Prostaglandin biosynthesis by midgut tissue isolated from the tobacco hornworm, Manduca sexta

We describe prostaglandin (PG) biosynthesis by isolated midgut preparations from tobacco hornworms, Manduca sexta. Microsomal-enriched midgut preparations yielded four PGs, PGA/B2, PGD2, PGE2 and PGF22? all of which were confirmed by analysis on gas chromatography-mass spectrometry (GC-MS). PGA and PGB are double bond isomers which do not resolve on TLC but do resolve by GC; for convenience, we use the single term PGA2 for this product. PGA2 was the major product under most conditions. The midgut preparations were sensitive to reaction conditions, including radioactive substrate, protein concentration (optimal at 1 mg/reaction), reaction time (optimal at 0.5 min), temperature (optimal at 22°C), buffer pH (highest at pH 6), and the presence of a co-factor cocktail composed of reduced glutathione, hydroquinine and hemoglobin. In vitro PG biosynthesis was inhibited by two cyclooxygenase inhibitors, indomethacin and naproxen. Subcellular localization of PG biosynthetic activity in midgut preparations, determined by ultracentrifugation, revealed the presence of PG biosynthetic activity in the cytosolic and microsomal fractions, although most activity was found in the cytosolic fractions. This is similar to other invertebrates, and different from mammalian preparations, in which the activity is exclusively associated with the microsomal fractions. Midgut preparations from M. sexta pupae, adult cockroach, Periplaneta americana, and corn ear worms, Helicoverpa zea, also produced the same four major PG products. We infer that insect midguts are competent to biosynthesize PGs, and speculate they exert important, albeit unrevealed, actions in midgut physiology. © 2002 Elsevier Science Ltd. All rights reserved.NEB-17-054Thanks to Dr Ralph Howard and Dr Juan Cibrian for reading and commenting on a draft of this work. This is paper no. 13,358, Nebraska Agricultural Research Division and contribution no. 1102 of the Department of Entomology. This work was supported by a fellowship from KahramanMaras Sutcu Imam University to H. Tunaz, a fellowship from TUBITAK-NATO to K. Büyükgüzel and by the Agricultural Research Division, University of Nebraska (NEB-17-054)