Diuresis in a desert beetle? hormonal control of the malpighian tubules ofOnymacris plana (Coleoptera: Tenebrionidae)

Summary Malpighian tubules of a desert tenebrionid beetle,Onymacris plana, have been studied as isolated preparations. Under control conditions tubules of female beetles secreted fluid at an average rate of 3.3 nl/min, but this rate was increased 20–25 times by a diuretic hormone (DH).Homogenates of the brain, corpora cardiaca (CC) and prothoracic ganglion induced striking increases in tubule secretion rates, which sometimes exceeded 100 nl/min. The increased rates were sustained for 3 h without renewal of the medium. Diuretic activity was also present in the other thoracic ganglia. High K treatment caused release of DH from the CC only.Exogenous cyclic AMP (1 mM) stimulated the isolated tubules ofO. plana, but to a lesser extent than the DH. The cationic composition of the secreted fluid resembled that of most other insect tubules, with high K and low Na concentrations. Stimulation with DH doubled the Na concentration.The DH was not inactivated by the tubules themselves, but was destroyed by contact with the haemolymph. An inactivation mechanism is vital in the apparently contradictory situation of a desert beetle possessing a diuretic hormone. The role of the cryptonephric system during diuresis is unknown.Abbreviations DH diuretic hormone - cAMP adenosine 3:5-cyclic monophosphoric acid - CC corpora cardiaca     

The cyclic nucleotide phosphodiesterases of spinach chloroplasts and microsomes

Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1_c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 10⁵ and 3.7 × 10⁵). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1_c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1_m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 10⁵. Electrophoresis separated 1_m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 10⁵ and 1.28 × 10⁵). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1_c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1_m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1_c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1_m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1_c but not 1_m. Electrophoresis after dissociation indicates that 1_c and 1_m are both enzyme complexes. After dissociation, the 1_c complex but not that of 1_m could be reassociated. The ribonuclease of the 1_m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1_c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1_m complex being concerned with RNA catabolism.     

Effects of biogenic amines and hormones on butterfly Malpighian tubules: Dopamine stimulates fluid secretion

Isolated Malpighian tubules of Papilio demodocus, the citrus swallowtail butterfly, were stimulated by biogenic amines as well as by cyclic AMP and the naturally occuring diuretic hormone. The greatest secretory response was obtained with 5-hydroxytryptamine, and smaller responses with dopamine and noradrenaline, but none of these amines could induce the maximal secretion rates obtained with cyclic AMP and diuretic hormone. Various other biogenic amines, hormones and pharmacological agents, including adrenaline, had no effect on Papilio tubules. The blocking agents cyproheptadine, phentolamine and propranolol did not alter the tubule response to biogenic amines.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Isolation and identification of a diuretic hormone from Zootermopsis nevadensis

A diuretic hormone (DH) was isolated from extracts of heads of Zootermopsis nevadensis, a dampwood termite. The peptide has 46 residues, M(r) = 5,328.2 Da, with the sequence TGAVPSLSIVNPLDVLRQRLLLEIARRRMRQSQDQIQANREMLQTI-NH(2,) showing it to be a CRF-related DH. This peptide increases cyclic AMP production in Malpighian tubules of Manduca sexta. We detected another factor in the head extracts which behaved as a more basic peptide on ion exchange chromatography. The latter factor also stimulated cyclic AMP production in the bioassay, but two large scale attempts to isolate this peptide were unsuccessful. We believe the second peptide is acid labile.     

Contact Inhibition of Transformed Cells Incompletely Restored by Dibutyryl Cyclic AMP

INCREASED levels of cyclic AMP have been found in normal cells as compared with malignant cells^1,2. Several types of malignant cells become morphologically similar to untransformed cells when incubated in media containing cyclic AMP or its derivative dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP)^3,4. Sheppard reported that 3T3 mouse fibroblasts, transformed by polyoma virus, grew to low saturation density and became less agglutinable with wheat germ agglutinin if theophylline and dibutyryl cyclic AMP were added to the medium⁵.     

Partial purification and properties of a multifunctional 3′,5′-cyclic nucleotide phosphodiesterase from Lactuca cotyledons

The extraction, partial purification and properties of a 3′,5′-cyclic nucleotide phosphodiesterase from lettuce cotyledons is described. Purification involved fractional precipation with (NH₄)₂SO₄, chromatography on Sephadex G-200, affinity chromatography on Affi-Gel Blue and non-denaturing polyacrylamide gel electrophoresis. The behaviour of the final enzyme preparation on SDS-polyacrylamide gel electrophoresis was examined and inidcated an M, of ca 62 000. The enzyme from 3′,5′-cyclic nucleotide phosphodiesterases previously isolated from plant tissues in that it exhibits activity towards pyrimidine as well as purine cyclic nucleotides. Furthermore, it hydrolyses cyclic CMP at a comparable rate to that with which it hydrolyses cyclic AMP and cyclic GMP. Both 3′- and 5′-AMP were released, with the 5′-nucleotide being the major product. Whereas the K_m with all three substrates remained constant during the purification procedure, V_max with cyclic AMP was lower than that for cyclic CMP but increased as purification proceeded. The effects were examined of a range of di- and trivalent metal ions on the enzyme activity. Fe³⁺ significantly stimulated the activity, more so when cyclic GMP was the substrate. Cu²⁺ inhibited the activity.     

Presence of Locusta diuretic hormone in endocrine cells of the ampullae of locust Malpighian tubules

This is an investigation of an endocrine cell type in the midgut of the migratory locust Locusta migratoria. This cell type is found in the posterior region of the midgut and is especially common in the ampullae through which Malpighian tubules drain into the gut at the midgut-hindgut junction. Strong Locusta diuretic hormone-like immunoreactivity in these cells was colocalized with FMRFamide- and substance P-like immunoreactivities. At the ultrastructural level, immunoreactivity for Locusta diuretic hormone was found in spherical granules (mean diameter of 450 nm), the contents of which showed variable electron density. Fractionation of a methanolic extract of the ampullae by reversed-phase high performance liquid chromatography revealed the presence of two peaks of Locusta diuretic hormone-like immunoreactive material, both of which stimulate cyclic AMP production by isolated Malpighian tubules. The more hydrophobic material is most likely Locusta diuretic hormone, which has the same retention time when chromatographed under identical conditions. Received: 15 September 1995 / Accepted: 16 February 1996     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

Immunocytochemical localisation and biological activity of diuretic peptides in the housefly, Musca domestica

The distribution of a CRF-related diuretic peptide (Musca-DP) and the diuretic/myotropic insect myokinins in the central nervous system of larval and adult houseflies was investigated using antisera raised against Locusta-DH and leucokinin-I, respectively. Two separate, small populations of immunoreactive neurons are present in the brain and fused thoracic-abdominal ganglion mass. There is no evidence for these immunoreactivities being colocalised either within single neurons or at neurohaemal release sites. Crude extracts of tissues containing immunoreactive material increase fluid secretion by isolated Malpighian tubules from adult flies. Diuretic activity is highest in tissues containing myokinin-immunoreactive material. Consistent with this observation, myokinin analogues produce a four- to five-fold increase in fluid secretion, which is more than twice the response to Musca-DP. These effects are mimicked by treatments that increase intracellular calcium and cyclic AMP, respectively. When tested at threshold concentrations, the two classes of diuretic peptide act synergistically to accelerate tubule secretion, and their separate localisation may be important for the precise control of diuresis.     

Comparison between rat and rabbit anticyclic AMP antibodies--specificity toward acyl derivatives of cyclic AMP

This paper deals with the specificity of the anti 3′,5′-cyclic AMP antibodies which can be obtained with 2′-O-succinyl cyclic AMP-albumin as an immunogen. The binding of the hapten and its analogs was measured by equilibrium dialysis. Rat and rabbit antibodies were compared. In both cases the best ligands for the anti-hapten antibodies are 2′-O-acylated derivatives of cyclic AMP: the dissociation constants are below 10^?10m. Cyclic AMP itself and its 6 N, 2′-O-diacylated derivatives are recognized less efficiently; their dissociation constants lie around 10^?8m, similar to that of natural cyclic AMP binding proteins. Other nucleotides lacking either adenine or the 3′,5′-phosphate ring are not recognized. Three different populations of antibodies were detected by a more detailed analysis of the equilibrium curves.     

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase

The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN₆,O²′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Isolation and characterization of a diuretic peptide from Acheta domesticus. Evidence for a family of insect diuretic peptides.

A diuretic peptide (Acheta-DP) has been isolated from extracts of whole heads of the house cricket, Acheta domesticus. The native peptide increases both cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro to an extent comparable with those responses obtained with supra-maximal amounts of crude extracts of corpora cardiaca. The primary structure of Acheta-DP was established as a 46-residue amidated peptide: TGAQSLSIVAPLDVLRQRLMNELNRRRMRELQGSRIQQNRQLLTSI-NH2. Acheta-DP has 41% sequence identity with a diuretic peptide isolated from Manduca sexta, providing direct evidence for the presence of a family of diuretic peptides in insects.     

A cyclic AMP binding-protein from barley seedlings

A protein fraction extracted from barley seedlings was shown to bind 3′:5′-cyclic AMP. The binding effect is real and not due to interference with the standard binding-protein assay used. Evidence is presented that this is a specific binding-protein; even at high concentrations other protein fractions from the same source showed no affinity for cyclic AMP. None of a range of cyclic and non-cyclic nucleotides that were examined exhibited a degree of binding with the protein comparable to that with cyclic AMP. The cyclic AMP/binding protein complex has a K_d of 8 nM. This complex eluted at an identical position in the elution sequence from a Sephadex G-150 column as the uncomplexed binding-protein. The barley binding-protein is in a fraction which also exhibits the enzymic activities of glucose 6-phosphatase, ATPase, 5′-nucleotidase, and fructose 1,6-diphosphatase.     

Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

In vivo preparation of (32P) adenosine 3',5'-cyclic monophosphate

A simple method for the preparation of [³²P]adenosine 3′,5′-cyclic monophosphate (cyclic AMP) is described. A culture of Escherichia coli mutant deficient in cyclic AMP receptor protein is incubated with [³²P]orthophosphate of known specific activities (up to 4000 Ci/mole) for several cell doublings. 10¹² cells of this mutant excrete approximately 1.4 μmoles of cyclic AMP/hr. The extracellular cyclic AMP can be purified by adsorption to charcoal, chromatography on an alumina plate, and paper chromatography.     

Cyclic AMP-induced tyrosinase synthesis in Neurospora crassa

Cyclic AMP induces the synthesis of tyrosinase in $\text{Neurospora crassa}$. Adenine, adenosine, 3′-AMP, 5′-AMP, and 2′,3′-cyclic AMP have no inductive effect while 8-bromocyclic AMP and dibutyryl cyclic AMP are good inducers. Caffeine and theophylline, inhibitors of cyclic AMP phosphodiesterase, also induce tyrosinase. A possible relationship between cyclic AMP induction and previously reported induction by cycloheximide is suggested.     

Isolation and characterization of CRF-related diuretic hormones from the whitelined sphinx moth Hyles lineata

We have isolated and characterized two diuretic hormones (DH), Hylli-DH41 and Hylli-DH30, from extracts of whole heads of the lepidopteran Hyles lineata. We monitored the isolation by measuring the ability of fractions to affect levels of cyclic AMP production by Malpighian tubules of Manduca sexta maintained in vitro. These DH are related to a family of vertebrate neuropeptides which includes sauvagine, corticotropin-releasing factor (CRF), and urotensin I. Both Hylli-DH41 (RMPSLSIDLPMSVLRQKLSLE KERKVQALRAAANRNFLNDI-NH2) and Hylli-DH30 (SFSVNPAVEILQHRYMEKVAQNNRNFLNRV-NH2) show extremely high similarity with two DH from the tobacco hornworm M. sexta. This is not surprising because both H. lineata and M. sexta are sphingid moths. The discovery of these DH provides a third example of two CRF-related DH occurring in one insect species.     

The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata

We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH(46) and Dippu-DH(31), on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH(46), or slight inhibitory activity, in the case of DH(31).