Gary L. Pool

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes.

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19 degrees C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5 degrees C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A2 is involved in the heat shock response.

Rat eustachian tube synthesizes disaturated phosphatidylcholine

Otitis media results when the eustachian tube fails to adequately ventilate the middle ear. A surface tension-lowering substance may be required for normal tube opening, especially in young children with poorly developed naso-pharyngeal musculature. We report here that rat eustachian tube epithelium synthesizes disaturated phosphatidylcholine, which is recognized as the surface tension-lowering substance of pulmonary surfactant.

Spontaneous and protein-catalyzed transfer of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) between phospholipid bilayers

1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor or alkylacetyl-GPC), a bioactive phospholipid that possesses hypotensive, platelet-aggregating and inflammatory properties, is known to be secreted by a variety of cell types. The biological activity of alkylacetyl-GPC is related to a precise chemical structure that implicates interaction with proteins. We have studied the spontaneous and protein-catalyzed transfer of alkylacetyl-GPC between phospholipid vesicles and have demonstrated the following: 1. There are at least two transferable pools of alkylacetyl-GPC in sonicated phospholipid vesicles. 2. These two pools differ in the rate at which they dissociate from the vesicles; one pool equilibrates between donor and acceptor vesicles instantaneously while the other pool is transferred much more slowly. 3. Dialysis of alkylacetyl-GPC between phospholipid vesicles through the aqueous phase is slow. 4. A protein fraction derived from rat lung cytosol catalyzes the transfer of the nonequilibrating pool of alkylacetyl-GPC between phospholipid vesicles; this transfer is superimposed on the spontaneous transfer and is unchanged in experiments using vesicles from which the rapidly equilibrating pool has been removed.

Lung phosphatidylcholine transfer in six vertebrate species: Correlations with surfactant parameters

We have examined phosphatidylcholine transfer activity in lung-soluble fractions from six vertebrate species. There is a significant correlation between the amount of phosphatidylcholine transfer activity and both the alveolar surface area and surface active material. This suggested that phosphatidylcholine exchange proteins have a role in the lung surfactant system.

Castration, gonadal hormones and Sidman avoidance acquisition.

Abstract The effects of hormones on the acquisition of a leverpress Sidman avoidance task were investigated. Rats were assigned to six groups: (a) castration with estradiol treatment; (b) castration with estradiol plus progesterone treatment; (c) castration with progesterone treatment; (d) castration with testosterone treatment; (e) castration with saline treatment; (f) sham castration with saline treatment. Shocks, responses and interresponse times were recorded during a two-hour acquisition session. Following acquisition, blood was drawn and serum hormone levels were determined by radioimmunoassay. Consistent with previous reports, estradiol-treated rats showed a nonsignificant trend toward superior acquisition of the task. Estradiol, relative to sham treatment, produced a significant reduction in individual variation in the acquisition of the avoidance task.

Effects of ovariectomy and hormone replacement of DRL behavior in the rat

Abstract Fifty-two female rats were sham-operated or ovariectomized. Groups of ovariectomized animals were sham-implanted or implanted with estradiol, progesterone or a combination of estrogen and progesterone. Assays were conducted to determine serum hormone concentrations. Animals were trained on a DRL-20 schedule for 30 days. During DRL, ovariectomized animals showed an increased response rate relative to intact animals. However, DRL performance data indicated ovariectomized rats did not differ significantly from intact female rats or hormone replacement groups on measures of DRL efficiency or on the number of earned reinforcers.

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.

Acylation of lyso platelet-activating factor by splenocytes of the rainbow trout,Oncorhyncus mykiss

In mammalian systems, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) is rapidly inactivated by a deacetylation/reacylation system that produces 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine which is highly enriched in arachidonic acid. There is some evidence that n−3 fatty acids may have an impact on this system in humans but the nature of this impact is unclear. In rainbow trout, n−3 fatty acids are known to be essential dietary components which are derived through the food chain. Substantial quantities of n−3 fatty acids are found in trout membrane phospholipids. We show here that in sharp contrast to mammalian cells, trout cells acylate lyso platelet-activating factor, alkyl-GPC, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, (lyso-PAF) with a high degree of specificity for n−3 fatty acids. When [3H]lysoPAF was incubated with these cells, only three molecular species of alkylacylglycerophosphocholine were produced, and 92% contained n−3 fatty acids. Since isolated membranes yielded similar results, it appears that the acylation proceedsvia a coenzyme A-independent transacylase as found in mammalian systems.

Lipid Mediator Mechanisms in Fish

Abstract Salmonids synthesize platelet-activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), and much of the interest in the potential use of lipids to mediate physiological events in fish metabolism is focused on this compound. In sharp contrast to mammalian cells, salmonid cells acylate lysoPAF with a high degree of specificity for omega-3 fatty acids. Polyunsaturated fatty acids in the lipids in tissues of rainbow trout Oncorhynchus mykiss are compartmentalized differently than in mammalian tissues. On extracellular challenge by PAF, trout leukocytes exhibit both chemotaxis and respiratory burst responses. During the metabolism of PAF in rainbow trout leukocytes, acylation of lysoPAF appears highly selective for docosahexaenoic acid (C22:6 n-3) and eicosapentaenoic acid (C20:5 n-3) and is localized in the endoplasmic reticulum. The acyl moiety in the sn-2 position of the acylation product 1-O-alkyl-2-acyl-glycero-3-phosphocholine may be removed by hydrolysis to produce lysoPAF in the...

Effects of castration and hormone replacement on Sidman avoidance acquisition in the rat

The effects of castration and hormone replacement on the acquisition of a leverpress Sidman avoidance task were examined. Sixty male rats were assigned randomly to one of five groups: (1) castration with estradiol treatment, (2) castration with progesterone treatment, (3) castration with estradiol and progesterone treatment, (4) castration with saline treatment, and (5) sham castration with saline treatment. Serum hormone concentrations were determined by radioimmunoassay. While serum assays indicated an increase of at least 600% to 3,300% for progesterone and estrogen levels, respectively, the results did not reveal any significant castration or hormone effects on the acquisition of the avoidance task.