the HAO and LAO components, were not altered. The effects of ethanol, added in vitro, on the HAO component were decreased in ethanol-tolerant animals. The selective effect of chronic ethanol ingestion on (Na+,K+)ATPase activity indicates the specificity of action of ethanol in the CNS.

Effects of short-chain alcohols and norepinephrine on brain (Na+, K+)ATPase activity

(Na+,K+)ATPase activity in synaptic membranes from whole brains of mice was inhibited by a series of short-chain aliphatic alcohols (ethanol through pentanol). The relationship of inhibitory potency to alcohol chain length and to alcohol membrane:water partition coefficient suggested that the inhibitory effect of the alcohols does not depend totally on their interaction with neuronal membrane lipids. Although partitioning into the membranes is important for this inhibitory effect, a direct interaction of alcohol with the enzyme protein may also be involved in the inhibition. Norepinephrine did not significantly potentiate inhibition of (Na+,K+)ATPase activity by low concentrations of ethanol in preparations of either mouse or rat brain. Thus, under our conditions, ethanol, at levels which can be reached in vivo, only slightly inhibited enzyme activity, and the possible importance of this inhibition in mediating the in vivo acute or chronic effects of ethanol on the CNS remains open to question.

Schistosoma mansoni: the role of calcium in the stimulation of cercarial proteinase release.

We investigated the role of calcium mobilization in the induction of proteinase release from cercarial preacetabular glands. Proteinase release was measured by the ability of cercariae to break down a 3H-labeled proline extracellular fibroblast matrix and calcium influx was measured using 45Ca2+. The role of calcium in the activation of cercarial proteinase was examined by investigating the effects of calcium addition and removal on linoleate-induced matrix degradation, the ability of various calcium modulators (Verapamil, fendiline, nifedipine, SK-525A, BAY K-8644, Ryanodine, and SK-7171A) to stimulate or inhibit linoleate-induced proteinase release, the ability of calcium modulators directly to induce cercarial proteinase release, and the ability of various stimulants of proteinase release to induce calcium influx or efflux from cercariae. The results of these studies indicate that proteinase release is dependent on external calcium concentration, voltage-operated channels are either nonexistent in cercariae or have a minimal role in overall calcium influx, and that activation of Ca2+ influx can be caused by both free fatty acids and calcium modulators by a hypothesized receptor-operated channel. Although calcium uptake is important in cercarial proteinase release, it is not the only factor involved. Calcium uptake alone does not guarantee that proteinase will be secreted. On the other hand, if Ca2+ influx does not occur, proteinase will not be secreted.

Penetration of Schistosoma mansoni cercariae into a living skin equivalent.

We evaluated the use of a living skin equivalent (LSE) as a suitable membrane for Schistosoma mansoni cercarial penetration. LSE is a living artificial skin composed of a dermal layer containing human dermal fibroblasts embedded in a collagen lattice and an epidermal layer consisting of differentiated human keratinocytes. The keratinocytes differentiate into a stratum corneumlike layer, whereas the dermal-epidermal junction forms a layer similar, but not identical to, the basement membrane. We exposed LSE to 50 cercariae for 0, 3, 6, 20, and 30 hr at 37 C, and the percentage of penetration was evaluated by counting cercariae remaining on the LSE surface. No cercarial penetration was observed in the first 15 min of exposure; however, penetration was detected at all other times. Maximum penetration rates were observed at 20 hr (80%). In other experiments LSE was pretreated topically with 0 or 4 micrograms/cm2 linoleic acid, then exposed to between 800 and 1,000 cercariae for 18-20 hr at 37 C. LSE pretreated with linoleate had significantly higher penetration rates than untreated membranes (81% +/- 2.51% vs. 65.9% +/- 6.97%, P = 0.03). Increasing linoleate concentrations from 10 to 40 micrograms/cm2 gradually decreased the ability of cercariae to penetrate the membrane. Some LSE membranes also were processed for light microscopy, and we present photomicrographs showing schistosomulae within the epidermal and dermal layers of the LSE. We conclude that despite the time it takes for cercariae to penetrate LSE, these membranes may allow investigators to examine, in vitro, host-parasite interactions at the level of the skin.

Influence of a menhaden oil diet on cercarial penetration of Schistosoma mansoni

The ability of a menhaden oil (MO) diet to influence cercarial penetration into mouse tail skin was evaluated. Male CD-1 mice 4-6 wk old (15.2 g average weight) were fed a 0, 10%, or 20% MO-supplemented diet for 2 wk. After this time mice were infected with either 65 +/- 3 or 145 +/- 3 [35S]methionine/cysteine-labeled cercariae for 1 hr by tail immersion. Twenty-four hours and 7 days later groups of mice were killed and their tail skin removed and autoradiographed. At 24 hr postinfection, mice fed a 20% MO diet had significantly higher cercarial penetration than controls and 10% MO diets (56% +/- 5.2 vs. 44% +/- 2.9, P = 0.02, 1-tailed t-test). After 7 days mice fed a 20% MO diet retained more radioactive foci than controls or 10% MO diets (21% +/- 2.0 vs. 15% +/- 1.3, P = 0.01, 1-tailed t-test).