Biomedical subjects
Publications and source records attributed to K W Beyenbach.
The Ussing model has long provided the conceptual foundation for understanding epithelial transport mechanisms energized by the Na(+)-K(+)-ATPase. Plasma membranes may also use the vacuolar (V-type) H(+)-ATPase as the primary energy source of membrane and epithelial transport. A pure electrogenic pump, the V-type H(+)-ATPase energizes not only membranes it inhabits but also other transport pathways via electrical coupling.
Stellate cells of Aedes aegypti Malpighian tubules were investigated using patch-clamp methods to probe the route of transepithelial Cl(-) secretion. Two types of Cl(-) channel were identified in excised, inside-out apical membrane patches. The first Cl(-) channel, type I, had a conductance of 24 pS, an open probability of 0.816+/-0.067, an open time of 867+/-114 ms (mean +/- s.e.m., four patches) and the selectivity sequence I(-)>Cl(-)(much greater than) isethionate>gluconate. The I(-)/Cl(-)>>isethionate>gluconate. The I(-)Cl(-) permeability ratio was 1.48, corresponding to Eisenman sequence I. The type I Cl(-) channel was blocked by 2,2'-iminodibenzoic acid (DPC) and niflumic acid (2-[3-(trifluoromethyl)anilo]nicotinic acid). The removal of Ca(2+) from the Ringer's solution on the cytoplasmic side had no effect on channel activity. The second Cl(-) channel, type II, had a conductance of 8 pS, an open probability of 0.066+/-0.021 and an open time of 7.53+/-1.46 ms (mean +/- s.e.m., four patches). The high density and halide selectivity sequence of the type I Cl(-) channel is consistent with a role in transepithelial Cl(-) secretion under control conditions, but it remains to be determined whether these Cl(-) channels also mediate transepithelial Cl(-) secretion under diuretic conditions in the presence of leucokinin.
Of all known vertebrate tissues, the kidneys of fish are the champions of Mg transport. They can switch from Mg conservation in fresh water to Mg wasting in seawater. High rates of tubular transport and the ability to alternate between Mg reabsorption and secretion make fish kidneys the model of choice investigating the mechanisms of transepithelial and membrane Mg transport and its regulation by extracellular hormones and intracellular messengers. Studies in isolated proximal tubules indicate active transepithelial Mg transport that requires metabolic energy for both tubular reabsorption and secretion. Whether active transport is primary and mediated by a Mg-pump, or secondary and mediated via cotransport or antiport, is unknown. In fresh water fish, the active transport pathway appears to include a Mg-channel located in brush-border membranes of proximal tubules. Although plasma Mg concentrations are well protected, a primary hormone controlling Mg balance has yet to be identified in any animal. Moreover, the mechanisms of intracellular Mg homeostasis, especially in epithelial cells with high Mg throughput, are unknown. New methods, including Mg imaging and genetic/molecular approaches promise to unravel Mg transport mechanisms in teleost renal tubules.
Principal cells of the Malpighian tubule of the yellow fever mosquito were studied with the methods of two-electrode voltage clamp (TEVC). Intracellular voltage (V(pc)) was -86.7 mV, and input resistance (R(pc)) was 388.5 kOmega (n = 49 cells). In six cells, Ba(2+) (15 mM) had negligible effects on V(pc), but it increased R(pc) from 325.3 to 684.5 kOmega (P < 0.001). In the presence of Ba(2+), leucokinin-VIII (1 microM) increased V(pc) to -101.8 mV (P < 0.001) and reduced R(pc) to 340.2 kOmega (P < 0.002). Circuit analysis yields the following: basolateral membrane resistance, 652. 0 kOmega; apical membrane resistance, 340.2 kOmega; shunt resistance (R(sh)), 344.3 kOmega; transcellular resistance, 992.2 kOmega. The fractional resistance of the apical membrane (0.35) and the ratio of transcellular resistance and R(sh) (3.53) agree closely with values obtained by cable analysis in isolated perfused tubules and confirm the usefulness of TEVC methods in single principal cells of the intact Malpighian tubule. Dinitrophenol (0.1 mM) reversibly depolarized V(pc) from -94.3 to -10.7 mV (P < 0.001) and reversibly increased R(pc) from 412 to 2,879 kOmega (P < 0.001), effects that were duplicated by cyanide (0.3 mM). Significant effects of metabolic inhibition on voltage and resistance suggest a role of ATP in electrogenesis and the maintenance of conductive transport pathways.
The effects of bafilomycin A(1), a blocker of V-type H(+)-ATPases, were investigated in Malpighian tubules of Aedes aegypti. Bafilomycin A(1) reduced rates of transepithelial fluid secretion and the virtual short-circuit current (vI(sc)) with an IC(50) of approximately 5 micromol l(-)(1). As vI(sc) decreased, the electrical resistance increased across the whole epithelium and across the apical membrane, indicating effects on electroconductive pathways. Bafilomycin A(1) had no effect when applied from the tubule lumen, pointing to the relative impermeability of the apical membrane to bafilomycin A(1). Thus, bafilomycin A(1) must take a cytoplasmic route to its blocking site in the proton channel of the H(+)-ATPase located in the apical membrane of principal cells. The inhibitory effects of bafilomycin A(1) were qualitatively similar to those of dinitrophenol in that voltages across the epithelium (V(t)), the basolateral membrane (V(bl)) and the apical membrane (V(a)) depolarized towards zero in parallel. Moreover, V(bl )always tracked V(a), indicating electrical coupling between the two membranes through the shunt. Electrical coupling allows the H(+)-ATPase to energize not only the apical membrane, but also the basolateral membrane. Furthermore, electrical coupling offers a balance between electroconductive entry of cations across the basolateral membrane and extrusion across the apical membrane to support steady-state conditions during transepithelial transport.
Since the aglomerular toadfish (Opsanus tau) experiences a natriuresis following transfer to 10% seawater, we examined the role of natriuretic peptides in the acclimation of toadfish to hypo-osmotic media. Gel filtration chromatography of acid extracts of toadfish heart and kidney identified a broad peak of atrial natriuretic peptide-like immunoreactivity in both tissues, with maximal immunoreactivity in fractions coeluting with human alpha-atrial natriuretic peptide. Using a homologous bioassay to measure changes in aortic ring tension, both vasorelaxing and, surprisingly, vasoconstricting bioactivities were identified in gel fractions of heart extract. No significant vasorelaxing activity was identified in kidney extract or fractions. Instead, a potent vasoconstricting activity was observed, with maximal activity in gel fractions with an estimated MW greater than 1000 Da. Levels of atrial natriuretic peptide immunoreactivity in plasma from the caudal vein were very low in seawater toadfish and were unchanged 12 h after transfer of toadfish to 10% seawater. We conclude, that natriuretic peptides are present in the heart and kidney of toadfish. However, atrial natriuretic peptide-like peptides of cardiac origin circulating to the kidney via the caudal vein do not appear responsible for the natriuresis that ensues upon the transfer of toadfish to 10% seawater. In the absence of glomeruli, this tubular natriuresis may be regulated by natriuretic peptides present in the kidney.
The mechanism of action of synthetic Culex corticotropin-releasing factor (CRF)-like diuretic peptide (CCRF-DP) was investigated in isolated, perfused Malpighian tubules of the yellow fever mosquito, Aedes aegypti. Low concentrations of CCRF-DP (10(-10) and 10(-9) M) caused depolarizing oscillations of the lumen-positive transepithelial voltage (Vt) in Malpighian tubules, whereas high concentrations (10(-8) and 10(-7) M) first depolarized and then transiently hyperpolarized Vt; CCRF-DP always lowered transepithelial resistance (Rt), regardless of voltage depolarization or hyperpolarization. The short-circuit current (Isc), an electrical estimate of active transepithelial transport of Na and K, remained unchanged at low concentrations of CCRF-DP, but Isc more than doubled at high concentrations. These effects of CCRF-DP suggest dose-dependent sites of action: low concentrations of CCRF-DP affect the paracellular pathway, and high concentrations affect both paracellular and transcellular pathways.
The mechanism of action of synthetic CCRF-DP, the corticotropin-releasing factor (CRF)-related diuretic peptide of the salt marsh mosquito Culex salinarius, was investigated in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. A low concentration of CCRF-DP (10(-9)mol l-1) caused a small but insignificant increase in transepithelial secretion of NaCl and fluid, but significantly reduced transepithelial voltage and resistance without a change in short-circuit current, pointing to the stimulation of passive Cl- transport through the paracellular pathway as the principal mechanism of a mild diuresis. Significant changes in voltage and resistance but not in short-circuit current were duplicated by the ionophore A23187 (0.4 micromol l-1), suggesting Ca2+ as a second messenger at 10(-9)mol l-1 CCRF-DP. A high concentration of CCRF-DP (10(-7)mol l-1) significantly increased transepithelial secretion of NaCl and fluid and significantly increased short-circuit current, pointing to the stimulation of active Na+ transport through the transcellular pathway as the mechanism of a strong diuresis. This effect was mimicked by dibutyryl-cAMP, suggesting cAMP as a second messenger at 10(-7)mol l-1 CCRF-DP. Dibutyryl-cGMP had no effects. These results suggest dose-dependent, receptor-mediated effects of CCRF-DP that target discrete transport pathways via discrete second messengers: low concentrations of CCRF-DP cause a mild diuresis, apparently via Ca2+-mediated effects on paracellular Cl- transport, and high concentrations cause a strong diuresis via cAMP-mediated effects on active transcellular Na+ transport in addition to the effects on the paracellular pathway.
Sites of renal Mg transport were identified in seawater killifish (Fundulus heteroclitus) using a Cameca model IMS-3f ion microscope. Killifish were given an intraperitoneal injection of the stable isotope 26Mg (99.5% enrichment) to stimulate and trace renal Mg excretion. We identified two sites of 26Mg transport in frozen freeze-dried cryosections of kidney: the proximal tubule, known to secrete Mg, and the collecting duct, heretofore not known to handle Mg. In epithelial cells of the proximal tubule, the punctate distribution of injected 26Mg suggests transcytotic excretion of Mg in bound form. In collecting ducts, a subpopulation of Mg/Ca-rich cells was identified with high accumulations of injected 26Mg. Here, the punctate distribution of 26Mg decreased from the apical to the basal region of the cells, revealing a transcytotic gradient of apparently bound Mg. Since proximal tubules of fish are implicated with Mg secretion, Mg/Ca-rich cells in the collecting duct may reabsorb Mg, thereby providing the usual two-step of renal regulation, now also for Mg.
Isolated renal proximal tubules of sea water fish net secrete fluid in vitro. The principal electrolytes in secreted fluid are Na, Cl, Mg and S. Transepithelial voltages may be lumen-negative or -positive by a few millivolts, and transepithelial resistances are low partly due to high paracellular Na and Cl permeabilities. Transepithelial electrochemical potentials indicate secretion of Mg into the tubule lumen by active transport. As Mg concentration in secreted fluid rises, Na concentration falls. Surprisingly, these observations of fluid secretion are made in glomerular and aglomerular proximal tubules, suggesting a fundamental mechanism common to both. Central to this commonality appears to be their behavior as open Donnan systems. Mg actively secreted into the tubule lumen from which it cannot diffuse back into the peritubular medium causes the transepithelial secretion of diffusible Na and Cl. Water follows by osmosis. Since there is flow out of the distal end of the tubule Donnan equilibrium is not attained. Instead, a dynamic Donnan system is maintained, driven by active transport of Mg. A mathematical model of tubular electrolyte and fluid secretion confirms the operation of this open, dynamic Donnan system in aglomerular and glomerular proximal tubules.
The mechanism of tubular Mg transport was investigated in membrane vesicles (MV) of trout kidneys prepared by differential centrifugation with sucrose. MV consisted largely of brush-border membranes, as indicated by high enrichments of brush-border membrane enzymes. Although measured transport of 28 Mg included a binding component, most membrane transport was into or out of an osmotically active space. There was no evidence for amiloride-sensitive Na/Mg exchange, nor was Mg uptake affected by the carboxyl group reagents trimethyloxonium tetrafluoroborate, glycine methyl ester.HCl-1-ethyl-3- (3-dimethyl-aminopropyl)carbodiimide, and N,N'-dicyclohexyl carbodiimide or the Ca channel modulators D-600, verapamil, diltiazem, and BAY K 8644. However, Mg uptake increased in the presence of inside-negative voltages generated by inward gradients of the permeant anions NO3, SCN, and Cl or by outward gradients of K (plus valinomycin). Alkaline-earth cations displayed the selectivity sequence VII (Mg > Ca > Sr > Ba) for cis-inhibition of 28 Mg uptake. Mg efflux was trans-inhibited by La and Gd, and Mg uptake was cis-inhibited by Mn. The sulfhydryl group reagents p- chloromercuribenzoic acid and p-chloromercuriphenylsulfonate stimulated Mg uptake and efflux. These results reveal an electrodiffusive pathway for Mg transport in trout renal MV.
Brush-border membrane vesicles (BBMV) enriched with alkaline phosphatase (8.1-fold) and gamma-glutamyl transpeptidase (11.5-fold) were prepared from the rainbow trout kidney. D-[3H]glucose uptake was stimulated by inward Na gradients but not by K, choline, Li, N-methyl-D-glucamine, or mannitol gradients. Na-dependent glucose uptake displayed overshoot in voltage-polarized vesicles (VPV; negative inside) but not in short-circuited vesicles (SCV). Recognition of carbons 2 and 3 of the glucopyranose ring was essential for glucose uptake. Phlorizin inhibited Na-dependent D-glucose uptake with an inhibition constant of 11.4 microM. The Michaelis-Menten constant of glucose was 0.58 mM in VPV and increased to 1.49 mM in SCV, whereas that for sodium was 193 mM in VPV and similar in SCV. Maximum velocity of Na was reduced in SCV. The Hill coefficient was 1 for both Na and glucose in VPV and SCV. Our studies indicate a single Na-D-glucose cotransporter that transports Na and glucose with a 1:1 stoichiometry and voltage-dependent kinetics. The transporter shares functional properties with both mammalian transporters SGLT1 and SGLT2.
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.
Isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti spontaneously secrete NaCl, KCl and water across an epithelium of modest transepithelial resistance (40-80 omega cm2) 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 omega cm2, and renders the basolateral and apical membrane voltages nearly equipotential (approximately -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 omega cm2. Bilateral reductions of the Cl concentration in tubule lumen and peritubular bath fully restore Rsh to 55.8 omega cm2 in 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.
That the kidneys of marine fish have powerful renal mechanisms for the excretion of magnesium (Mg) from the body has been known since the early 1930s, but it took another 40 years before the first renal Mg transport model was suggested by Natochin and Gusev. Since rates of net renal sodium (Na) reabsorption were closely correlated with rates of net renal Mg secretion in scorpion fish, Natochin and Gusev proposed tubular Na/Mg exchange transport. However, confirmation of Na/Mg exchange in other fish kidneys has been elusive. Detailed renal clearance studies in sea water rainbow trout have shown that bladder reabsorption of Na and water, the process which concentrates Mg in the bladder, accounts for much of Natochin's original observation. Nevertheless, studies of isolated perfused proximal tubules of the flounder and killifish do show inverse relationships between the concentrations of Na and Mg in the tubule lumen, consistent with Na/Mg exchange. Unfortunately, large paracellular Na permeabilities in renal proximal tubules do not clarify whether paracellular Na fluxes of Na/Mg exchange transport across the brush border membrane are responsible for generating inverse concentrations of Na and Mg in the tubule lumen. These uncertainties have led the authors to their present use of brush border membrane vesicles to look for evidence of Na/Mg exchange transport.
In the course of electrophysiological studies of Malpighian tubules of the mosquito Aedes aegypti, we have found unusual effects of 2,4-dinitrophenol (DNP) that offer new insights into the electrogenic and conductive properties of the tubule. DNP (10(-4)M) depolarized the basolateral membrane voltage from -58.0 to -3.3 mV, and it depolarized the apical membrane voltage from 110.6 to 8.9 mV. In parallel the transepithelial electrical resistance increased from 11.4 to 16.8 k omega.cm, and the fractional resistance of the apical membrane increased from 0.32 to 0.57. On the assumption that measures of transepithelial resistance in the presence of DNP approach the shunt resistance, the experimental results indicate the following characteristics for the equivalent circuit of the tubule: 1) a shunt resistance that is approximately one-half the transcellular resistance, 2) low and high electromotive forces, respectively, at the basolateral and apical membranes of principal cells, 3) an electrogenic pump at the apical membrane, and 4) a basolateral membrane voltage that is due mostly to the voltage developed by current flow across the basolateral membrane resistance.
A population of proximal tubules when isolated from the glomerular kidneys of seawater-adapted (SW) and freshwater-adapted (FW) killifish (Fundulus heteroclitus) spontaneously secrete fluid. Regardless of SW or FW adaptation, Na and Cl are the dominant electrolytes in secreted fluid. Mg concentrations in fluid secreted by both tubules are significantly greater than those in the peritubular bath, and Mg concentrations are inversely related to Na concentrations. Proximal tubules from either SW or FW fish exhibit low transepithelial voltage (-1 to -2 mV) and low transepithelial resistances (20-30 omega.cm2) typical of other vertebrate proximal tubules. Transepithelial diffusion potentials for Na, Cl, Mg, and SO4 suggest that the paracellular pathway is Na selective and impermeable to divalent ions. Consideration of transepithelial electrochemical potential differences for Na, Cl, Mg, and SO4 suggests active transport of Mg, SO4, and Cl in proximal tubules isolated from SW- and FW-adapted fish. The similarities in the functional properties of secretory proximal tubules isolated from SW- and FW-adapted killifish are striking and raise questions about the in vivo role of these tubules in the renal adaptations to seawater and freshwater.
The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.