Klaus W. Beyenbach

The V-type H+ ATPase: molecular structure and function,physiological roles and regulation

SUMMARY It was nearly 30 years before the V-type H+ ATPase was admitted to the small circle of bona fide transport ATPases alongside F-type and P-type ATPases. The V-type H+ ATPase is an ATP-driven enzyme that transforms the energy of ATP hydrolysis to electrochemical potential differences of protons across diverse biological membranes via the primary active transport of H+. In turn, the transmembrane electrochemical potential of H+ is used to drive a variety of (i) secondary active transport systems via H+-dependent symporters and antiporters and (ii) channel-mediated transport systems. For example, expression of Cl- channels or transporters next to the V-type H+ ATPase in vacuoles of plants and fungi and in lysosomes of animals brings about the acidification of the endosomal compartment, and the expression of the H+/neurotransmitter antiporter next to the V-type H+ ATPase concentrates neurotransmitters in synaptic vesicles. First found in association with endosomal membranes, the V-type H+ ATPase is now also found in increasing examples of plasma membranes where the proton pump energizes transport across cell membranes and entire epithelia. The molecular details reveal up to 14 protein subunits arranged in (i) a cytoplasmic V1 complex, which mediates the hydrolysis of ATP, and (ii) a membrane-embedded V0 complex, which translocates H+ across the membrane. Clever experiments have revealed the V-type H+ ATPase as a molecular motor akin to F-type ATPases. The hydrolysis of ATP turns a rotor consisting largely of one copy of subunits D and F of the V1 complex and a ring of six or more copies of subunit c of the V0 complex. The rotation of the ring is thought to deliver H+ from the cytoplasmic to the endosomal or extracellular side of the membrane, probably via channels formed by subunit a. The reversible dissociation of V1 and V0 complexes is one mechanism of physiological regulation that appears to be widely conserved from yeast to animal cells. Other mechanisms, such as subunit-subunit interactions or interactions of the V-type H+ ATPase with other proteins that serve physiological regulation, remain to be explored. Some diseases can now be attributed to genetic alterations of specific subunits of the V-type H+ ATPase.

Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules

Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.

Transport mechanisms of diuresis in Malpighian tubules of insects

SUMMARY We have studied Malpighian tubules of Aedes aegypti using a variety of methods: Ramsay fluid secretion assay, electron probe analysis of secreted fluid, in vitro microperfusion and two-electrode voltage clamp. Collectively, these methods have allowed us to elucidate transepithelial transport mechanisms under control conditions and in the presence of diuretic peptides. Mosquito natriuretic peptide (MNP), a corticotropin-releasing factor (CRF)-like diuretic peptide, selectively increases transepithelial secretion of NaCl and water, meeting the NaCl loads of the blood meal. The intracellular messenger of MNP is cAMP, which increases the Na+ conductance and activates the Na+/K+/2Cl--cotransporter in the basolateral membrane of principal cells. Leucokinin non-selectively increases transepithelial NaCl and KCl secretion, which may deal with hemolymph volume expansions or reduce the flight pay load upon eclosion from the aquatic habitat. The non-selective NaCl and KCl diuresis stems from the increase in septate junctional Cl- conductance activated by leucokinin using Ca2+ as second messenger. Fundamental to diuretic mechanisms are powerful epithelial transport mechanisms in the distal segment of the Malpighian tubules, where transepithelial secretion rates can exceed the capacity of mammalian glomerular kidneys in the renal turnover of the extracellular fluid compartment. In conjunction with powerful epithelial transport mechanisms driven by the V-type H+-ATPase, diuretic hormones enable hematophagous and probably also phytophagous insects to deal with enormous dietary loads, thereby contributing to the evolutionary success of insects.

The Developmental, Molecular, and Transport Biology of Malpighian Tubules

Molecular biology is reaching new depths in our understanding of the development and physiology of Malpighian tubules. In Diptera, Malpighian tubules derive from ectodermal cells that evaginate from the primitive hindgut and subsequently undergo a sequence of orderly events that culminates in an active excretory organ by the time the larva takes its first meal. Thereafter, the tubules enlarge by cell growth. Just as modern experimental strategies have illuminated the development of tubules, genomic, transcriptomic, and proteomic studies have uncovered new tubule functions that serve immune defenses and the breakdown and renal clearance of toxic substances. Moreover, genes associated with specific diseases in humans are also found in flies, some of which, astonishingly, express similar pathophenotypes. However, classical experimental approaches continue to show their worth by distinguishing between -omic possibilities and physiological reality while providing further detail about the rapid regulation of the transport pathway through septate junctions and the reversible assembly of proton pumps.

Regulation of epithelial shunt conductance by the peptide leucokinin.

SummaryIsolated Malpighian tubules of the yellow fever mosquito Aedes aegypti spontaneously secrete NaCl, KCl and water across an epithelium of modest transepithelial resistance (40–80 Ωcm2) and high transepithelial voltage (30–70 mV, lumen positive). Transepithelial electrochemical potentials indicate that Na and K are secreted by active and Cl by passive transport mechanisms. The addition of synthetic leucokinin-VIII (LK-VIII, insect myotropic peptide) to the peritubular bath significantly increases the rates of transepithelial NaCl, KCl and water secretion. In parallel, LK-VIII depolarizes the transepithelial voltage from 59.3 to 5.7 mV, decreases the transepithelial resistance from 57.7 to 9.9 Ωcm2, and renders the basolateral and apical membrane voltages nearly equipotential (∼ −90 mV). Unilateral step changes of the [Cl] in the peritubular bath or tubule lumen elicit small transepithelial Cl diffusion potentials in the absence of LK-VIII but large transepithelial Cl diffusion potentials, up to 85% of Nernst equilibrium potentials, in the presence of LK-VIII. In Malpighian tubules treated with dinitrophenol for estimates of the shunt resistance Rsh, LK-VIII reduces Rsh from 52.5 to 5.8 ωcm2. Bilateral reductions of the Cl concentration in tubule lumen and peritubular bath fully restore Rsh to 55.8 Ωcm2in the presence of LK-VIII. LK-VIII has no effects when presented from the luminal side. These results suggest that LK-VIII increases the Cl conductance of the epithelial shunt via a receptor located at the basolateral side of the epithelium.

Dynamic changes in flow rate and composition of urine during the post-bloodmeal diuresis inAedes aegypti (L.)

Summary1.The urine of blood-fed mosquitos was collected and analyzed for elemental composition and osmolality.2.Peak rates of urine flow averaged 4.9 droplets/min at 6 min following the beginning of the bloodmeal; peak flow urine contained, in mM, Na 178, K 4, and Cl 132, and the urine was approximately isosmotic to hemolymph.3.As urine flow rates fell, the [Na] of the urine decreased and the [K] increased. Urine osmolality declined, measuring less than 100 mOsm/kg in some samples, as compared to 354 mOsm/kg measured in pre-bloodmeal hemolymph.4.When urine flow rates had fallen to less than 10% of peak flow rates, urine again became approximately isosmotic to hemolymph, still containing Na and K as the principal cations.5.Approximately 40% each of the water, Na and Cl contained in the plasma fraction of the bloodmeal was excreted during the 1–2h period following the bloodmeal. This excretion represents only 20–30% of the total loads ingested in the bloodmeal.6.The data are consistent with dynamic changes in the functioning of both the Malpighian tubules and hindgut during the post-bloodmeal diuresis.

Vacuolar-type proton pumps in insect epithelia

SUMMARY Active transepithelial cation transport in insects was initially discovered in Malpighian tubules, and was subsequently also found in other epithelia such as salivary glands, labial glands, midgut and sensory sensilla. Today it appears to be established that the cation pump is a two-component system of a H+-transporting V-ATPase and a cation/nH+ antiporter. After tracing the discovery of the V-ATPase as the energizer of K+/nH+ antiport in the larval midgut of the tobacco hornworm Manduca sexta we show that research on the tobacco hornworm V-ATPase delivered important findings that emerged to be of general significance for our knowledge of V-ATPases, which are ubiquitous and highly conserved proton pumps. We then discuss the V-ATPase in Malpighian tubules of the fruitfly Drosophila melanogaster where the potential of post-genomic biology has been impressively illustrated. Finally we review an integrated physiological approach in Malpighian tubules of the yellow fever mosquito Aedes aegypti which shows that the V-ATPase delivers the energy for both transcellular and paracellular ion transport.

Differential effects of secretagogues on Na and K secretion in the Malpighian tubules ofAedes aegypti (L.)

Summary1.Rates of fluid secretion were measured in isolated Malpighian tubules from adult female mosquitoes and the concentrations of Na, K and Cl in the secreted fluid were measured by electron probe analysis under control conditions and in the presence of the secretagogues dibutyryl cAMP or head extract.2.Under control conditions tubules secreted fluid at an average rate of 0.8 nl/min. The principal solutes in secreted fluid were Na at 94 mM, K at 91 mM and Cl at 161 mM.3.Dibutyryl cAMP (1 mM) stimulated the rate of fluid secretion ∼250% (to 2.9 nl/min), increased the [Na] of secreted fluid to 178 mM, decreased the [K] to 17 mM, and did not change the [Cl]. Consequently, the net effect of dibutyryl cAMP was a highly significant stimulation of Na, Cl, and water secretion with no significant change in the secretion of K.4.An aqueous extract of mosquito heads (0.1 head/μl) also stimulated the rate of fluid secretion ∼250% (to 2.8 nl/min), but introduced no major changes in the composition of secreted fluid with the exception of a slight, though significant decrease in the [K] from 91 to 73 mM. Consequently, the net effect of the head extract was the highly significant stimulation of Na, K, Cl, and water secretion.5.The different effects of dibutyryl cAMP and head extract on the secretion of cations suggest that Na secretion and K secretion can be regulated independently of one another. In vivo, such a separate regulation of Na and K secretion might be important in the maintenance of hemolymph Na and K concentrations, especially following a blood meal.

The V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti: localization and activity.

SUMMARY The V-type H+-ATPase is thought to provide the driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. To confirm the presence of this proton pump in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we used several antibodies raised against the V-type H+-ATPase of Manduca sexta. Western blot analysis confirmed the presence of the V-type H+-ATPase in Malpighian tubules of Aedes aegypti. In situ immunostaining identified the V-type H+-ATPase at the apical membrane of the mitochondrion-rich brush border of principal cells. The V-type H+-ATPase was not found in stellate cells. Measurements of ATPase activity revealed that bafilomycin-sensitive and NO3--sensitive ATPase activity accounted for 50–60% of total ATPase activity in crude extracts of Malpighian tubules. No significant ouabain- or vanadate-sensitive Na+/K+-ATPase activity was detected. These results support the conclusion reached previously in electrophysiological studies that the mechanisms for transepithelial electrolyte secretion in the Aedes Malpighian tubules rely on the V-type H+-ATPase as the principal energizer of epithelial transport. Measures of transepithelial Na+ and K+ secretion and estimates of the H+ flux mediated by the V-type H+-ATPase suggest a 1:1 stoichiometry for Na+/H+ and K+/H+ exchange transport across the apical membrane.

Culekinin depolarizing peptide: a mosquito leucokinin-like peptide that influences insect Malpighian tubule ion transport

A peptide termed culekinin depolarizing peptide (CDP) was isolated from approximately 1.2 million mosquitos (94% Culex salinarius). The peptide was isolated on the basis of a rapid myotropic assay that utilized a hindgut preparation from Leucophaea maderae and a transepithelial voltage assay that used mosquito Malpighian tubules from Aedes aegypti. A 15% trifluoroacetic acid extraction from the mosquitos, two solid phase extraction steps, and six HPLC steps resulted in the isolation of 9.7 nmol of CDP. This value corresponds to approximately 8 fmol/mosquito. Edman degradation indicated the following sequence for CDP: Asn-Pro-Phe-His-Ser-Trp-Gly-NH2. The sequence was confirmed as the suspected C-terminal amide form of the peptide, since native and synthetic CDP had identical chemical and biological properties. CDP is a member of the leucokinin family of neuropeptides. The leucokinins have been found in three other insect species (Leucophaea maderae, Acheta domesticus and Locusta migratoria) where these peptides were isolated by their myotropic properties alone. CDP shares a C-terminal sequence homology (i.e., Phe-X-Ser-Trp-Gly-NH2) with the rest of the leucokinins. CDP corresponds to the strongest tubule depolarizing activity in the C. salinarius extract. These findings agree with previous structure-activity studies that suggest that mosquitos would contain a leucokinin-like factor that had Phe-His-Ser-Trp-Gly-NH2 as the C-terminal pentapeptide. This is the first leucokinin isolated from blood feeding or holometabolous insects.