Gorging response of Glossina palpalis palpalis to ATP analogues

ABSTRACT. The potencies of seventeen analogues of ATP as gorging inducers for Glossina palpalis palpalis were evaluated. The ranking for effective dose that induced half the flies to gorge (ED₅₀) was: A tetra P 5 ATP=2'd ATP ADP=2'd ADP > AMP-PNP > 3'd ATP 2'3'dd ATP > AMP-PCP > adenosine 5' triphosphate 2',3'dialdehyde AMP-CPP >> AMP. Females detect ATP and its analogues better than males. The ED₅₀ of ATP was 5 × 10^-7 M for teneral females and 1.5 × 10^-6 M for males. According to the potency order of the ATP analogues, the G.p.palpalis gustatory receptors recognizing ATP can be classified as P_2y purinoceptors.     

The effect of putative diuretic factors on in vivo urine production in the mosquito,Aedes aegypti

Changes in total urine production were measured in the mosquito, Aedes aegypti, following injection of 5-hydroxytryptamine (5-HT), Culex salinarius diuresin, culekinin depolarizing peptides (CDP-I, II and III) or the A. aegypti leucokinin peptides (ALP-I, II and III). All stimulated total urine production in a dose dependent manner. 5-HT was the least potent in urine production experiments with ED(50) values nearly 100-fold higher than other diuretic agonists. Doses greater than 2x10(-4) &mgr;moles inhibited urine production, suggesting either the occurrence of receptor down regulation, more than one type of 5-HT receptor, or increases in hindgut resorption of urine. The ALPs had relatively low ED(50) values compared to the CDPs suggesting that the endogenous peptides may have higher receptor binding affinities. Injection of mosquitoes with polyclonal antisera raised against either ALP-I or C. salinarius diuresin significantly reduced the response to injections of the respective peptides. The evidence presented above suggests that mosquito leucokinins and the C. salinarius diuresin function in the neuroendocrine regulation of urine production in the mosquito.     

Effect of plasma components on the feeding response of the mosquito Aedes aegypti L. to adenine nucleotides

ABSTRACT. The gorging response of Aedes aegypti to the ATP dissolved in platelet-poor plasma is greater than that of ATP dissolved in 0.15 m NaCl. The plasma components NaHCO₃ and albumin account for the full effect of the potentiation. Phosphate or tris buffers do not duplicate the bicarbonate effect. In 0.15 m NaCl with bicarbonate but lacking albumin the concentrations inducing 50% feeding are 58 μM ATP, 140 μM ADP, 460 μM AMP and 1500 μM cAMP. Non-adenine nucleotides such as ITP and GTP and phytic acid, and 2,3-diphosphoglyceric acid had no activity.     

ATP analogues and other phosphate compounds as gorging stimulants for Rhodnius prolixus

Five analogues of ATP and six other non-nucleotide compounds with phosphate groups were tested as gorging stimulants for second-instar larvae of Rhodnius prolixus to determine the importance of the phosphate chain. Only molecules with terminal phosphate groups were potent. Insertion of an imido group (5′-Adenylylimidodiphosphate, AMP-PNP) or a methylene group (β, γ-Methylene adenosine 5′-triphosphate, AMP-PCP) between the β and γ phosphates of ATP reduced the potency compared to ATP by ratios of 1.8 and 25.5, respectively. Substituting ribose (Adenosine 5′-diphosphoribose, AMP-PR) for the γ phosphate group or an amidate or a sulphate group (Adenosine 5′-phosphoramidate, AMP-N; Adenosine 5′-phosphosulphate, AMP-S) for the β and γ phosphate groups of ATP resulted in a complete loss of stimulatory activity.Some non-nucleotide phosphate compounds were potent phagostimulants. Pyrophosphate with an ED₅₀ of 64 μM had a potency ratio compared with ATP of 1:17. Methylene diphosphonic acid (ED₅₀ 680 μM) and even single phosphate ions (ED₅₀ 2.5 mM) had substantial potency. Two isomers of phosphoglyceric acid differ greatly in their ability to stimulate gorging; 2-PGA was active (ED₅₀ 160 μM) whereas 3-PGA had almost no activity.A summary of known phagostimulants to R. prolixus supports the hypothesis that ATP-like gorging stimulants act by forming a temporary binding to 3 sites on a receptor protein in the membrane of the chemosensory cell. The amino group on C₆ of adenine, the OH group on C₂ of ribose and the terminal phosphate group(s) determine potency, presumably by determining binding affinity. However, only the phosphate group appears essential to the chemosensory process.     

Feeding behaviour in response to blood fractions and chemical phagostimulants in the black-fly, Simulium venustum

ABSTRACT. Wild-caught black-flies ( Simulium venustum Say complex) were presented with diets at 37°C in an artificial feeding apparatus. Washed human red cells resuspended 1:1 in Ringer solution were potent phagostimulants, causing 89% of flies to gorge. Whole plasma was more potent (32% gorging) than platelet-poor plasma (2%). The ED₅₀ for red cells was 3.5%. Although ADP, contained in high-concentration in platelets, was confirmed as a more potent phagostimulant than ATP (ED₅₀ of 5πM V. 12μM), red cells were clearly more phagostimulatory than platelets, and with a potency more than adequate to trigger gorging in vivo. A high response to the ATP analogues, β, γ-methylene ATP and adenine phosphosulphate, supports the view that the phosphate chain is relatively unimportant in determining nucleotide potency to simuliids. The compounds phytic acid and 2,3-disphosphoglycerate, potent stimulants to Rhodnius prolixus , produced only moderate and no response, respectively at 1 mM; 5-hydroxytryptamine, another major constituent of platelets, also produced only a moderate response. Only flies caught while showing a characteristic probing behaviour would subsequently probe and feed in vitro; this 'blood-feeding mode' rapidly disappeared in the absence of stimuli eliciting actual probing, but for flies in this state a small temperature rise was sufficient stimulus for probing.     

Sensitivity of the mosquito Aedes aegypti (Culicidae) labral apical chemoreceptors to phagostimulants

The feeding of Aedes aegypti (L.) on blood and nectar is induced by phagostimulants: adenine nucleotides and sugars respectively. This work examines the responses of the four chemoreceptor cells in the labral apical sensilla to these phagostimulants. The apical chemoreceptors can detect the presence of adenine nucleotides. This part of the response is in good agreement with the gorging behavior. The output of the chemoreceptors cannot distinguish among different adenine nucleotides or among their concentrations (0.01-1 mmol/l), whereas gorging behavior is affected by the identity of adenine nucleotides and by their concentrations. Hence the gorging behavior cannot be driven by the output of these chemoreceptors alone. To the presence of adenine nucleotides Cell 2 was the only cell that responded with high frequencies, while the response of Cell 4 was almost abolished. The response of Cell 2 to ATP depended on the mosquito's physiological state. This dependence accorded well with the gorging behavior; Cell 2 responded with a higher frequency to ATP in the gorging state, than when not in a gorging state. The responses to sucrose and fructose constituted the only case recorded in which all these chemoreceptors failed to respond. This depression of response implies that other chemoreceptors must be present as sugar detectors.     

Feeding responses of the horsefly, Tabanus nigrovittatus, to physical factors, ATP analogues and blood fractions

ABSTRACT. Wild-caught female horseflies, Tabanus nigrovittatus Macq. (Diptera: Tabanidae), were presented solutions of seven analogues of ATP in 0.15 m NaCl, or various blood fractions, either as free liquids at 22 or 38C or covered with a Parafilm M membrane at 38C. Warming the diet, so that it can stimulate the insects' heat receptors, or presenting it warmed and covered with a membrane, which the flies can pierce and thus deploy their mouthparts as they would when blood-feeding, enhances the response to gorging stimulants. ADP (ED₅₀ 45 μM) was the most potent chemical phagostimulant. There were no significant differences between the potencies of AMP, A(TETRA)P, AMP-PCP, AMP-PNP or AMP-S, which were 3.5-5 times less potent than ADP. Cyclic AMP had no phagostimulatory activity at concentrations of 400 or 1000 μM. The ED₅₀ for washed red blood cells (RBC) in saline was 4.5% (one tenth the concentration found in blood). RBC-free plasma caused only 10% gorging but plasma with 0.5% RBC caused 61% gorging, indicating synergism between RBC and plasma.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

Reduced peptide bond pseudopeptide analogues of secretin. A new class of secretin receptor antagonists.

The ability to assess the importance of secretin in various physiological processes is limited by the lack of specific potent antagonists. Recently, reduced peptide bond (psi) analogues of bombesin or substance P in which the -CONH- bond is replaced by -CH2NH- are reported to be receptor antagonists. To attempt to develop a new class of secretin receptor antagonists, we have adopted a similar strategy with secretin and sequentially altered the eight NH2-terminal peptide bonds, the biological active portion of secretin. In guinea pig pancreatic acini, secretin caused a 75-fold increase in cyclic AMP (cAMP). Secretin inhibited 125I-secretin binding with a half-maximal effect at 7 nM. Each of the psi analogues inhibited 125I-secretin binding. [psi 4,5]Secretin was the most potent, causing the half-maximal inhibition at 4 microM, and was 2-fold more potent than the [psi 1,2]secretin; 7-fold more than [psi 3,4]secretin, [psi 5,6]secretin, and [psi 8,9]secretin; 9-fold more than [psi 7,8]secretin; 13-fold more potent [psi 6,7]secretin, and 17-fold more than [psi 2,3]secretin. Secretin caused a half-maximal increase in cAMP at 1 nM. At concentrations up to 10 microM, [psi 2,3]secretin, [psi 4,5]secretin, and [psi 8,9]secretin did not alter cAMP whereas [psi 1,2]secretin and [psi 6,7]secretin caused a detectable increase in cAMP at 10 nM, [psi 7,8]secretin at 300 nM, [psi 5,6]secretin at 1 microM, and [psi 3,4]secretin at 10 microM. The [psi 4,5], [psi 2,3], and [psi 8,9] analogues of secretin each inhibited 1 nM secretin-stimulated cAMP as well as [psi 3,4]secretin, which functioned as a partial agonist. [psi 4,5]Secretin was the most potent, causing half-maximal inhibition at 3 microM whereas [psi 8,9]secretin was 6-fold less potent, and [psi 2,3]secretin and [psi 3,4]secretin were 17-fold less potent. [psi 4,5]Secretin inhibited secretin-stimulated cAMP and binding of 125I-secretin in a competitive manner. [psi 4,5]Secretin did not interact with cholecystokinin, bombesin, calcitonin gene-related peptide, or cholinergic receptors but did interact with receptors for vasoactive intestinal peptide, causing half-maximal inhibition at 72 microM and thus had a 18-fold higher affinity for secretin than vasoactive intestinal peptide receptors. These results indicate that reduced peptide bond analogues of the NH2 terminus of secretin represent a new class of secretin receptor antagonists. It is likely that in the future even more potent members of this class can be developed which may be useful to investigate the role of secretin in various physiological processes.     

The glycoprotein toxin of Bacillus thuringiensis subsp. israelensis indicates a lectinlike receptor in the larval mosquito gut

The mosquito-active protein crystals produced by Bacillus thuringiensis subsp. israelensis contain covalently attached aminosugars which are critical for their larvicidal activity. The 50% lethal concentrations toward Aedes aegypti larvae were increased up to 10-fold by mild periodate treatment, up to 40-fold by forming the protein crystals in the presence of tunicamycin, and up to 7-fold by the presence during the mosquito bioassays of N-acetylglucosamine or its trimer, triacetylchitotriose. Periodate-treated crystals and crystals formed in the presence of tunicamycin had greatly reduced binding capacities for wheat germ agglutinin, an N-acetylglucosamine-specific lectin. These results suggest that the B. thuringiensis subsp. israelensis glycoprotein toxin binds to a lectinlike receptor in the larval mosquito gut. Furthermore, the distinct lectin-binding patterns exhibited by diptera-active versus lepidoptera-active B. thuringiensis crystals suggest that host specificity for the microbial insecticides is determined, in part, by the carbohydrate portion of their glycoprotein crystals.     

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.     

The glycoprotein toxin of Bacillus thuringiensis subsp. israelensis indicates a lectinlike receptor in the larval mosquito gut.

The mosquito-active protein crystals produced by Bacillus thuringiensis subsp. israelensis contain covalently attached aminosugars which are critical for their larvicidal activity. The 50% lethal concentrations toward Aedes aegypti larvae were increased up to 10-fold by mild periodate treatment, up to 40-fold by forming the protein crystals in the presence of tunicamycin, and up to 7-fold by the presence during the mosquito bioassays of N-acetylglucosamine or its trimer, triacetylchitotriose. Periodate-treated crystals and crystals formed in the presence of tunicamycin had greatly reduced binding capacities for wheat germ agglutinin, an N-acetylglucosamine-specific lectin. These results suggest that the B. thuringiensis subsp. israelensis glycoprotein toxin binds to a lectinlike receptor in the larval mosquito gut. Furthermore, the distinct lectin-binding patterns exhibited by diptera-active versus lepidoptera-active B. thuringiensis crystals suggest that host specificity for the microbial insecticides is determined, in part, by the carbohydrate portion of their glycoprotein crystals.     

Role of the nicotinic acid group in NAADP receptor selectivity

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to be an intracellular Ca2+-releasing messenger in a wide variety of systems to date. Its actions are both potent and highly specific despite differing structurally from the endogenous cellular co-factor and its precursor, NADP, only in the substitution of a hydroxyl for the amine group at the 3' position of the pyridine ring. This substitution allows NAADP to bind to a membrane-localized binding site in sea urchin egg homogenates with an IC50 at least 1000-fold greater than that of NADP as measured by competition radioligand binding assays. This suggests that the NAADP receptor protein must include certain features in the NAADP binding site that regulate this specificity. In order to investigate this interaction, we synthesised a series of NAADP analogues differing from NAADP at the 3' position of the pyridine ring that included both simple carboxylic acid analogues as well as a series of chemical isosters. We then investigated both their affinity for the NAADP binding site in sea urchin egg homogenates and their ability to activate the NAADP sensitive Ca2+ channel. We hereby show that a negative charge at the 3' position is an important determinant of affinity but the protein displays a large tolerance for the size of the group. Furthermore, the protein does not easily accommodate multiple charged groups or large uncharged groups.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Structure-activity relationship of synthetic truncated analogues of vasoactive intestinal peptide (VIP): an enhancement in the activity by a substitution with arginine

In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.     

Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, we derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of [3H]-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing m1-m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for m1 receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at m1 receptors, and a 4-fold greater potency at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.     

ATP-induced activation of purified rat hepatic glucocorticoid receptors

We have utilized unactivated rat hepatic glucocorticoid receptor complexes purified to near homogeneity by a three-step scheme which includes affinity chromatography, gel filtration and anion exchange chromatography, to demonstrate for the first time that ATP can interact directly with the receptor protein in stimulating activation. This stimulation is reflected by an increase in DNA-cellulose binding as well as by a shift in the elution profile of the purified receptor complexes from DEAE-cellulose. A concentration of 10 mM Na2MoO4 is able to block both of these effects. ATP stimulates activation in a dose-dependent manner (maximally at 10 mM), and elicits maximal activation within 30 min at 15 degrees C. There appears to be no nucleotide specificity since GTP, CTP and UTP, as well as ADP and GDP also stimulate activation. All of these observations closely parallel data obtained from similar activation experiments performed with crude rat hepatic receptors. ATP does not appear to stimulate activation of receptors (crude or purified) by initiating a phosphorylation reaction since hydrolysis-resistant analogues of ATP are also effective. Pyrophosphate (PPi) is as effective as ATP in promoting receptor activation, since it elicits similar increases in DNA-cellulose binding, shifts in elution patterns from DEAE-cellulose, and dose-response relationships. None of the compounds tested stimulate activation indirectly by pH or ionic strength effects. Despite the fact that high ATP concentrations (3-4-fold higher than those present in vivo) are necessary to stimulate maximal activation, a physiological role of ATP in directly regulating in vivo activation of glucocorticoid receptors cannot be ruled out.     

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 (EC₅₀) was 17 nM. The ¹²⁵I-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. ¹²⁵I-LPA bound to the 54 kDa protein(s) with a K_d value of 13±3 nM in agreement with the EC₅₀ 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.     

Potencies of combined doses of nucleotides as gorging stimulants for Rhodnius prolixus

The four nucleotides ATP, UTP, deoxyATP, and A(TETRA)P (adenosine tetraphosphate) were tested singly and in combinations for their potency in eliciting the gorging response of Rhodnius prolixus. All mixtures of ATP and dATP, and ATP and UTP, were more potent by a factor of 1.25 to 1.9 than predicted from their potencies when tested singly. No significant synergism or inhibition was seen with combinations of UTP and dATP, or ATP and A(TETRA)P. Lack of competitive inhibition suggests that physical fit between stimulating molecule and chemoreceptive protein rather than enzymatic modification of the stimulant is the mechanism of chemoreception of these compounds. The slight synergism seen is explained by competitive inhibition of a salivary ATPase.