Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria.

In this study a Ca(2+) sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca(2+)-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca(2+) sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca(2+) transient. Cytoplasmic (Fura-2) and mitochondrial Ca(2+) (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca(2+) loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca(2+) sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K(d) of approximately 220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca(2+) transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca(2+) release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by approximately 65% and increased the time to 50% decay, whilst cytosolic Ca(2+) transients were unchanged. The mitochondrial Ca(2+) uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca(2+). The mitochondrial-targeted Ca(2+) probe indicates sustained and transient phases of mitochondrial Ca(2+) signal, which are dependent on cytoplasmic Ca(2+) levels and require a functional mCU.

Metabolomic profiling of Drosophila using liquid chromatography Fourier transform mass spectrometry.

Hydrophilic interaction chromatography (HILIC) interfaced with an Orbitrap Fourier transform mass spectrometer (FT-MS) was used to carry out metabolomic profiling of the classical Drosophila mutation, rosy (ry). This gene encodes a xanthine oxidase/dehydrogenase. In addition to validating the technology by detecting the same changes in xanthine, hypoxanthine, urate and allantoin that have been reported classically, completely unsuspected changes were detected in each of the tryptophan, arginine, pyrimidine and glycerophospholipid metabolism pathways. The rosy mutation thus ramifies far more widely than previously detected.

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.

Characterization of the Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family.

The NHE family of Na+/H+ exchangers is believed to play an essential role in animals, but may play an additional, specialised epithelial role in insects. The pharmacological sensitivity of the Drosophila melanogaster Malpighian tubule to a range of amiloride derivatives was shown to be consistent with an effect on an exchanger, rather than a Na+ channel. Consistent with this, no degenerin/epithelial Na+ channel (ENaC) genes could be detected in Malpighian tubules by reverse transcriptase/polymerase chain reaction (RT-PCR). Using a low-stringency homology searching, three members of the NHE family were identified in the genomic sequence of Drosophila melanogaster, although only two genes were represented as expressed sequence tags. All three genes (DmNHE1 at cytological position 21B1, DmNHE2 at 39B1 and DmNHE3 at 27A1) were found by RT-PCR to be widely expressed, and one (DmNHE2) was shown to have multiple transcripts. The putative translations of the three genes mark them as distantly related members of the family, inviting the possibility that they may serve distinct roles in insects.

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.

A novel Drosophila alkaline phosphatase specific to the ellipsoid body of the adult brain and the lower Malpighian (renal) tubule.

Two independent Drosophila melanogaster P(GAL4) enhancer-trap lines revealed identical GAL4-directed expression patterns in the ellipsoid body of the brain and in the Malpighian (renal) tubules in the abdomen. Both P-element insertions mapped to the same chromosomal site (100B2). The genomic locus, as characterized by plasmid rescue of flanking DNA, restriction mapping, and DNA sequencing, revealed the two P(GAL4) elements to be inserted in opposite orientations, only 46 bp apart. Three genes flanking the insertions have been identified. Calcineurin A1 (previously mapped to 21E-F) lies to one side, and two very closely linked genes lie to the other. The nearer encodes Aph-4, the first Drosophila alkaline phosphatase gene to be identified; the more distant gene [l(3)96601] is novel, with a head-elevated expression, and with distant similarity to transcription regulatory elements. Both in situ hybridization with Aph-4 probes and direct histochemical determination of alkaline phosphatase activity precisely matches the enhancer-trap pattern reported by the original lines. Although the P-element insertions are not recessive lethals, they display tubule phenotypes in both heterozygotes and homozygotes. Rates of fluid secretion in tubules from c507 homozygotes are reduced, both basally, and after stimulation by CAP(2b), cAMP, or Drosophila leucokinin. The P-element insertions also disrupt the expression of Aph-4, causing misexpression in the tubule main segment. This disruption extends to tubule pigmentation, with c507 homozygotes displaying white-like transparent main segments. These results suggest that Aph-4, while possessing a very narrow range of expression, nonetheless plays an important role in epithelial function.

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.

The multifunctional Drosophila melanogaster V-ATPase is encoded by a multigene family.

In animals, V-ATPases are believed to play roles in the plasma membrane, as well as endomembrane. To understand these different functions, it is necessary to adopt a genetic approach in a physiologically tractable model organism. For this purpose, Drosophila melanogaster is ideal, because of the powerful genetics associated with the organism and because of the unusually informative epithelial phenotype provided by the Malpighian tubule. Recently, the first animal "knockouts" of a V-ATPase were described in Drosophila. The resulting phenotypes have general utility for our understanding of V-ATPase function and suggest a screen for novel subunits and associated proteins. Genome project resources have accelerated our knowledge of the V-ATPase gene family size and the new Drosophila genes vhaSFD, vha100-1, vha100-2, vha100-3, vha16-2, vha16-3, vha16-4, vhaPPA1, vhaPPA2, vhaM9.7.1, and vhaM9.7.2 are described. The Drosophila V-ATPase model is thus well-suited to both forward and reverse genetic analysis of this complex multifunctional enzyme.

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.

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.

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.

Psychosocial interventions for patients with chronic obstructive pulmonary disease.

Depression, hopelessness, and pessimism are common behaviors seen in patients with chronic obstructive pulmonary disease (COPD). Healthcare personnel have traditionally focused on therapies to treat the physiologic effects of this debilitating disease. The psychologic problems associated with COPD that can affect a patient's personal and social quality of life are not often addressed. This article reviews current research on these problems and suggests nursing interventions that can assist in meeting patient-centered outcomes.

Functional domains are specified to single-cell resolution in a Drosophila epithelium.

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P[GAL4] enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary ("stellate") cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an "identified cell" system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

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.

Site-selected mutagenesis of the Drosophila second chromosome via plasmid rescue of lethal P-element insertions.

This paper describes a fast and efficient approach to correlating cloned genes with mutant phenotypes in Drosophila. We make use of a large collection D. melanogaster lines with recessive lethal insertions of a P[lacW] transposon on their second chromosome. Within this collection there clearly must be many insertions corresponding to Drosophila genes that have been cloned and characterized, e.g., via homology with cloned mammalian genes, but for which mutant phenotypes have yet to be identified. We have made use of the fact that P[lacW] contains a plasmid replicon to establish a collection of rescued plasmids containing genomic DNA flanking the sites of transposon insertion. Plasmids representing a total of 1836 lines were independently rescued and pooled in batches of 10 and 100. Pools of 100 plasmids were screened by hybridization with cDNAs corresponding to cloned second chromosome loci. Hybridizing pools were then narrowed down to single plasmids by a process of subdivision and rehybridization, and corresponding mutant lines were obtained. The success rate was better than one in four. This rate would undoubtedly be improved by the use of genomic DNA probes.

Characterisation of vha26, the Drosophila gene for a 26 kDa E-subunit of the vacuolar ATPase.

A Drosophila melanogaster gene and cDNA for the E-subunit of the V-ATPase were characterised. The deduced product has 226 amino acids and a molecular mass of 26.1 kDa. The gene is a single copy at 83B1-4 on chromosome 3R. The coding sequence is punctuated by three introns which do not align with those in Neurospora. The gene is ubiquitously expressed as an mRNA of 2.3 kb. but at lower levels in pupae.

The Drosophila melanogaster gene vha14 encoding a 14-kDa F-subunit of the vacuolar ATPase.

A Drosophila melanogaster (Dm) cDNA (vha14) encoding the 14-kDa F-subunit of the vacuolar H(+)-ATPase (V-ATPase) has been cloned via homology with the corresponding Manduca sexta (Ms) gene. Its deduced translation product is a 124-amino-acid polypeptide sharing 90% identity with the Ms polypeptide and 50% identity with an analogous polypeptide of Saccharomyces cerevisiae, and a more distant similarity to a subunit of the Na(+)-transporting ATPase of Enterococcus hirae. Homology was also found with expressed sequence tags from man, Arabidopsis thaliana, Caenorhabditis elegans and C. briggsiae, Oryza sativa and Plasmodium falciparum, indicating that the subunit is phylogenetically conserved. The Dm gene (vha14) is present as a single copy at cytological position 52B on the second chromosome, and gives rise to an mRNA species of 0.65 kb. Expression of the latter shows relatively little variation during development, or between adult head, thorax and abdomen, suggesting that the F-subunit is a relatively ubiquitous component of the V-ATPase.

Separate control of anion and cation transport in malpighian tubules of Drosophila Melanogaster.

Microelectrode measurements of basal, apical and transepithelial potentials in the Malpighian tubules of Drosophila melanogaster were obtained under a range of conditions in order to investigate whether each of the three main second messenger systems known to act in the tubules (cyclic AMP, cyclic GMP and Ca2+) acted specifically on either cation or anion transport, or whether they activated both systems. Ion-selective microelectrode determinations of K+ concentration and pH of secreted fluid allowed the role of each signalling system to be analysed further. Stimulation with cyclic nucleotides markedly alters the potential profile across principal cells through the selective activation of an apical electrogenic V-ATPase. By contrast, manipulation of extracellular chloride levels, combined with stimulation with leucokinin, does not affect the potential profile across the principal cells, showing that chloride must pass through another route. The cell-permeant Ca2+ chelator BAPTA-AM was shown to suppress the action of leucokinins (insect peptides that induce rapid fluid secretion), but not those of cyclic AMP, the neuronally derived insect peptide cardioacceleratory peptide 2b (CAP2b) or its intracellular messenger cyclic GMP. This shows that leucokinins act through Ca2+ and not through cyclic nucleotides and that the cyclic nucleotide pathways do not co-activate the intracellular Ca2+ pathway to exert their effects. Taken together, these results show that leucokinin acts through intracellular Ca2+, independently of cyclic AMP or cyclic GMP, to raise the chloride permeability of the epithelium. By contrast, either cyclic AMP or cyclic GMP (upon CAP2b stimulation) acts on the electrogenic cation-transporting apical V-ATPase, with only a negligible effect on anion conductance and without perturbing intracellular [Ca2+]. There is thus a clear functional separation between the control pathways acting on cation and anion transport in the tubules. Given the evidence from D. melanogaster and other species that chloride does not pass through the principal cells, we speculate that these two pathways may also be physically separated within cell subtypes of the tubules.