Alan S. Bowman

Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum

Dopamine-induced saliva from ticks fed [3H]arachidonic acid contained the radiolabelled prostaglandins E2, F2 alpha, D2, and B2, the latter probably derived from PGE2 owing to the alkalinity of tick saliva. Prostaglandin synthetase (PGS) activity in the salivary gland homogenate from the lone star tick, Amblyomma americanum, could not be detected by standard radiometric methodologies successfully employed for tissues from many animal species, including numerous arthropods. Modifications to the assay conditions had no effect. The presence of a PGS-inhibitor in the salivary glands was ruled out. It is postulated that the PGS in A. americanum salivary glands may be considerably different from that found in other animals, including vertebrate hosts.

Prostaglandin E2 in the salivary glands of the female tick, Amblyomma americanum (L.): calcium mobilization and exocytosis

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the ticks saliva during feeding.

Origin of arachidonic acid in the salivary glands of the lone star tick, Amblyomma, americanum.

The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabolites of various radiolabeled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phospholipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, steric, and oleic acids. [3H]oleic acid, [14C]linoleic acid, [14C]gamma-linolenic acid and [14C]eicosatrienoic acids were incorporated into salivary gland phospholipids but underwent little change including elongation and/or desaturation to arachidonate. Ingested [3H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arachidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal.

Distribution of arachidonic acid among phospholipid subclasses of lone star tick salivary glands.

The subclass composition of choline- and ethanolamine-containing phospholipids was determined by analysis of acyl-linked fatty acids released by base hydrolysis of diradylglycerobenzoates formed from lone star tick salivary gland diacyl, alkylacyl and alkenylacyl phospholipids. The diacyl subclass comprises 87% of all choline-containing phospholipids, while th alkylacyl subclass comprises c. 9% and the alkenylacyl subclass c. 4%. The diacyl subclass comprises 72-77% of ethanolamine-containing phospholipids and about 14 and 13% of this subclass of phospholipid are alkylacyl and alkenylacyl lipids, respectively. Arachidonic acid (20:4) is the most abundant fatty acid (28% of all fatty acids) esterified in the alkylacyl form of phosphatidylcholine (PC) and it comprises 17% of the fatty acids in alkenylacyl-PC. The alkylacyl form of phosphatidylethanolamine (PE) is also rich in 20:4 (24%) while the alkenylacyl-PE subclass contains only 9% 20:4. Despite the relatively high amounts of 20:4 within the ether-linked phospholipids, the majority of the salivary gland 20:4 (> 83%) is found in the diacyl phospholipid subclass because of the preponderence of this subclass in tick salivary glands. Isolated salivary glands incorporated [3H]-20:4 primarily (> 98%) into the sn-2 position of diacyl PC > PE, with some incorporation into triglycerides. Continued incubation in the absence of labeled 20:4 demonstrated remodeling of [3H]-20:4 from PC into PE, and from the diacyl subclass to the alkylacyl subclass in the choline containing phospholipids.

Uptake, incorporation and redistribution of arachidonic acid in isolated salivary glands of the lone star tick

The ability of isolated salivary glands from the lone star tick, Amblyomma americanum, to take up, incorporate and redistribute [3H]arachidonic acid was examined. Uptake of arachidonic acid was concentration dependent--a single salivary gland incorporated up to approximately 2.8 micrograms arachidonic acid in 60 min. Over 90% of the [3H]arachidonate entering the glands was esterified and found only in the phospholipid (approximately 80%) and triglyceride (approximately 10%). Essentially no radioactivity was associated with the diglyceride fraction and none with phosphatidic acid indicating de novo phospholipid synthesis was negligible. Phospholipid synthesis via acylation of lysophospholipids (the Lands pathway) was indicated by the rapidity of the synthesis (< 2 min) and the sensitivity to sulfhydryl-blocking agents. Within the phospholipids, [3H]arachidonate was incorporated only into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Initially [3H]arachidonate was incorporated primarily into PC, but as the incubation proceeded PE contained an increasing proportion of the label. The proportion of [3H]arachidonate incorporated into triglyceride increased at higher media concentrations of arachidonic acid. The roles of lysophosphatide acyltransferase, transacylase and diglycerol acyltransferase in the distribution of arachidonate in tick salivary glands are discussed.

ALTERATION OF ARACHIDONATE LEVELS IN TICK SALIVARY GLANDS BY DIETARY MODIFICATION OF HOST BLOOD LIPIDS

Tick saliva contains prostaglandins of the 2-series, believed to facilitate bloodmeal acquisition. Because ticks cannot synthesize the prostaglandin precursor, arachidonic acid, investigations were undertaken to study the uptake, incorporation, and distribution of arachidonic acid in the salivary glands of the lone star tick in vitro and in vivo. Uptake of [3H]arachidonate by isolated salivary glands was reduced in the presence of low concentrations of arachidonic or eicosapentaenoic acids, but much higher, non-physiological concentrations of oleic and linoleic acids were required to inhibit [3H]arachidonate uptake. The incorporation of [3H]arachidonate into triglycerides increased at high concentrations of arachidonic or eicosapentaenoic acid, but not at any concentration of oleic or linoleic acid. Eicosatetraynoic acid greatly inhibited [3H]arachidonic acid. Guinea pigs fed hydrogenated coconut oil, safflower/primrose oil, or fish oil exhibited altered blood lipids; notably increased levels of eicosapentaenoic acid when fed fish oil. Salivary gland lipids in ticks fed on these hosts were also altered. Ticks parasitizing fish oil-fed guinea pigs contained high levels of eicosapentaenoic acid with a 30% reduction in arachidonate levels. The results demonstrated that eicosapentaenoic acid in the host diet had profound effects on arachidonate assimilation by tick salivary glands, which could lead to altered prostaglandin content in tick saliva.