David Petzel

Immunohistological localization of regulatory peptides in the midgut of the female mosquito Aedes aegypti.

The midgut of the female mosquitoAedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatotropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.

Inhibition of eicosanoid biosynthesis modulates basal fluid secretion in the Malpighian tubules of the yellow fever mosquito (Aedes aegypti)

Abstract Inhibition of eicosanoid biosynthesis in in vitro preparations of Malpighian tubules isolated from adult females of the yellow fever mosquito Aedes aegypti substantially reduced basal fluid secretion rates. The phospholipase A 2 inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the cyclooxygenase inhibitor indomethacin far more effectively reduced basal fluid secretion than the epoxygenase inhibitor SKF-525A. The lipoxygenase inhibitor esculetin had no effect on basal fluid secretion. These findings indicate that products of cyclooxygenase are involved in regulating basal fluid secretion in Malpighian tubules. The effects of indomethacin were expressed in a dose-dependent manner, further indicating that eicosanoids are physiologically involved in fluid secretion. The effects of cyclooxygenase inhibition on reduction of basal secretion rates have not prevented adenosine 3′,5′-cyclic monophosphate (cAMP) from exerting its secretagogue effect. These results strongly support the hypothesis that eicosanoids, especially prostaglandins, are involved in Malpighian tubule function in A. aegypti . Recognition that eicosanoids may be involved in regulating basal fluid secretion rates introduces a previously unrecognized tier of regulatory physiology into insect renal function.

Arachidonic acid and prostaglandin E2 in malpighian tubules of female yellow fever mosquitoes

The fatty acid compositions of Malpighian tubules from adult females of the mosquito Aedes aegypti were determined for total lipids, phospholipids, triacylglycerols and three phospholipid fractions, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol/phosphatidylserine (PI/PS). The prostaglandin precursor arachidonic acid (20:4n-6) occurred in total lipids and phospholipids, but not triacylglycerols. Within phospholipids, nearly all of the 20:4n-6 was detected in PC, with only traces in PE, and none was detected in PI/PS. Isolated Malpighian tubules incorporated exogenous radioactive 20:4n-6 into tissue phospholipids and diacylglycerols, with most of the radioactivity recovered in diacylglycerol. These data indicate selective incorporation of 20:4n-6 into tissue lipids. PGE2 was detected in Malpighian tubule whole mounts by immunohistochemical staining. These findings support the idea that prostaglandins are physiologically active in mosquito Malpighian tubules.

Eicosanoid biosynthesis inhibitors modulate basal fluid secretion rates in the Malpighian tubules of the ant, Formica polyctena

Abstract Inhibition of eicosanoid biosynthesis in in vitro preparations of Malpighian tubules isolated from adult ants, Formica polyctena , reduced basal fluid secretion rates. Inhibition of total eicosanoid biosynthesis with 100 μM 5,8,11,14-eicosatetraynoic acid (ETYA) and inhibition of prostaglandin biosynthesis with 100 μM indomethacin strongly reduced basal fluid secretion. The lipoxygenase inhibitor esculetin and the epoxygenase inhibitor SKF-525A did not influence fluid secretion rates during its application, although it blunted the cAMP effect somewhat after washout. These findings indicate that prostaglandins are involved in regulating fluid secretion rates in ant Malpighian tubules. Although stimulation by cAMP was somewhat reduced, the influence of ETYA and indomethacin on fluid secretion rates did not prevent adenosine 3′,5′-cyclic monophosphate from exerting its secretagogue effect. This indicates that the eicosanoid biosynthesis inhibitors acted in a physiological way on the Malpighian tubules. The eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid, is present in phospholipids of Malpighian tubules. This finding indicates that substrate for prostaglandin biosynthesis is available.

Characterization of gill Na/K-ATPase activity and ouabain binding in Antarctic and New Zealand nototheniid fishes

The effects of thermal acclimation in two Nototheniid species, the stenothermal Antarctic Trematomous bernacchii and the eurythermal New Zealand Notothenia angustata, were investigated. Serum osmolality, gill Na/K-ATPase activity, sodium pump density and ouabain affinity were determined. Both fish were acclimated at their upper and lower viable thermal temperatures. Warm acclimation (+4 degrees C) of the T. bernacchii significantly decreased their serum osmolality from 550 to 450 mOsm/kg compared to cold-acclimation (-1.5 degrees C) and this was accompanied by a two-fold increase in gill Na/K-ATPase activity. Warm-acclimation (+14 degrees C) of N. angustata did not significantly change their serum osmolality from 330 mOsm/kg or gill Na/K-ATPase activity compared to the cold-acclimated (+4 degrees C) N. angustata. Using [(3)H]ouabain binding techniques, the B(max) and K(d) values of gill Na/K-ATPase enzymes were determined. No difference in the B(max) or K(d) of the warm-acclimated T. bernacchii accounted for the increase in Na/K-ATPase activity. We conclude that the change in gill Na/K-ATPase activity in the warm-acclimated T. bernacchii is not mediated by an increase in the number of enzyme sites and is not reflected in a change in ouabain affinity for Na/K-ATPase.

The Effect of Fluid Replacement on Endurance Performance

The purpose of this study was to examine the effects of fluid replacement on power output (PO), rating of perceived exertion (RPE), heart rate (HR), body weight (BW), urine osmolarity (Uosm), and urine electrolyte concentrations ([UNa+], [UK+], [UCl-]) in physically active men (n = 4) and women (n = 7). The participants were asked to generate their highest possible PO during 60 minutes of cycling under 3 randomized conditions: ingestion of (a) no fluid (trial 1); (b) 1200 ml of distilled water (trial 2); and (c) 1200 ml of Gatorade (trial 3). BW and urine volume (Vu) were measured before and after the ride to determine sweat rate [(SR = ΔBW + Vfluid intake + Vu)/time]. The results indicated that there were no significant differences between trials for PO (123–127 W), RPE (14), HR (140–142 b·min−1), and SR (11.9–12.4 ml·min−1). However, [UNa+] was significantly (p < 0.05) lower postexercise for all 3 trials, and [UCl-] was significantly reduced following trials 2 and 3. There was a significant increase (p < 0.001) in BW postexercise for trials 2 and 3 when compared with the no-fluid trial; however, the effects of water and Gatorade were similar. These results suggest that fluid replacement during 1 hour of moderately intense cycling does not enhance performance in physically active men and women who are normally hydrated.

Identification of mRNA and protein expression of the Na/K-ATPase α1-, α2- and α3-subunit isoforms in Antarctic and New Zealand nototheniid fishes

Abstract Antarctic nototheniids living in waters below 0 °C have a serum osmolality nearly double that of temperate nototheniids. Warm acclimation of Antarctic nototheniids to +4 °C decreases serum osmolality by 25% and nearly doubles gill Na/K-ATPase activity without an increase in Na/K-ATPase number or a change in the enzymes affinity for ouabain. However, the increased activity may result from a change in gill Na/K-ATPase α-subunit isoform composition. Four known Na/K-ATPase α-subunit isoforms have been identified in other species, however, identification of the isoforms in teleosts has been incomplete. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and cloning, we have identified partial cDNA sequences of the Na/K-ATPase α1-, α2- and α3-subunit isoforms in the Antarctic nototheniid Trematomus bernacchii . Using isoform-specific primers, the mRNA for all three isoforms was found in T. bernacchii brain, gill, heart, trunk kidney and muscle. Using isoform-specific antibodies, the Na/K-ATPase α1-, α2- and α3-subunit isoforms were found in the tissues of both T. bernacchii and Notothenia angustata , a temperate nototheniid. This is the first report detecting all three isoforms at the nucleotide and protein level in teleosts. Further experiments will determine if the gill T. bernacchii Na/K-ATPase α-subunit isoform composition changes during warm acclimation.

Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule

The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tyr], had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC(50)) was 17 nM. The (125)I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues. (125)I-LPA bound to the 54 kDa protein(s) with a K(d) value of 13+/-3 nM in agreement with the EC(50) for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.