Effects of pH on apical calcium entry and active calcium transport in rabbit cortical collecting system

1994 ◽  
Vol 266 (4) ◽  
pp. F620-F627 ◽  
Author(s):  
R. J. Bindels ◽  
A. Hartog ◽  
S. L. Abrahamse ◽  
C. H. Van Os
Keyword(s):  
Calcium Transport ◽  
Apical Membrane ◽  
Channel Blockers ◽  
Intracellular Acidification ◽  
Apical Side ◽  
Collecting Ducts ◽  
Effects Of Ph ◽  
Active Calcium ◽  
Permeable Supports ◽  
Collecting System

Rabbit connecting tubules and cortical collecting ducts were isolated by immunodissection and cultured on permeable supports. The monolayers actively transported Ca2+ with a net transcellular rate of 92 +/- 3 nmol.h-1.cm-2. Methoxyverapamil, felodipine, diltiazem, omega-conotoxin GVIA, and omega-agatoxin IVA when added to the apical side had no effect on Ca2+ absorption. Neither hyperpolarization nor depolarization of the apical membrane affected Ca2+ transport rates significantly. Stepwise lowering of the apical pH (pHa) from 8.0 to 5.6 gradually inhibited Ca2+ transport from 88 +/- 5 to 7 +/- 2 nmol.h-1.cm-2. Measuring the intracellular pH (pHi) with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein revealed that lowering the pHa from 8.0 to 5.6 decreased pHi from 7.8 to 6.7. To determine whether inhibition of Ca2+ absorption results from intracellular acidification, pHi was lowered using an NH4Cl pulse while extracellular pH was kept constant. Intracellular acidification from 7.4 +/- 0.2 to 6.9 +/- 0.1 reduced Ca2+ absorption by 26 +/- 6% only. In addition, lowering of the basolateral pH to 6.2 resulted in a pHi of 6.8 +/- 0.1, without affecting Ca2+ absorption rates. In conclusion, the basal Ca2+ influx mechanism in the apical membrane is most likely a voltage-independent Ca2+ transporter, insensitive to Ca2+ channel blockers, but strongly inhibited by apical acidification.

Na+ transport in isolated rat CCD: effects of bradykinin, ANP, clonidine, and hydrochlorothiazide

1991 ◽  
Vol 260 (1) ◽  
pp. F86-F95 ◽  
Author(s):  
A. J. Rouch ◽  
L. Chen ◽  
S. L. Troutman ◽  
J. A. Schafer
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Potential Difference ◽  
Na Channel ◽  
Bathing Solution ◽  
Channel Blockers ◽  
Na Transport ◽  
Luminal Membrane ◽  
Collecting Ducts ◽  
Basolateral Membrane Potential

We examined the effects of bradykinin (BK), atrial natriuretic peptide (ANP), hydrochlorothiazide (HCTZ), and clonidine on Na+ transport in isolated perfused cortical collecting ducts from rats treated with deoxycorticosterone. Arginine vasopressin was present in the bathing solution at 220 pM. Clonidine (1 microM, bathing solution) depolarized transepithelial potential difference (PDT) from -11.9 +/- 2.0 (SE) to -7.4 +/- 1.7 mV (P less than 0.001), hyperpolarized basolateral membrane potential difference (PDbl) from -85 +/- 1 to -87 +/- 1 mV (P less than 0.01), and increased the fractional resistance of the apical membrane (FRa) from 0.81 +/- 0.02 to 0.86 +/- 0.02 (P less than 0.03), indicating that it inhibited the Na+ conductance of the luminal membrane. BK (1 or 10 nM) or ANP (10 nM) in the bathing solution had no effect on PDT, PDbl, or FRa. BK, ANP, or 0.1 mM luminal HCTZ also had no effect on lumen-to-bath 22Na+ flux (J1----b), whereas we showed previously that clonidine inhibits J1----b by 30% (L. Chen, M. Paris, S. K. Williams, M. C. Reif, and J. A. Schafer. Kidney Int. 37: 366, 1990). Luminal addition of Na+ channel blockers amiloride (10 microM) or benzamil (1 microM) reduced J1----b to a level not significantly different from bath-to-lumen 22Na+ flux measured previously (M. Reif, S. L. Troutman, and J. A. Schafer. J. Clin. Invest. 77: 1291-1298, 1986), and neither BK nor HCTZ had any further effect. These results show that transcellular Na+ transport occurs exclusively through the apical membrane amiloride-sensitive channel, and this conductance is inhibited by clonidine but not by BK, ANP, or HCTZ.

scholarly journals Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions

2001 ◽  
Vol 280 (4) ◽  
pp. L646-L658 ◽  
Author(s):  
Lucky Jain ◽  
Xi-Juan Chen ◽  
Semra Ramosevac ◽  
Lou Ann Brown ◽  
Douglas C. Eaton
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Culture Conditions ◽  
Alveolar Type ◽  
Type Ii ◽  
Biophysical Properties ◽  
Air Interface ◽  
The Individual ◽  
Permeable Supports

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6 ± 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a P Na/ P K >80 that is inhibited by submicromolar concentrations of amiloride ( K 0.5 = 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (α, β, and γ) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits α-, β-, and γ-ENaC.

Active calcium transport in the skin of the frog Rana pipiens: kinetics and seasonal rhythms.

1995 ◽  
Vol 198 (4) ◽  
pp. 967-974
Author(s):  
D F Stiffler
Keyword(s):  
Calcium Transport ◽  
Rana Pipiens ◽  
Dorsal Skin ◽  
Electrochemical Gradient ◽  
Fish Gill ◽  
Seasonal Rhythms ◽  
Active Calcium ◽  
Net Flux ◽  
Ventral Skin ◽  
Freshwater Environments

The frog Rana pipiens takes up Ca2+ against an electrochemical gradient from dilute external solutions that are similar to natural freshwater environments. The influx is dependent upon external [Ca2+] and is saturable. Kinetic analysis yielded a Km of 0.625 mmol l-1 and a Jmax of 38 nmol cm-2h-1. These kinetic variables suggest that both the affinity and capacity are smaller than those for Na+ and Cl- transport in the skin of the same species. They are also smaller than those for Ca2+ transport in fish gill. A significant portion (20-25%) of the Ca2+ entering a frog remains in Ca(2+)-rich layers of the skin, with ventral skin containing about three times as much Ca2+ as dorsal skin. There are seasonal rhythms in Ca2+ exchange: although Ca2+ influx does not vary significantly over the year, efflux is minimal in July, while net flux, which is negative most of the year, appears to be positive in July. Since these fluxes do not include dietary calcium, one cannot conclude that feeding frogs are in negative Ca2+ balance.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

Forskolin-induced HCO3- current across apical membrane of the frog corneal epithelium

1990 ◽  
Vol 259 (2) ◽  
pp. C215-C223 ◽  
Author(s):  
O. A. Candia
Keyword(s):  
Corneal Epithelium ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Pump Current ◽  
Gas Composition ◽  
Apical Side ◽  
Disulfonic Stilbene ◽  
Stimulation Of

Forskolin (and other Cl- secretagogues) does not affect the very small Na(+)-originated short-circuit current (Isc) across frog corneal epithelium bathed in Cl- free solutions. However, forskolin in combination with increased PCO2 bubbling of the solutions (5-20% CO2) stimulated Isc proportionally to PCO2 to a maximum of approximately 8 microA/cm2. This current could be eliminated and reinstated by sequentially changing the gas composition of the bubbling to 100% air and 20% CO2-80% air. The same effects were observed when PCO2 changes were limited to the apical-side solution. Stroma-to-tear HCO3- movement was deemed unlikely, since the increase in Isc was observed with a HCO3(-)-free solution on the stromal side and CO2 gassing limited to the tear side. From the effects of ouabain and tryptamine, at least 80% of the Isc across the basolateral membrane can be accounted for by the Na+ pump current plus K+ movement from cell to bath. Methazolamide also inhibited Isc. Current across the apical membrane cannot be attributed to an electronegative Na(+)-HCO3- symport given the insensitivity of Isc to a disulfonic stilbene and the fact that stroma-to-tear Na+ fluxes did not increase on stimulation of Isc. The tear-to-stroma Na+ flux also remained unaltered, negating an increased apical bath-to-cell Na+ flow. The forskolin-20% CO2 manipulation produced a depolarization of the intracellular potential, a reduction in the apical-to-basolateral resistance ratio, and a decrease in transepithelial resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

A Novel Pharmacological Probe Links the Amiloride-Insensitive NaCl, KCl, and NH4Cl Chorda Tympani Taste Responses

2001 ◽  
Vol 86 (5) ◽  
pp. 2638-2641 ◽  
Author(s):  
John A. DeSimone ◽  
Vijay Lyall ◽  
Gerard L. Heck ◽  
Tam-Hao T. Phan ◽  
Rammy I. Alam ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Apical Membrane ◽  
Cetylpyridinium Chloride ◽  
Taste Receptor ◽  
Chorda Tympani ◽  
Intracellular Acidification ◽  
Cellular Origin ◽  
Receptor Cells ◽  
Nacl Concentration

Chorda tympani taste nerve responses to NaCl can be dissected pharmacologically into amiloride-sensitive and -insensitive components. It is now established that the amiloride-sensitive, epithelial sodium channel acts as a sodium-specific ion detector in taste receptor cells (TRCs). Much less is known regarding the cellular origin of the amiloride-insensitive component, but its anion dependence indicates an important role for paracellular shunts in the determination of its magnitude. However, this has not precluded the possibility that undetected apical membrane ion pathways in TRCs may also contribute to its origin. Progress toward making such a determination has suffered from lack of a pharmacological probe for an apical amiloride-insensitive taste pathway. We present data here showing that, depending on the concentration used, cetylpyridinium chloride (CPC) can either enhance or inhibit the amiloride-insensitive response to NaCl. The CPC concentration giving maximal enhancement was 250 μM. At 2 mM, CPC inhibited the entire amiloride-insensitive part of the NaCl response. The NaCl response is, therefore, composed entirely of amiloride- and CPC-sensitive components. The magnitude of the maximally enhanced CPC-sensitive component varied with the NaCl concentration and was half-maximal at [NaCl] = 62 ± 11 (SE) mM. This was significantly less than the corresponding parameter for the amiloride-sensitive component (268 ± 71 mM). CPC had similiar effects on KCl and NH4Cl responses except that in these cases, after inhibition with 2 mM CPC, a significant CPC-insensitive response remained. CPC (2 mM) inhibited intracellular acidification of TRCs due to apically presented NH4Cl, suggesting that CPC acts on an apical membrane nonselective cation pathway.

Model explaining the relation between distal nephron Li+ reabsorption and urinary Na+ excretion in rats

1998 ◽  
Vol 274 (3) ◽  
pp. F445-F452 ◽  
Author(s):  
Michael Shalmi ◽  
Thomas Jonassen ◽  
Klaus Thomsen ◽  
Jonathan D. Kibble ◽  
Peter Bie ◽  
...  
Keyword(s):  
Urinary Excretion ◽  
Apical Membrane ◽  
Fractional Excretion ◽  
Distal Nephron ◽  
Conscious Rats ◽  
Angiotensin Iii ◽  
Collecting Ducts ◽  
Induced Changes ◽  
Different Levels

Li+ may be reabsorbed via an amiloride-sensitive mechanism in the collecting ducts of rats administered a low-Na+ diet. This was investigated by measuring the increase in fractional urinary excretion of Li+(FELi) in response to amiloride in conscious rats at two different levels of plasma Li+ concentration and after administration of bendroflumethiazide (BFTZ), angiotensin III (ANG III), and aldosterone (Aldo). The results confirmed that amiloride increased (FELi) in rats on a low-Na+ diet (20 ± 1 to 35 ± 1%, means ± SE), whereas no increase was observed in rats on a normal Na+ diet (37 ± 1 to 38 ± 1%). The lithiuretic effect of amiloride was 1) abolished by preadministration of BFTZ (32 ± 1 to 33 ± 2%) to Na+-deprived rats and 2) increased by ANG III (27 ± 3 to 33 ± 2%) and Aldo (25 ± 2 to 37 ± 2%) in Na+-replete rats. Amiloride-induced changes in FELiwere independent of plasma Li+concentration but inversely related to the fractional excretion of Na+ and the amiloride-sensitive excretion of K+. These results are compatible with the hypothesis that a low tubular Na+ concentration reduces end-tubular Na+ reabsorption and results in hyperpolarization of the apical membrane, thus favoring Li+ uptake into the cells.

Influence of ATP on sarcoplasmic reticulum function of vascular smooth muscle

1982 ◽  
Vol 242 (3) ◽  
pp. C242-C249 ◽  
Author(s):  
G. D. Ford ◽  
M. L. Hess
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Uptake Activity ◽  
Active Calcium ◽  
Active Preparation

A vesicular fraction isolated from bovine aorta and enriched in fragmented sarcoplasmic reticulum (FSR) exhibited active calcium transport and ATPase activity. By use of a hypotonic NaHCO3 extraction solution, an active preparation was isolated that retained activity for up to 4 days. A small but significant (P less than 0.05) Ca2+-stimulated, Mg2+-dependent ATPase associated with calcium transport was demonstrated with a specific activity of 0.33 mumol inorganic phosphate (Pi).mg-1.min-1. The basal Mg2+ ATPase demonstrated Michaelis-Menten kinetics [Km(Mg2+-ATP) = 0.44 +/- 0.01 X 10(-3) M; Vmax = 2.22 +/- 0.01 mumolPi.mg-1.min-1]. The Ca2+-stimulated, Mg2+-ATPase demonstrated apparent substrate inhibition (Ks approximately 10 mM) with no evidence for end-product (ADP) or excess added Ca2+ contributing to this inhibition. Oxalate-supported active calcium uptake velocities also exhibited quantitatively similar substrate inhibition. These results suggest that FSR from vascular smooth muscle contains either two enzymes or one enzyme with two isomeric forms, one of which is associated with the calcium uptake activity of this structure and the other of unknown function.

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