Resolution of apical from basolateral membrane of shark rectal gland

1986 ◽  
Vol 251 (5) ◽  
pp. C721-C726 ◽  
Author(s):  
W. P. Dubinsky ◽  
L. B. Monti
Keyword(s):  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Apical Membrane ◽  
Specific Activity ◽  
Basolateral Membrane ◽  
Anion Transport ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Crude Homogenate ◽  
Shark Rectal Gland

Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane.

Cl- secretion by cultured shark rectal gland cells. II. Effects of forskolin on cellular electrophysiology

1991 ◽  
Vol 260 (4) ◽  
pp. C824-C831 ◽  
Author(s):  
W. M. Moran ◽  
J. D. Valentich
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Electrical Potential ◽  
Ringer Solution ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Electrophysiological Properties ◽  
Shark Rectal Gland ◽  
Membrane Electrical Potential ◽  
Cl Conductance

Employing microelectrode techniques we have assessed the cellular electrophysiological properties of shark rectal gland (SRG) cells in primary culture. In the absence of secretagogues a 10-fold reduction in the Cl- concentration of the apical superfusate shark Ringer solution had little effect on either apical membrane electrical potential difference (Va) or fractional resistance (fRa), indicating little, if any, apical membrane Cl- conductance. Superfusing the basolateral surface with high-K+ shark Ringer solution (K+ increased 10-fold) depolarized the basolateral membrane electrical potential difference (Vb) by 43 mV, indicating that this barrier is largely K+ conductive. In addition, basolateral Ba2+ (5 mM) depolarized Vb by 12 mV and reduced fRa from 0.92 to 0.58, results consistent with a K(+)-conductive basolateral membrane in unstimulated SRG cells. Basolateral forskolin (10(-6) M) depolarized Va by 25 mV and caused a dramatic reduction in fRa from 0.97 to approximately 0.10. Under these conditions, a 10-fold decrease in apical superfusate Cl- concentration depolarized Va by 37 mV, revealing an adenosine 3',5'-cyclic monophosphate-induced apical membrane Cl- conductance. The time course of the forskolin-induced changes in Va and Vb suggests that the basolateral membrane K+ conductance increased and maintained the driving force for apical Cl- exit, as in other Cl(-)-secreting epithelia. These electrophysiological properties compare favorably with those of the perfused SRG tubule and indicate that SRG primary cultures are a suitable model for Cl(-)-secreting epithelia.

Selective permeability barrier to urea in shark rectal gland

2005 ◽  
Vol 289 (1) ◽  
pp. F83-F89 ◽  
Author(s):  
Joshua D. Zeidel ◽  
John C. Mathai ◽  
John D. Campbell ◽  
Wily G. Ruiz ◽  
Gerard L. Apodaca ◽  
...  
Keyword(s):  
Activation Energy ◽  
Epithelial Cells ◽  
Water Flow ◽  
Water Permeability ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Water Flux ◽  
Rectal Gland ◽  
Basolateral Membranes ◽  
Urea Permeability

Elasmobranchs such as the dogfish shark Squalus acanthius achieve osmotic homeostasis by maintaining urea concentrations in the 300- to 400-mM range, thus offsetting to some degree ambient marine osmolalities of 900–1,000 mosmol/kgH2O. These creatures also maintain salt balance without losing urea by secreting a NaCl-rich (500 mM) and urea-poor (18 mM) fluid from the rectal gland that is isotonic with the plasma. The composition of the rectal gland fluid suggests that its epithelial cells are permeable to water and not to urea. Because previous work showed that lipid bilayers that permit water flux do not block flux of urea, we reasoned that the plasma membranes of rectal gland epithelial cells must either have aquaporin water channels or must have some selective barrier to urea flux. We therefore isolated apical and basolateral membranes from shark rectal glands and determined their permeabilities to water and urea. Apical membrane fractions were markedly enriched for Na-K-2Cl cotransporter, whereas basolateral membrane fractions were enriched for Na-K-ATPase. Basolateral membrane osmotic water permeability (Pf) averaged 4.3 ± 1.3 × 10−3 cm/s, whereas urea permeability averaged 4.2 ± 0.8 × 10−7 cm/s. The activation energy for water flow averaged 16.4 kcal/mol. Apical membrane Pf averaged 7.5 ± 1.6 × 10−4 cm/s, and urea permeability averaged 2.2 ± 0.4 × 10−7 cm/s, with an average activation energy for water flow of 18.6 kcal/mol. The relatively low water permeabilities and high activation energies argue strongly against water flux via aquaporins. Comparison of membrane water and urea permeabilities with those of artificial liposomes and other isolated biological membranes indicates that the basolateral membrane urea permeability is fivefold lower than would be anticipated for its water permeability. These results indicate that the rectal gland maintains a selective barrier to urea in its basolateral membranes.

Propionate induces cell swelling and K+ accumulation in shark rectal gland

1989 ◽  
Vol 257 (2) ◽  
pp. C377-C384 ◽  
Author(s):  
G. M. Feldman ◽  
F. N. Ziyadeh ◽  
J. W. Mills ◽  
G. W. Booz ◽  
A. Kleinzeller
Keyword(s):  
Atpase Activity ◽  
Propionic Acid ◽  
Exchange Process ◽  
Squalus Acanthias ◽  
Cell Swelling ◽  
Rectal Gland ◽  
Structural Elements ◽  
Acid Diffusion ◽  
Shark Rectal Gland ◽  
Solute Accumulation

Small organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter. The Na+-H+ exchange process tends to correct intracellular pH (pHi), and thus it maintains a favorable gradient for propionic acid diffusion and allows propionate to accumulate. Activation of the Na+-H+ antiport also facilitates Na+ entry into the cell and Nai accumulation. At the same time Na+-K+-ATPase activity, unaffected by propionate, replaces Nai with Ki, whereas the K+ leak rate, decreased by propionate, allows Ki to accumulate. As judged by 86Rb+ efflux, the reduction in K+ leak was not due to propionate-induced cell acidification or reduction in Cli concentration. Despite inducing cell swelling, propionate did not disrupt cell structural elements and F actin distribution along cell membranes.

Intracellular chloride activities in the isolated perfused shark rectal gland

1983 ◽  
Vol 245 (5) ◽  
pp. F640-F644
Author(s):  
M. J. Welsh ◽  
P. L. Smith ◽  
R. A. Frizzell
Keyword(s):  
Basolateral Membrane ◽  
Apical Cell ◽  
Electrical Potential ◽  
Electrochemical Potential ◽  
Potential Difference ◽  
Rectal Gland ◽  
Electrical Potential Difference ◽  
Apical Cell Membrane ◽  
Shark Rectal Gland ◽  
Electrochemical Equilibrium

The isolated, perfused shark rectal gland secretes Cl when stimulated with adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of secretion, we used Cl-selective and conventional (KCl-filled) microelectrodes to measure the intracellular Cl activity (aClc). Under nonsecreting conditions, the electrical potential difference across the basolateral membrane (psi b) was -78 m V and aClc was 57 mM, a value seven times greater than predicted for electrochemical equilibrium across the basolateral membrane. When theophylline and 8-bromo-cAMP were added to the perfusate, the transglandular electrical potential difference doubled and the rate of fluid secretion increased 20-fold; however, neither psi b nor aClc changed. During both nonsecreting and secreting conditions the intracellular accumulation of Cl results in an electrochemical potential difference favoring Cl exit across the apical cell membrane. The constancy of aClc despite the variation in secretion rate suggests that stimulation is associated with an equivalent enhancement of net Cl movement across both the apical and basolateral membranes. When stimulated glands were perfused with Na-free (choline) Ringer, secretion was abolished and aClc fell toward the value predicted for electrochemical equilibrium. These findings suggest that the "uphill" step in Cl secretion lies at the basolateral membrane, where cellular Cl accumulation probably involves secondary active transport; i.e., Cl entry is driven by an inwardly directed electrochemical potential difference for Na.

Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport

1977 ◽  
Vol 233 (4) ◽  
pp. F298-F306 ◽  
Author(s):  
P. Silva ◽  
J. Stoff ◽  
M. Field ◽  
L. Fine ◽  
J. N. Forrest ◽  
...  
Keyword(s):  
Chloride Transport ◽  
Sodium Concentration ◽  
Chloride Secretion ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Tentative Hypothesis ◽  
Shark Rectal Gland ◽  
Electrochemical Equilibrium

The isolated rectal gland of Squalus acanthias was stimulated to secrete chloride against an electrical and a chemical gradient when perfused in vitro by theophylline and/or dibutyryl cyclic AMP. Chloride secretion was depressed by ouabain which inhibits Na-K-ATPase. Thiocyanate and furosemide also inhibited chloride secretion but ethoxzolamide, a carbonic anhydrase inhibitor, did not. Chloride transport was highly dependent on sodium concentration in the perfusate. The intracellular concentration of chloride averaged 70-80 meq/liter in intact glands, exceeding the level expected at electrochemical equilibrium and suggesting active transport of chloride into the cell. These features suggest a tentative hypothesis for chloride secretion by the rectal gland in which the uphill transport of chloride into the cytoplasm is coupled through a membrane carrier to the downhill movement of sodium along its electrochemical gradient. The latter is maintained by the Na-K-ATPase pump while chloride is extruded into the duct by electrical forces.

scholarly journals AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland (Squalus acanthias)

2018 ◽  
Vol 314 (4) ◽  
pp. C473-C482
Author(s):  
Rugina I. Neuman ◽  
Juliette A. M. van Kalmthout ◽  
Daniel J. Pfau ◽  
Dhariyat M. Menendez ◽  
Lawrence H. Young ◽  
...  
Keyword(s):  
Feedback Inhibition ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Squalus Acanthias ◽  
Catalytic Subunit ◽  
Rectal Gland ◽  
Apical Side ◽  
Shark Rectal Gland ◽  
Amp Activated Protein Kinase

The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

Mode of activation of salt secretion by C-type natriuretic peptide in the shark rectal gland

1999 ◽  
Vol 277 (6) ◽  
pp. R1725-R1732 ◽  
Author(s):  
Patricio Silva ◽  
Richard J. Solomon ◽  
Franklin H. Epstein
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Squalus Acanthias ◽  
Rectal Gland ◽  
Stimulatory Action ◽  
Kinase C ◽  
Shark Rectal Gland ◽  
Pkc Activation ◽  
Stimulation Of

We studied the modes of activation of the salt-secreting rectal gland of the spiny dogfish, Squalus acanthias, by the native cardiac peptide CNP. The stimulatory action of CNP in isolated perfused glands is inhibited by 10 mM procaine, presumably by blocking release of vasoactive intestinal peptide (VIP) from nerves. Procaine reduces the slope of the dose-response curve of human CNP and that of shark CNP (each P < 0.0001). CNP increases short-circuit current in cultured rectal gland cells from 4.8 ± 1.6 to 27.0 ± 7.8 μA/cm2. It also stimulates the secretion of chloride in isolated perfused glands in the presence of 10 mM procaine from 72 ± 31 to 652 ± 173 μeq ⋅ h−1 ⋅ g−1. These results suggest that CNP has a direct cellular action not mediated by the neural release of VIP. The residual stimulation of perfused glands in the presence of procaine was almost completely inhibited by staurosporine [10 nM; an inhibitor of protein kinase C (PKC)] from 652 ± 173 to 237 ± 61 μeq ⋅ h−1 ⋅ g−1. Although CNP stimulates guanylyl cyclase in shark rectal gland, chloride secretion of perfused glands was not elicited by 8-bromoadenosine-cGMP (8-BrcGMP) alone nor by the activator of PKC phorbol ester. The combination of PKC activation and 8-BrcGMP infusion, however, stimulated chloride secretion in perfused glands from 94 ± 30 to 506 ± 61 μeq ⋅ h−1 ⋅ g−1, a level comparable to that observed in glands blocked with procaine. Several parallel pathways appear to be synergistic in activating chloride secretion stimulated by CNP in the rectal gland.

Missense mutations in Na+:HCO3− cotransporter NBC1 show abnormal trafficking in polarized kidney cells: a basis of proximal renal tubular acidosis

2005 ◽  
Vol 289 (1) ◽  
pp. F61-F71 ◽  
Author(s):  
Hong C. Li ◽  
Peter Szigligeti ◽  
Roger T. Worrell ◽  
Jeffrey B. Matthews ◽  
Laura Conforti ◽  
...  
Keyword(s):  
Renal Tubular Acidosis ◽  
Membrane Fraction ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Kidney Cells ◽  
Missense Mutations ◽  
Wild Type ◽  
Membrane Expression ◽  
Potential Recording ◽  
Renal Tubular

The kidney Na+:HCO3− cotransporter NBC1 is located exclusively on the basolateral membrane of kidney proximal tubule cells and is responsible for the reabsorption of majority of filtered bicarbonate. Two well-described missense mutations in NBC1, R510H and S427L, are associated with renal tubular acidosis (RTA). However, the exact relationship between these mutations and NBC1 dysregulation remains largely unknown. To address this question, cDNAs for wild-type kidney NBC1 and its mutants R510H and S427L were generated, fused in frame with NH2 terminally tagged GFP, and transiently expressed in Madin-Darby canine kidney cells. In parallel studies, oocytes were injected with the wild-type and mutant NBC1 cRNAs and studied for membrane expression and activity. In monolayer cells grown to polarity, the wild-type GFP-NBC1 was exclusively localized on the basolateral membrane domain. However, GFP-NBC1 mutant R510H was detected predominantly in the cytoplasm. GFP-NBC1 mutant S427L, on the other hand, was detected predominantly on the apical membrane with residual cytoplasmic retention and basolateral membrane labeling. In oocytes injected with the wild-type or mutant GFP-NBC1 cRNAs, Western blot analysis showed that wild-type NBC1 is predominantly localized in the membrane fraction, whereas NBC1-R510H mutant was predominantly expressed in the cytoplasm. NBC1-S427L mutant was mostly expressed in the membrane fraction. Functional analysis of NBC1 activity in oocytes by membrane potential recording demonstrated that compared with wild-type GFP-NBC1, the GFP-NBC1 mutants H510R and S427L exhibited significant reduction in activity. These findings suggest that the permanent isolated proximal RTA in patients with H510R or S427L mutation resulted from a combination of inactivation and mistargeting of kidney NBC1, with H510R mutant predominantly retained in the cytoplasm, whereas S427L mutant is mistargeted to the apical membrane.

scholarly journals The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells

1992 ◽  
Vol 283 (2) ◽  
pp. 553-560 ◽  
Author(s):  
J A Ellis ◽  
M R Jackman ◽  
J P Luzio
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Fraction ◽  
Apical Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Basolateral Membrane ◽  
Subcellular Fractionation ◽  
Integral Membrane Proteins ◽  
Basolateral Plasma Membrane

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5′-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.

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