Biomedical subjects

Klaus W Beyenbach

Publications and source records attributed to Klaus W Beyenbach.

11 recordsLinked to original sources

PubMed2006

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

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.

Animals↗

PubMed2004

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium.

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was -6.8 mV (apical side), transepithelial resistance was 1,050 Omega.cm(2), and short-circuit current (I(sc)) was 8.1 microA/cm(2). Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kOmega.cm(2), respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of alpha-epithelial Na(+) channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na(+)-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na(+) flux (19 nmol.min(-1).cm(-2)) was two times as large as the reverse flux, and net transepithelial Na(+) flux was nearly double the amiloride-sensitive I(sc). In plain Ringer solution, the amiloride-sensitive I(sc) went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl(-)-dependent I(sc) consistent with the stimulation of transepithelial Cl(-) secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na(+) absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl(-) secretion activated by cAMP.

Amiloride↗

PubMed2004

Kidneys sans glomeruli.

The evolution of the vertebrate kidney records three occasions, each separated by about 50 million years, when fish have abandoned glomeruli to produce urine by tubular mechanisms. The recurring dismissal of glomeruli suggests a mechanism of aglomerular urine formation intrinsic to renal tubules. Indeed, the transepithelial secretion of organic solutes and of inorganic solutes such as sulfate, phosphate, and magnesium can all drive secretory water flow in renal proximal tubules of fish. However, the secretion of NaCl via secondary active transport of Cl is the primary mover of secretory water flow in, surprisingly, proximal tubules of both glomerular and aglomerular fish. In filtering kidneys, the tubular secretion of solute and water is overshadowed by reabsorptive transport activities, but secretion progressively comes to light as glomerular filtration decreases. Thus the difference between glomerular and aglomerular urine formation is more a difference of degree than of kind. At low rates of glomerular filtration in seawater fish, NaCl-coupled water secretion serves to increase the renal excretory capacity by increasing the luminal volume into which waste, excess, and toxic solutes can be secreted. The reabsorption of NaCl and water in the distal nephron and urinary bladder concentrates unwanted solutes for excretion while minimizing renal water loss. In aglomerular fish, NaCl-coupled water secretion across proximal tubules replaces glomerular filtration to increase renal excretory capacity. A review of the literature suggests that tubular secretion of NaCl and water is an early function of the vertebrate proximal tubule that has been retained throughout evolution. Active transepithelial Cl secretion takes place in gall bladders studied as models of the mammalian proximal tubule and in proximal tubules of amphibians and apparently also of mammals. The tubular secretion of Cl is also observed in mammalian distal tubules. The evidence consistent with and for Cl secretion in, respectively, proximal and distal tubules of the mammalian kidney calls for a reexamination of basic assumptions in renal physiology that may lead to new opportunities for managing some forms of renal disease.

Adaptation, Physiological↗

PubMed2004

Effects of leucokinin-VIII on Aedes Malpighian tubule segments lacking stellate cells.

The diuretic peptide leucokinin is known to increase fluid secretion in Malpighian tubules of the yellow fever mosquito Aedes aegypti by increasing a transepithelial Cl(-) conductance. The present study sought to examine whether stellate cells provided this transepithelial conductance in Aedes Malpighian tubules as they do in Drosophila Malpighian tubules. Aedes Malpighian tubule segments with and without stellate cells were perfused in vitro for measurements of the transepithelial voltage (V(t)), resistance (R(t)) and Cl(-) diffusion potentials (DP(Cl)). In 11 tubule segments containing both principal cells and stellate cells, 1 micro mol l(-1) leucokinin-VIII added to the peritubular bath immediately and significantly decreased V(t) from 39.3+/-14.3 mV to 2.3+/-0.7 mV, decreased R(t) from 12.4+/-2.6 kOmegacm to 2.4+/-0.3 kOmegacm, and increased DP(Cl) from 8.2+/-1.2 mV to 42.1+/-5.4 mV. These effects of leucokinin-VIII were qualitatively and quantitatively similar in six tubule segments containing no stellate cells; V(t) decreased from 37.8+/-7.0 mV to 3.4+/-0.6 mV, R(t) decreased from 8.8+/-2.1 kOmegacm to 1.7+/-0.2 kOmegacm, and DP(Cl) increased from 5.8+/-2.6 mV to 50.0+/-2.1 mV. Thus, stellate cells are not required for signaling or mediating the effects of leucokinin in Malpighian tubules of Aedes aegypti. The results further support previous observations that principal cells signal the effects of leucokinin to increase the Cl(-) conductance of the paracellular pathway through septate (or tight) junctions.

Aedes↗

PubMed2004

Mechanisms of K+ transport across basolateral membranes of principal cells in Malpighian tubules of the yellow fever mosquito, Aedes aegypti.

The mechanisms of K(+) entry from the hemolymph into principal cells of Malpighian tubules were investigated in the yellow fever mosquito, Aedes aegypti. The K(+) channel blocker Ba(2+) (5 mmol l(-1)) significantly decreased transepithelial (TEP) fluid secretion (V(s)) from 0.84 nl min(-1) to 0.37 nl min(-1) and decreased the K(+) concentration in secreted fluid from 119.0 mmol l(-1) to 54.3 mmol l(-1) with no change in the Cl(-) concentration. Even though the Na(+) concentration increased significantly from 116.8 mmol l(-1) to 144.6 mmol l(-1), rates of TEP ion secretion significantly decreased for all three ions. In addition, Ba(2+) had the following significant electrophysiological effects: it depolarized the TEP voltage (V(t)) from 19.4 mV to 17.2 mV, increased the TEP resistance (R(t)) from 6.4 kOhmscm to 6.9 kOhmscm, hyperpolarized the basolateral membrane voltage of principal cells (V(bl)) from -75.2 mV to -88.2 mV and increased the cell input resistance from 363.7 kOhms to 516.3 kOhms. These effects of Ba(2+) reflect the block of K(+) channels that, apparently, are also permeable to Na(+). Bumetanide (100 micro mol l(-1)) had no effect on TEP fluid secretion and electrical resistance but significantly decreased TEP K(+) secretion, consistent with the inhibition of electroneutral Na(+)/K(+)/2Cl(-) cotransport. TEP Na(+) secretion significantly increased because other Na(+) entry pathways remained active. Bumetanide plus Ba(2+) completely inhibited TEP electrolyte and fluid secretion, with fast and slow kinetics reflecting the Ba(2+) block of basolateral membrane K(+) channels and the inhibition of Na(+)/K(+)/2Cl(-) cotransport, respectively. The single and combined effects of Ba(2+) and bumetanide suggest that (1) K(+) channels and Na(+)/K(+)/2Cl(-) cotransport are the primary mechanisms for bringing K(+) into cells, (2) K(+) channels mediate a significant Na(+) influx, (3) Na(+) has as many as four entry pathways and (4) the mechanisms of TEP K(+) and Na(+) secretion are coupled such that complete block of TEP K(+) renders the epithelium unable to secrete Na(+).

Aedes↗

PubMed2004

The mechanism of action of the antidiuretic peptide Tenmo ADFa in Malpighian tubules of Aedes aegypti.

The mechanism of action of Tenebrio molitor antidiuretic factor 'a' (Tenmo ADFa) was explored in isolated Malpighian tubules of Aedes aegypti. In the Ramsay assay of fluid secretion, Tenmo ADFa (10(-9) mol l(-1)) significantly inhibited the rate of fluid secretion from 0.94 nl min(-1) to 0.44 nl min(-1) without significant effects on the concentrations of Na+, K+ and Cl- in secreted fluid. In isolated perfused tubules, Tenmo ADFa had no effect on the transepithelial voltage (Vt) and resistance (Rt). In principal cells of the tubule, Tenmo ADFa had no effect on the basolateral membrane voltage (Vbl) and the input resistance of principal cells (Rpc). Tenmo ADFa significantly increased the intracellular concentration of cyclic guanosine monophosphate (cGMP) from 2.9 micromol l(-1) (control) to 7.4 micromol l(-1). A peritubular [cGMP] of 20 micromol l(-1) duplicated the antidiuretic effects of Tenmo ADFa without inducing electrophysiological effects. In contrast, 500 micromol l(-1) cGMP significantly depolarized V(bl), hyperpolarized Vt, and reduced Rt and Rpc, without increasing antidiuretic potency beyond that of 20 micromol l(-1) cGMP. A plot of peritubular cGMP concentration vs Vbl revealed a steep dose-response between 300 micromol l(-1) and 700 micromol l(-1) with an EC50 of 468 micromol l(-1). These observations suggest a receptor- and cGMP-mediated mechanism of action of Tenmo ADFa. Tenmo ADFa and physiological concentrations of cGMP (< 20 micromol l(-1)) reduce the rate of isosmotic fluid secretion by quenching electroneutral transport systems. The inhibition reveals that as much as 50% of the normal secretory solute and water flux can stem from electrically silent mechanisms in this highly electrogenic epithelium.

Aedes↗

PubMed2003

Regulation of tight junction permeability with switch-like speed.

PURPOSE OF REVIEW: The case is made that tight junctions can undergo large reversible conductance changes in a matter of seconds and yet preserve their permselectivity. The diuretic peptide leucokinin transforms (renal) Malpighian tubules of the yellow fever mosquito from a moderately tight epithelium to a leaky epithelium by increasing the chloride-conductance of the paracellular shunt pathway. The nine-fold increase in the paracellular chloride-conductance brings about a non-selective stimulation of transepithelial sodium chloride and potassium chloride secretion, as expected from a conductance increase in the pathway taken by the counterion of sodium and potassium. RECENT FINDINGS: The leucokinin signaling pathway consists in part of a receptor coupled G-protein, phospholipase C, inositol-1,4,5-trisphosphate, and increased intracellular calcium concentration that bring about the increase in the paracellular, tight junction chloride-conductance. As the conductance of the tight junction pathway increases it becomes more selective for the transepithelial passage of chloride. SUMMARY: Epithelial cells in Malpighian tubules taper to tight junctions at their lateral edges exposing them directly to apical and serosal solutions. Furthermore, evolutionary pressures to excrete salt and water at high rates without the aid of glomerular filtration have led to powerful mechanisms of tubular secretion, capable of diuresis when the mosquito is challenged with the volume expansion of a blood meal. The tubular diuresis is mediated in part by increasing the paracellular chloride conductance. Thus, anatomical and physiological specializations in Malpighian tubules combine to yield the evidence for the dynamic hormonal regulation of the tight junction pathway.

Animals↗

PubMed2003

The dependence of electrical transport pathways in Malpighian tubules on ATP.

The relationship between the intracellular ATP concentration [ATP](i) and the electrical properties of principal cells was investigated in Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Under control conditions, [ATP](i) was 0.91 mmol l(-1), the input resistance of the principal cell (R(pc)) was 334.1 k Omega, and the basolateral membrane was marked by a large K(+)-conductance and a membrane voltage (V(bl)) of -75.8 mV. Peritubular cyanide (CN, 0.3 mmol l(-1)) reduced [ATP](i) to 0.08 mmol l(-1) in less than 2 min; however, V(bl) dropped to -8 mV and R(pc) increased to 3150.8 k Omega in 8 min, while the K(+)-conductance of the basolateral membrane disappeared. Upon washout of CN, V(bl) and R(pc) returned to control values within 2 min, and the basolateral membrane recovered its K(+)-conductance. The recovery of normal [ATP](i) took 15 min. Dose-dependence and EC(50) values for the CN-inhibition of V(bl) and the increase in R(pc) were strikingly similar (184.0 micromol l(-1) and 164.4 micromol l(-1)). Similar effects of metabolic inhibition were observed with dinitrophenol (DNP), but the EC(50) values were 50.3 micromol l(-1) and 71.7 micromol l(-1) for the effects on V(bl) and R(pc), respectively. Barium, a blocker of K(+)-channels, significantly hyperpolarized V(bl) to -89.1 mV and increased R(pc) to 769.4 k Omega under control conditions, but had no effects during metabolic inhibition. These results illustrate a temporal relationship between [ATP](i) and electrogenic and conductive transport pathways in principal cells that is consistent with the role of ATP as an integrator of transport steps at apical and basolateral membranes of the cell. When [ATP](i) drops to levels that are 10% of control, the V-type H(+)-ATPase is inhibited, preventing the extrusion of K(+) to the tubule lumen. At the same time, basolateral membrane K(+)-channels close, preventing the entry of K(+) from the hemolymph. Intracellular K(+) homeostasis is thus protected during metabolic inhibition, allowing the cell to re-establish K(+) transport when ATP is synthesized again.

Adenosine Triphosphate↗

PubMed2003

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

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 NO(3)(-)-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.

Aedes↗

PubMed2003

Transport mechanisms of diuresis in Malpighian tubules of insects.

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.

Aedes↗

PubMed2002

Leucokinin activates Ca(2+)-dependent signal pathway in principal cells of Aedes aegypti Malpighian tubules.

The role of Ca(2+) in mediating the diuretic effects of leucokinin-VIII was studied in isolated perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Peritubular leucokinin-VIII (1 microM) decreased the transepithelial resistance from 11.2 to 2.6 kOmega. cm, lowered the transepithelial voltage from 42.8 to 2.7 mV, and increased transepithelial Cl(-) diffusion potentials 5.1-fold. In principal cells of the tubules, leucokinin-VIII decreased the fractional resistance of the basolateral membrane from 0.733 to 0.518. These effects were reversed by the peritubular Ca(2+)-channel blocker nifedipine, suggesting a role of peritubular Ca(2+) and basolateral Ca(2+) channels in signal transduction. In Ca(2+)-free Ringer bath, the effects of leucokinin-VIII were partial and transient but were fully restored after the bath Ca(2+) concentration was restored. Increasing intracellular Ca(2+) with thapsigargin duplicated the effects of leucokinin-VIII, provided that peritubular Ca(2+) was present. The kinetics of the effects of leucokinin-VIII is faster than that of thapsigargin, suggesting the activation of inositol-1,4,5-trisphosphate-receptor channels of intracellular stores. Store depletion may then bring about Ca(2+) entry into principal cells via nifedipine-sensitive Ca(2+) channels in the basolateral membrane.

Aedes↗