cGMP signalling in a transporting epithelium.

The biochemical aspects of cGMP signalling are well known, although in vivo roles of cGMP have only been recently discovered through work in genetic model organisms. The Drosophila melanogaster Malpighian (renal) tubule has been used to address the roles of cGMP in epithelial function. Here, we describe some of this work and outline recent progress in understanding the organotypic function of novel phosphodiesterases encoded by the D. melanogaster genome.

Insect renal tubules constitute a cell-autonomous immune system that protects the organism against bacterial infection.

Innate immunity is a widespread and important defence against microbial attack, which in insects is thought to originate mainly in the fat body. Here we demonstrate that the fluid-transporting Malpighian (renal) tubule of Drosophila melanogaster constitutes an autonomous immune-sensing tissue utilising the nitric oxide (NO) signalling pathway. Reverse transcriptase PCR (RT-PCR) shows that tubules express those genes encoding components of the Imd pathway. Furthermore, isolated tubules bind and respond to lipopolysaccharide (LPS), by upregulating anti-microbial peptide (diptericin) gene expression and increased bacterial killing. Excised, LPS-challenged tubules, as well as tubules from LPS-infected flies, display increased NO synthase (NOS) activity upon immune challenge. Targetted expression of a Drosophila NOS (dNOS) transgene to only principal cells of the tubule main segment using the GAL4/UAS system increases diptericin expression. In live flies, such targetted over-expression of dNOS to tubule principal cells confers increased survival of the whole animal upon E. coli challenge. Thus, we describe a novel role of Malpighian tubules in immune sensing and insect survival.

Model organisms: new insights into ion channel and transporter function. L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster.

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel (cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.

Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster.

The leucokinin (LK) family of neuropeptides has been found widely amongst invertebrates. A member of this family was purified from adults of the fruit fly Drosophila melanogaster. The peptide sequence for Drosophila leucokinin (DLK) was determined as Asn-Ser-Val-Val-Leu-Gly-Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly-amide, making it the longest member of the family characterized to date. Synthetic DLK peptide was shown to act to stimulate fluid secretion in D. melanogaster Malpighian (renal) tubules by approximately threefold, with an EC(50) of approximately 10(-)(10 )mol l(-)(1), and a secondary effect at approximately 10(-)(7 )mol l(-)(1). DLK also acted to elevate intracellular [Ca(2+)] in the Malpighian tubules by approximately threefold, with an EC(50) of 10(-)(10) to 10(-)(9 )mol l(-)(1). Responses were detected in stellate cells and occasionally in principal cells, although at no concentration tested did [Ca(2+)] in the principal cell increase significantly above background. In stellate cells, DLK produced a biphasic rise in intracellular [Ca(2+)] from resting levels of 80-100 nmol l(-)(1), with a transient peak being followed by a slower rise that peaked at 200-300 nmol l(-)(1) after 3 s, then decayed over approximately 10 s. The wide range of concentrations over which DLK acts suggests the involvement of more than one receptor. The genomic sequence encoding the DLK peptide has been identified, and the gene has been named pp. The gene resides at cytological location 70E3-70F4 of chromosome 3L. The localisation of this first Drosophila LK gene in a genetic model permits a genetic analysis of the locus.

Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells.

Anion conductance across the Drosophila melanogaster Malpighian (renal) tubule was investigated by a combination of physiological and transgenic techniques. Patch-clamp recordings identified clusters of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive "maxi-chloride" channels in a small domain of the apical membrane. Fluid secretion assays demonstrated sensitivity to the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, and niflumic acid. Electrophysiological analysis showed that the calcium-mediated increase in anion conductance was blocked by the same agents. Vibrating probe analysis revealed a small number of current density hot spots, coincident with "stellate" cells, that were abolished by low-chloride saline or the same chloride channel blockers. GAL-4-targeted expression of an aequorin transgene revealed that the neurohormone leucokinin elicits a rapid increase in intracellular calcium levels in stellate cells that precedes the fastest demonstrable physiological effect. Taken together, these data show that leucokinins act on stellate cells through intracellular calcium to increase transcellular chloride conductance through channels. As electrogenic cation conductance is confined to principal cells, the two pathways are spatially segregated in this tissue.

Genetic linkage study of a major susceptibility locus (D2S125) in a British population of non-insulin dependent diabetic sib-pairs using a simple non-isotopic screening method.

A polymorphic microsatellite marker (D2S125) was recently reported to show significant linkage to non-insulin dependent diabetes mellitus (NIDDM) in a population of Mexican-American affected sib-pairs. We have used a simple non-isotopic screening technique employing the polymerase chain reaction (PCR) with a biotinylated primer to study the genetic linkage and allele frequency distribution of the D2S125 marker in a population of 109 British NIDDMs (62 possible affected sib-pairs). The analysis provided no evidence for linkage of the D2S125 marker in the British subjects (MLS = 0.029, P > 0.05). The PCR screening method used proved to be a convenient and reliable alternative to the radiolabelling of PCR products.

Cell-type specific calcium signalling in a Drosophila epithelium.

Calcium is a ubiquitous second messenger that plays a critical role in both excitable and non-excitable cells. Calcium mobilisation in identified cell types within an intact renal epithelium, the Drosophila melanogaster Malpighian tubule, was studied by GAL4-directed expression of an aequorin transgene. CAP2b, a cardioactive neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. Furthermore, the data suggest a role for calcium signalling in the modulation of the nitric oxide signalling pathway in this epithelium. The GAL4-targeting system allows general application to studies of cell-signalling and pharmacology that does not rely on invasive or cytotoxic techniques.

Neuropeptide stimulation of the nitric oxide signaling pathway in Drosophila melanogaster Malpighian tubules.

Activation of the nitric oxide (NO) and guanosine 3', 5'-cyclic monophosphate (cGMP) signaling pathway stimulates fluid secretion by the Drosophila melanogaster Malpighian tubule. The neuropeptide cardioacceleratory peptide 2b (CAP2b) has been previously shown to stimulate fluid secretion in this epithelium by elevating intracellular cGMP levels. Therefore, it was of interest to investigate if CAP2b acts through NO in isolated tubules and thus presumably through stimulation of a tubule NO synthase (NOS). We show here by reverse-transcription polymerase chain reaction that Drosophila NOS (dNOS) is expressed in Malpighian tubules. Biochemical assays of NOS activity in whole tubules show that CAP2b significantly stimulates NOS activity. Additionally, fluid secretion and cyclic nucleotide assays show that CAP2b-induced elevation of intracellular cGMP levels and fluid secretion rates are dependent on the activation of a soluble guanylate cyclase. Treatment of tubules with a specific NOS inhibitor abolishes the CAP2b-induced rise in intracellular cGMP levels. These data indicate that CAP2b stimulates NOS and therefore, endogenous NO production, which, in turn, stimulates a soluble guanylate cyclase. This is the first demonstration of stimulation of an endogenous NOS by a defined peptide in Drosophila.

Molecular genetic analysis of V-ATPase function in Drosophila melanogaster.

V-ATPases are phylogenetically widespread, highly conserved, multisubunit proton pumps. Originally characterised in endomembranes, they have been found to energise transport across plasma membranes in a range of animal cells and particularly in certain epithelia. While yeast is the model of choice for the rapid generation and identification of V-ATPase mutants, it does not allow their analysis in a plasma membrane context. For such purposes, Drosophila melanogaster is a uniquely suitable model. Accordingly, we have cloned and characterised genes encoding several V-ATPase subunits in D. melanogaster and, using P-element technology, we have succeeded in generating multiple new alleles. Reporter gene constructs reveal ubiquitous expression, but at particularly high levels in those epithelial thought to be energised by V-ATPases, and several of the alleles have lethal recessive phenotypes characterised by epithelial dysfunction. These results, while providing the first gene knockouts of V-ATPases in animals, also illustrate the general utility of D. melanogaster as a model for the genetic analysis of ion transport and its control in epithelia.

Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype.

Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes. A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA (= l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions.

CAP2b, a cardioacceleratory peptide, is present in Drosophila and stimulates tubule fluid secretion via cGMP.

A cardioacceleratory peptide, CAP2b, identified originally in the lepidopteran Manduca sexta, stimulates fluid secretion by Malpighian tubules of the dipteran Drosophila melanogaster. High-performance liquid chromatography analyses of adult D. melanogaster reveal the presence of a CAP2b-like peptide, that coelutes with M. sexta CAP2b and synthetic CAP2b and that has CAP2b-like effects on the M. sexta heart. CAP2b accelerates fluid secretion in tubules stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) but has no effect on tubules stimulated by guanosine 3',5'-cyclic monophosphate (cGMP), implying that it acts through the latter pathway. By contrast, the action of leucokinin is additive to both cAMP and cGMP but not to thapsigargin, suggesting that leucokinin acts by the elevation of intracellular calcium. CAP2b stimulation elevates tubule cGMP levels but not those of cAMP. By contrast, leucokinin has no effect on levels of either cyclic nucleotide. Both CAP2b and cGMP increase transepithelial potential difference, suggesting that stimulation of vacuolar-adenosinetriphosphatase action underlies the corresponding increases in fluid secretion. Overall, the results show that a Drosophila CAP2b-related peptide acts to stimulate fluid secretion by Malpighian tubules through the cGMP-signaling pathway.

A novel role for the nitric oxide-cGMP signaling pathway: the control of epithelial function in Drosophila.

The nitric oxide (NO) signaling pathway plays major roles in the vertebrate vascular, nervous, and immune systems. Here we present evidence that all the elements in the NO pathway are present in, and act to control epithelial fluid secretion by, the Malpighian tubules of an insect, Drosophila melanogaster. This finding will allow both a physiological and a molecular genetic dissection of the NO pathway in the same tissue.

Endosalpingosis as a cause of chronic pelvic pain.

Endosalpingosis, a condition characterized by ectopic oviduct epithelium, is diagnosed histologically by the appearance of benign ciliated and nonciliated columnar cells in an abnormal location. Endosalpingosis is typically without symptoms. Our unusual case report shows symptomatic endosalpingosis first seen with chronic pelvic pain. Various aspects of this disease will also be discussed.

Deterioration in left ventricular performance during early coronary angioplasty after thrombolysis. Evidence for myocardial salvage.

To investigate the effects of controlled coronary artery reocclusion after successful thrombolysis, we studied 15 patients during early elective angioplasty of the patent infarct-related artery. Eight patients underwent left anterior descending artery dilation, and the other 7 had right coronary artery dilation. In 13 cases, ST-segment elevation developed during balloon occlusion. In all 15 cases, intravenous digital subtraction left ventriculography during balloon inflation showed that the ejection fraction decreased at least 5% (mean decrease, from 60% to 47%), despite preexisting Q waves overlying the infarct territory in 5 patients. Balloon inflation resulted in decreased apical segmental shortening in all 8 patients who underwent left anterior descending artery dilation; likewise, balloon inflation produced impairment of inferior-wall contraction in all 7 patients who had right coronary artery dilation. In this setting, a deterioration in left ventricular performance indicates that the restoration of coronary patency with thrombolysis has resulted in myocardial salvage. In patients with Q waves, such deterioration suggests that this electrocardiographic abnormality does not necessarily indicate a completed infarction.

The effects of p21N-ras expression in NIH-3T3 cells upon cyclic AMP metabolism.

The effects of overexpression of p21N-ras upon cyclic AMP metabolism have been examined in the inducible T15 cell line. In cells overexpressing the N-ras gene product, beta-adrenergic stimulation of cyclic AMP generation was reduced. The reduction was more pronounced the longer the ras gene was expressed and in chronically transformed cells a reduction in forskolin-stimulated cyclic AMP generation was also observed. The transformed cells exhibited a reduction in beta-adrenergic binding sites, but no change in the apparent EC50 for agonist induced cyclic AMP generation. Treatment of the cells with dibutyryl cyclic AMP induced a dose-dependent inhibition of proliferation, with the transformed cells being more sensitive than the control cells.

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Bombesin stimulation of T15 cells in which the inducible N-ras oncogene is overexpressed caused elevated production of inositol phosphates compared to uninduced cells [Wakelam, Davies, Houslay, McKay, Marshall & Hall (1986) Nature (London) 323, 173-176]. This elevated response is shown here to result from increased generation of inositol 1,4,5-trisphosphate leading to an elevated release of intracellular stored Ca2+. Single-cell analysis of Ca2+ release showed that the elevated response is not a consequence of an increased fraction of responding cells. These amplifications are consistent with p21N-ras acting like a guanine nucleotide coupling protein in this cell line.

Identification of the pertussis and cholera toxin substrates in normal and N-ras transformed NIH3T3 fibroblasts and an assessment of their involvement in bombesin-stimulation of inositol phospholipid metabolism.

Cholera and pertussis toxin-sensitive G-proteins were examined using specific immunological probes in wild type NIH3T3 cells and in clones of these cells containing the N-ras gene attached to a promotor where expression either was (T15+) or was not (T15-) induced. The major pertussis toxin sensitive-polypeptide had the immunological characteristics of Gi2. Two distinct forms of Gs alpha (45 and 42 kDa) were identified. Long term over-expression of p21N-ras (T15+ cells) did not alter the levels of Gi2 alpha or of Gs alpha. Pretreatment of NIH3T3 or T15 cells with either pertussis toxin or cholera toxin led to the complete in situ ADP-ribosylation of the respective G-proteins. Modification of Gi2 by pertussis toxin, however, had no inhibitory effect on the ability of bombesin to stimulate the production of inositol phosphates in any of these cells lines. Treatment of these cells with cholera toxin elicited a potent inhibition of the bombesin-stimulated production of inositol phosphates. This could be mimicked, however, by other agents which increase intracellular cyclic AMP concentrations. Cholera toxin treatment did not produce a significant alteration in the number of bombesin receptors on the cell surface. These results suggest that, in the T15 cell line, enhanced coupling of bombesin receptors to a phospholipase C-mediated hydrolysis of inositol phospholipids is either produced directly by p21N-ras or that overexpression of this gene product leads to the enhanced expression or function of a cholera and pertussis toxin-insensitive G-protein which then mediates the effect.