Presence Of Cyclic AMP Research Articles

Effect of inhibition of axonemal dynein ATPases on the regulation of flagellar and ciliary waveforms in Leishmania parasites

Trypanosomes of the genus Leishmania swim by undulating motions of a single flagellum driven by axonemal dynein ATPases, essential for parasite survival and infectivity. The flagellum possesses two waveforms; flagellar (tip-to-base) responsible for forward movements and ciliary (base-to-tip) possibly responsible for reorientation in response to changes in surroundings. However, the role of dyneins in regulating the two waveforms remains unknown. Moreover, the unpredictable nature of the parasite ciliary waveform makes it difficult to study. We have previously reported a detergent-extracted, ATP-reactivated model ideal for investigating flagellar motility regulation in Leishmania that allows one to generate reactivated Leishmania flagella with constitutively beating ciliary waves in presence of cyclic-AMP. Here, using three dynein inhibitors [erythro-9-(2-hydroxy-3-nonyl) adenine, ciliobrevin A and vanadate] we investigated the role of dyneins in regulating the two waveforms of Leishmania. Using high speed videomicroscopy we observed differential inhibition of beat frequencies and waveforms of flagellar and ciliary beats in live (in vivo) and ATP-reactivated (in vitro) parasites. Beat frequency of flagellar waveform was more strongly reduced than ciliary waveform. Surprisingly, inhibition of the ciliary waveform led to an altered phenotype with the distal half of the flagellum paralysed. ATPase assays confirmed that dynein activity of flagellar cells was more strongly inhibited compared to ciliary cells irrespective of the mechanism of inhibition. Possibly the two different waveforms are an outcome of changes in the mechanical properties of axonemal dyneins present at the tip of the flagellum that contains a sliding resistive structure. Our study suggests that dyneins responsible for the two waveforms in Leishmania bear different structural and functional conformations. Moreover, during ciliary beating, there is heterogeneity along the flagellum.

High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer

Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a structural model for the cyclic nucleotide-modulated potassium channel homolog from Mesorhizobium loti, MloK1, determined from 2D crystals in the presence of lipids. Even though crystals diffract electrons toonly ∼10Å, using cryoelectron microscopy (cryo-EM) and recently developed computational methods, we have determined a 3D map of full-length MloK1 in the presence of cyclic AMP (cAMP) at ∼4.5Å isotropic 3D resolution. The structure provides a clear picture of the arrangement of the cyclic nucleotide-binding domains with respect to both the pore and the putative voltage sensor domains when cAMP is bound, and reveals a potential gating mechanism in the context of the lipid-embedded channel.

Involvement of the Global Crp Regulator in Cyclic AMP-Dependent Utilization of Aromatic Amino Acids by Pseudomonas putida

The phhAB operon encodes a phenylalanine hydroxylase involved in the conversion of L-phenylalanine into L-tyrosine in Pseudomonas putida. The phhAB promoter is transcribed by RNA polymerase sigma-70 and is unusual in that the specific regulator PhhR acts as an enhancer protein that binds to two distant upstream sites (-75 to -92 and -132 to -149). There is an integration host factor (IHF) binding site that overlaps the proximal PhhR box, and, consequently, IHF acts as an inhibitor of transcription. Use of L-phenylalanine is compromised in a crp-deficient background due to reduced expression from the phhAB promoter. Electrophoretic mobility shift assays and DNase I footprinting assays reveal that Crp binds at a site centered at -109 only in the presence of cyclic AMP (cAMP). We show, using circular permutation analysis, that the simultaneous binding of Crp/cAMP and PhhR bends DNA to bring positive regulators and RNA polymerase into close proximity. This nucleoprotein complex promotes transcription from phhA only in response to L-phenylalanine.

On the biochemical classification of yeast trehalases: Candida albicans contains two enzymes with mixed features of neutral and acid trehalase activities

Two enzymes endowed with trehalase activity are present in Candida albicans. The cytosolic trehalase (Ntc1p), displayed high activity in exponential phase regardless of the carbon source (glucose, trehalose or glycerol). Ntc1p activity was similar in neutral (pH 7.1) or acid (pH 4.5) conditions, strongly inhibited by ATP, weakly stimulated by divalent cations (Ca2+or Mn2+) and unaffected in the presence of cyclic AMP. The Ntc1p activity decreased in stationary phase, except in glycerol-grown cultures, but the catalytic properties did not change. In turn, the cell wall-linked trehalase (Atc1p) showed elevated activity in resting cells or in cultures growing on trehalose or glycerol. Although Atc1p is subjected to glucose repression, exhaustion of glucose in itself did not increased the activity. Significant Atc1p values could also be measured at neutral or acid pH, but Atc1p was insensitive to ATP, cyclic AMP and divalent cations. These results are in direct contrast with the current classification of yeast trehalases based on their optimum pH. They are also relevant in the light of the proposed use of trehalase inhibitors for the treatment of candidiasis.

Staphylococcus aureus ArcR Controls Expression of the Arginine Deiminase Operon

We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

Adenovirus-delivered DKK3/WNT4 and Steroidogenesis in Primary Cultures of Adrenocortical Cells

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17 alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11 beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.

Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus

The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

1445: Immunohistochemical Presence of Cyclic AMP and Cyclic GMP-Dependent Protein Kinases A and G in Human Corpus Cavernosum Andcavernous ARTERIES

1445: Immunohistochemical Presence of Cyclic AMP and Cyclic GMP-Dependent Protein Kinases A and G in Human Corpus Cavernosum Andcavernous ARTERIES

234 Immunohistochemical presence of cyclic AMP and cyclic GMP-dependent protein kinase A and G in human cavernous arteries and corpus cavernosum

234 Immunohistochemical presence of cyclic AMP and cyclic GMP-dependent protein kinase A and G in human cavernous arteries and corpus cavernosum

CREB in the pond snail Lymnaea stagnalis: cloning, gene expression, and function in identifiable neurons of the central nervous system.

The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L. stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity.

Immunohistochemical presence of cyclic AMP- and cyclic GMP- phosphodiesterase isoenzymes in human clitoris

Immunohistochemical presence of cyclic AMP- and cyclic GMP- phosphodiesterase isoenzymes in human clitoris

Identification and characterization ofapf1−in a non-pathogenic mutant of the rice blast fungusMagnaporthe griseawhich is unable to differentiate appressoria

In a cross between two isolates of the rice blast fungusMagnaporthe grisea, a spontaneous mutant was identified which was non-pathogenic on rice and did not differentiate appressoria on hydrophobic surfaces. The mutant was also non-pathogenic on rice when inoculated on wounded leaves, suggesting that its ability to grow in plant tissue was also impaired. Mutant spores were three-celled, with apical cells longer than those of parental isolates. Using one full sib-cross and one back-cross, we showed that all of the mutant phenotypes (non-pathogenicity on rice, appressorium deficiency and elongated spores) co-segregated as a single gene which was namedapf1. Comparison ofapf1−mutants with previously characterized appressorium-deficient mutants ofM. griseaprovided evidence thatapf1is a new gene involved in appressorium differentiation which has pleiotropic effects on plant colonisation and spore morphology. Significantly,apf1−mutants could not form appressoria in the presence of cyclic AMP, indicating thatAPF1acts downstream, or independently, of the cAMP signalling pathway required for appressorium differentiation and function inM. grisea.

Cyclic AMP-responsive Expression of the Surfactant Protein-A Gene Is Mediated by Increased DNA Binding and Transcriptional Activity of Thyroid Transcription Factor-1

Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form of PKA regulatory subunit RIalpha. We suggest that a PKA-induced increase of TTF-1 phosphorylation and TBE binding activity mediates cyclic AMP-induced expression of the SP-A gene in lung type II cells.

Activation of dopamine D4 receptor inhibits an L-type calcium current in cerebellar granule cells.

The functions of the D4 receptor, a newly cloned D2-like receptor, as well as the identity of cells expressing it, are still poorly defined. Using quantitative polymerase chain reaction we detected the messenger RNA of the D4, but not other D2-like receptor, in cultured granule cells from neonatal rat cerebellum. In these neurons, dopamine reduced high-voltage-activated calcium current, with a pharmacology corresponding to that of the D4 receptor. The response declined from one to three days, when calcium currents were mostly sensitive to nifedipine, to 15 days, when nifedipine-insensitive calcium currents were also present and D4 receptor messenger RNA had declined. The dopamine response was abolished after pretreatment of the cells by pertussis toxin, was potentiated and made irreversible by infusion of guanosine 5'-O-(3-thiotriphosphate) but persisted in the presence of cyclic AMP and isobutylmethylxanthine. These results indicate the presence in the neonatal cerebellum of a functional D4 receptor inhibiting an L-type calcium current, an action involving a Gi/Go protein but independent from adenylate cyclase inhibition.

Cyclic AMP- plus ATP-dependent modulation of the NADH oxidase activity of porcine liver plasma membranes

Plasma membranes of porcine liver, highly purified by aqueous two-phase partition, oxidized NADH in the absence of added external acceptors. The oxidation was resistant to cyanide and responded to nanomolar concentrations of ATP alone or ATP in the presence of cyclic AMP. Both the K m for NADH and the long-term activity of the oxidase were affected. Upon incubation at 37°C with cyclic AMP (0.1–10 nM) and ATP (1–100 nM), the NADH oxidase activity was inhibited. The inhibition was complex and due to an approx. 5-fold increase in the K m for NADH compared to the NADH oxidase of membranes incubated in the absence of cyclic AMP + ATP. The response to cAMP + ATP was rapid and occurred within seconds of ATP addition. The response was inhibited by the selective inhibitor of cyclic AMP-dependent protein kinase, H-89. Neither cyclic AMP alone nor ATP alone at nanomolar concentrations elicited a rapid response. However, 10 nM ATP alone did result in similar alteration of K m and V max as did ATP + 0.1 nM cyclic AMP. The response to ATP alone or in preparations depleted of cyclic AMP required higher ATP concentrations than with cAMP present or occurred more slowly with a lag of 1–2 min. The NADH oxidase activity of porcine plasma membranes after cyclic AMP + ATP treatment retained high activity with storage at 4°C, whereas that of unincubated or sham-incubated plasma membranes was reduced with time of storage at 4°C. In some but not all instances, NADH oxidase activity inactivated by incubation with NADH at 37°C or after storage at 4°C could be reactivated by incubation with cyclic AMP plus ATP. As with the alteration in K m , cyclic AMP alone was without effect and ATP alone was much less effective than the combination. The results demonstrate ATP-dependent modulation of the NADH oxidase activity of isolated plasma membranes at physiological concentrations of ATP. This modulation may have functional significance in mediating the hormone and growth factor responsiveness of the plasma membrane NADH oxidase activity.

Occurrence of GTP-binding proteins in the ascomycete Saccobolus platensis

We have previously shown that in the ascomycete Saccobolus platensis the presence of cyclic AMP can overcome the requirement for light to produce apothecia. With the aim to gain insight into the phototransduction mechanism the existence of G protein-like molecules was investigated. By using the GTP analog [ 35 S]GTP-γS we detected the existence of GTP-binding proteins; the dissociation constant for the binding of the nucleotide was calculated ( K d = 52 n M ); the binding was effectively competed by GTP and GDPPS and to a lesser extent by ATP. We also demonstrated by SDS-PAGE the existence of proteins of 48, 41, 26, and 15 kDa that are substrates of the cholera toxin-dependent ADP-ribosylation reaction. Proteins resolved by SDS-PAGE were identified by Western blotting with antisera specific for vertebrate G protein subunits; the antibody anti-α common revealed two main bands at 48 and 41 kDa; the AS/7 antibody (anti-α t , −α 1 , and −a i2 ) led to the identification of one band at 41 kDa; the antibody anti-β subunit revealed a polipeptide band at around 37 kDa. The antibody anti-α common also abolished the binding of [ 35 S]GTP-γS to crude membranes. The results presented in this paper provide evidence for the existence in S. platensis of proteins that proved to have a behavior similar to that of mammalian G proteins.

Schwann Cells Exhibit P2Y Purinergic Receptors that Regulate Intracellular Calcium and Are Up‐Regulated by Cyclic AMP Analogues

Schwann cells establish close contact with axons during development, and this is maintained throughout life. Signaling by neurotransmitters may play an important role in Schwann cell-axon interaction. Schwann cells were examined for the presence of neuroligland receptors that are linked to increases in levels of cytoplasmic calcium. Schwann cell cultures were prepared from neonatal rat sciatic nerve and, after 0.25, 1, 4, 7, and 14 days in vitro (DIV), loaded with the calcium indicator dye fura 2-AM. The influence of neuroligands on the cytosolic free calcium concentration ([Ca2+]i) was then examined at each time point using a video-based imaging system. Approximately 80-95% of all freshly isolated Schwann cells responded to 10 microM ATP with a three-fold rise in [Ca2+]i. Bradykinin, glutamate, and histamine had no or only partial and inconsistent responses. The ATP-induced calcium response disappeared within 4 DIV. Culturing cells in the presence of cyclic AMP (cAMP) analogues (which induce proliferation and differentiation in vitro) restored the ability of Schwann cells to respond to ATP with increased [Ca2+]i. In the presence of cAMP analogues the extent of recovery of ATP responsiveness was dependent on serum concentration. Fifty to ninety percent of cells regained calcium responsiveness to ATP when grown in medium containing cAMP analogues and 1% serum. These cells also exhibited immunoreactivity to P0 antibody, characteristic of the myelinating lineage. In contrast, only 15-30% of the Schwann cells regained calcium responsiveness when grown in medium containing cAMP analogues and 10% serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of the regulation of glycerol and maltose metabolism by IIAGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system in Salmonella typhimurium.

The amount of IIAGlc, one of the proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS), was modulated over a broad range with the help of inducible expression plasmids in Salmonella typhimurium. The in vivo effects of different levels of IIAGlc on glycerol and maltose metabolism were studied. The inhibition of glycerol uptake, by the addition of a PTS sugar, was sigmoidally related to the amount of IIAGlc. For complete inhibition of glycerol uptake, a minimal ratio of about 3.6 mol of IIAGlc to 1 mol of glycerol kinase (tetramer) was required. Varying the level of IIAGlc (from 0 to 1,000% of the wild-type level) did not affect the growth rate on glycerol, the rate of glycerol uptake, or the synthesis of glycerol kinase. In contrast, the growth rate on maltose, the rate of maltose uptake, and the synthesis of the maltose-binding protein increased two- to fivefold with increasing levels of IIAGlc. In the presence of cyclic AMP, the maximal levels were obtained at all IIAGlc concentrations. The synthesis of the MalK protein, the target of IIAGlc, was not affected by varying the levels of IIAGlc. The inhibition of maltose uptake was sigmoidally related to the amount of IIAGlc. For complete inhibition of maltose uptake by a PTS sugar, a ratio of about 18 mol of IIAGlc to 1 mol of MalK protein (taken as a dimer) was required.

Involvement of Type 2C Phosphatase in the Dephosphorylation of 26 kDa Phosphoprotein in Rat Parotid Acinar Cells

Posphorylation of three particulate proteins with molecular masses of 34, 26, and 22 kDa was stimulated in the presence of cyclic AMP / 3-isobutyl-1-methylxanthine in saponin-permeabilized rat parotid acinar cells. When the particulate fraction isolated from the cells labeled with [γ-32P]ATP was incubated at 30°C, dephosphorylation of the 26 kDa phosphoprotein occurred in the presence of Mg2+ or Mn2+. Okadaic acid had no effect on the Mg2+-dependent dephosphorylation of the 26 kDa phosphoprotein. Addition of the recombinant type 2C phosphatase, Mg2+-dependent and okadaic acid-insensitive phosphatase, caused a remarkable dephosphorylation of the 26 kDa phosphoprotein. These observations strongly suggest type 2C phosphatase is involved in the dephosphorylation of the 26 kDa phosphoprotein.

CONCEALED TRANSEPITHELIAL POTENTIALS AND CURRENT RECTIFICATION IN TSETSE FLY MALPIGHIAN TUBULES

1. Electrophysiological techniques have been applied to tsetse fly Malpighian tubules for the first time. 2. In either Cl- or SO42- Ringer, both non-perfused and perfused tubules displayed transtubular potentials (Vt) at or close to 0 mV. Exposure to cyclic AMP elicited a marked secretory response and, in SO42- Ringer, a sharp (lumen-positive) increase in Vt. In Cl- Ringer, despite more than double the secretory response, there was little or no change in Vt. 3. Replacing Cl- with SO42- Ringer, in the presence of cyclic AMP, promptly increased Vt. In perfused tubules, this occurred irrespective of the Cl- or SO42- composition of the perfusate. 4. In Cl- Ringer, the transepithelial resistance (Rtrans) was less than half that previously reported in Malpighian tubules of other species. Cyclic AMP reduced Rtrans still further, whether tubules were bathed in Cl- or SO42- Ringer. 5. Current­voltage (I/V) plots often displayed current rectification, both before and more frequently after exposure to cyclic AMP, thus permitting estimation of both the electromotive force of the Na+ transport mechanism (ENa) and of the shunt resistance (Rshunt). Both ENa and Rshunt were markedly lower in tubules bathed in Cl- than in SO42- Ringer. Cyclic AMP was without effect on ENa and Rshunt, in either Cl- or SO42- Ringer. 6. In terms of the equivalent electrical circuit, the secretory response to cyclic AMP was due solely to a fall in resistance of the active transport pathway (Rseries). The absence of an appreciable Vt, in Cl- Ringer, is consistent with an apical Cl- shunt.