Arachidonate Metabolism in Selected Tissues of the Lone Star Tick, Amblyomma americanum and Other Arthropods

Venkat Kumar Pedibhotla, University of Nebraska - Lincoln

Abstract

This dissertation presents the first direct demonstration of prostaglandins (PG) biosynthesis by selected tissues of the lone star tick, Amblyomma americanum. In vitro preparations of salivary glands and non-salivary internal tissues were capable of biosynthesizing four PGs, $\rm PGA\sb2/PGB\sb2,\ PGD\sb2,\ PGE\sb2,\ PGF\sb{2\alpha}.$ In both tissue preparations, $\rm PGA\sb2/PGB\sb2$ was the major product. Under standard assay conditions, 2 mg/ml protein were incubated at pH 8.0 for 2 min at 32$\sp\circ$C. PG biosynthesis by internal tissues was sensitive to incubation conditions. PG biosynthesis requires the presence of a co-factor cocktail composed of reduced glutathione, hydroquinone, and hemoglobin. The influence of freezing microsomal fractions of internal tissues on PG biosynthesis was also assessed. Internal tissue preparations were fractionated into cytosolic and microsomal preparations by ultracentrifugation. PG biosynthetic activity was detected in both fractions. I compared the capacity for PG biosynthesis in preparations made from fresh ticks and those that were stored intact at $-80\sp\circ$C for 3 months. Intact ticks stored at $-80\sp\circ$C yielded reduced PG biosynthesis by salivary glands. This elaborates the point that, unlike mammalian tissue preparations, tick tissue preparations are not stable to freezing without altering PG biosynthetic activity. Cyclooxygenase inhibitors, indomethacin and naproxen, inhibited salivary gland PG biosynthesis above 10 $\mu$M and 15 $\mu$M respectively. Immunoblot analysis of microsomal-enriched preparation from isolated salivary glands showed that anti-PGH synthase-1 and -2 bound to a 68 kDa protein. Anti-PGHS-2 also bound to a 68 kDa protein in microsomal-enriched fraction prepared from unfed male and female ticks. The subcellular distribution of PG biosynthetic activity in ticks is similar to other invertebrates, but quite different from mammals, in which PG biosynthetic activity is almost exclusively localized in the microsomal fractions. These findings indicate that PG biosynthesizing enzymes were endogenous to ticks and not acquired from their hosts. An improved method for detecting subnanogram quantities of PGs on fluorometric HPLC is described. In the past, PG derivatization using anthryldiazomethane (ADAM) has been performed. Typically, the reactions were conducted for 12-16 hours in a excess of ADAM. After the formation of derivatives the samples were then subjected to clean-up procedures, usually on SEP-PAK columns, before analysis on HPLC. These procedures have been improved by reducing the reaction times, and by changing the reaction conditions to eliminate the need for pre-column clean-up. In the improved method the entire derivatization is completed in less than 5 hours. The lower limit of detection on our fluorescent detector is around 60 pg. We applied the method in analysis of PGs in salivary secretions and salivary glands from the lone star tick, Amblyomma americanum. The efficiency of extracting and derivatizing PGs from the biological sample is 85-90%. PGs occur in tick saliva in the range of 2.0-24.0 ng/ml. Whereas ADAM reagent is very unstable, even at $-20\sp\circ$C, PG derivatives can be stored for at least a month at $-20\sp\circ$C for a month. This method is rapid, economical, and sensitive. (Abstract shortened by UMI.)

Subject Area

Biochemistry|Zoology|Physiology

Recommended Citation

Pedibhotla, Venkat Kumar, "Arachidonate Metabolism in Selected Tissues of the Lone Star Tick, Amblyomma americanum and Other Arthropods" (1996). ETD collection for University of Nebraska-Lincoln. AAI9703788.
https://digitalcommons.unl.edu/dissertations/AAI9703788

COinS