Permeable junctional complexes. The movement of lanthanum across rabbit gallbladder and intestine.

Ionic lanthanum has been used to study transepithelial ion permeation in in vitro rabbit gallbladder and intestine (ileum) by adding 1 mM La(3+) to only the mucosal bathing solution. Transepithelial fluid transport electrical potential differences (p.d.), and resistances were measured. During La(3+) treatment the gallbladder's rate of active solute-coupled fluid transport remained constant, the resistance increased, and the 2:1 NaCl diffusion p.d. decreased. Mucosa-to-serosa fluxes of (140)La(3+) were measured and indicate a finite permeability of the gallbladder to La(3+). La(3+) also increased the transepithelial resistance and p d. of ileum. Electron microscopic examination of La(3+)-treated gallbladder showed: (a) good preservation of the fine structure, (b) electron-opaque lanthanum precipitates in almost every lateral intercellular space, most frequently near the apical end of the lateral spaces close to or within the junctional complex, (c) lanthanum among the subjacent muscle and connective tissue layers, and (d) lanthanum filling almost the entire length of so-called "tight" junctions. No observations were made which unequivocally showed the penetration of lanthanum into the gallbladder cells. Electron micrographs of similar La(3+)-treated ilea showed lanthanum deposits penetrating the junctional complexes. These results coupled with other physiological studies indicate that the low resistance pathway for transepithelial ion permeation in gallbladder and ileum is through the tight junctions A division of salt-transporting epithelia into two main groups, those with "leaky" junctional complexes and those with tight junctional complexes, has been proposed.

Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells.

The time course for development of polarized function and morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH-sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na-dependent, amiloride-sensitive component of pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE-1 isoform of the Na/H exchanger was used to determine its morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti-ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in the cell membranes of the bullfrog gastric mucosa with Acid secretion.

The effective area, resistance, and configuration of the apical and basolateral cell membranes of the bullfrog gastric mucosa were studied as a function of acid secretion rate, by alternating-current impedance methods. The drop in transepithelial resistance with acid secretion is attributed to the great increase in apical membrane area (hence conductance) associated with tubulovesicles. There is no evidence of a change in basolateral membrane resistance or of apical membrane premeability per unit area.

Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.

We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, ISC, in microA/cm2) and apical (ap) Cl permeability (PCl) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. PCl and PK were measured by permeabilizing the bl or ap membrane with nystatin. PCl was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 35SO4-labeled glycosaminoglycan [GAG], a fluid-phase marker of Golgi-derived vesicles) was used to measure exocytosis at both ap and bl membranes. At rest, ISC = 3.6, PK = 0.8 x 10(-6), PCl = 2.1 x 10(-6) with SPQ and 2.4 x 10(-6) electrically, and there was constitutive GAG secretion (i.e., exocytosis) to both ap and bl sides (bl > 2 x ap). Carbachol (C) increased: ISC (delta = 18.6), PK (6.5x), PCl (1.8-2.9x), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (delta = 29), PCl (5.5-11x) and ap exocytosis (1.5-2x), but had no effect on PK or bl exocytosis. Synergistic effects on ISC occurred when C was added to F-treated cells but not vice versa, even though the characteristic effects of F+C on PCl, PK, and/or GAG secretion were identical to those exhibited when stimulants were added individually. Cl secretion results from coordinated activation of channels at ap and bl membranes, and exocytosis may play a role in these events.

Effects of extracellular pH on intracellular pH-regulation and growth in a human colon carcinoma cell-line.

Mechanisms of intracellular pH (pHi) regulation seem to be involved in cellular growth and cell division. Little is known about how extracellular acidosis, known to occur in central regions of solid tumors, or alkaline conditions affect pHi regulation in colonic tumors. pHi changes in the colonic adenocarcinoma cell-line SW-620 were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF, and proliferative activity was assessed by [3H]thymidine uptake. Resting pHi in Hepes-buffered solution was 7.53 +/- 0.01 (n = 36). Both 1 mM amiloride and Na(+)-free solution inhibited pHi recovery from acidification and decreased pHi in resting cells. In HCO3-/CO2-buffered media resting pH1 was 7.42 +/- 0.01 (n = 36). Recovery from acidification was Na(+)-dependent, CI(-)-independent, and only partially blocked by 1 mM amiloride. In the presence of amiloride and 200 microM H2DIDS pHi recovery was completely inhibited. In Na(+)-free solution pHi decreased from 7.44 +/- 0.04 to 7.29 +/- 0.03 (n = 6) and no alkalinization was observed in CI(-)-free medium. Addition of 5 microM tributyltin bromide (an anion/OH-exchange ionophore) caused pHi to decrease from 7.43 +/- 0.05 to 7.17 +/- 0.08 (n = 5). The effects of pH0 on steady-state pHi, pHi recovery from acidification and proliferative activity after 48 h were investigated by changing buffer [CO2] and [HCO3-]. In general, increases in pH0 between 6.7 and 7.4 increased pHi recovery, steady-state pHi and growth rates. In summary, SW-620 cells have a resting pHi > 7.4 at 25 degrees C, which is higher than other intestinal cells. Acid extrusion in physiological bicarbonate media is accomplished by a pHi-sensitive Na+/H+ exchanger and a pHi-insensitive Na(+)-HCO3-cotransporter, both of which are operational in control cells at the resting pHi. No evidence for activity of a CI-/HCO3- exchanger was found in these cells, which could account for the high pHi observed and may explain why the cells continue to grow in acidic tumor environments.

CFTR displays voltage dependence and two gating modes during stimulation.

The patch-clamp technique in conjunction with current noise analysis was employed to clarify the events underlying the regulation of the CFTR (cystic fibrosis transmembrane conductance regulator) during cAMP-dependent stimulation. 3T3 fibroblast cells expressing the CFTR were stimulated in cell-attached mode with forskolin. The number (N) of activated channels per patch ranged from 1 to approximately 100. In true single-channel recordings, CFTR's gating was best described by two open states (approximately 5 and approximately 100 ms) and three closed states (< or = 5, approximately 100, and approximately 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and approximately 50 Hz, respectively. Single-channel time constants were dependent on voltage. The fastest closed state increased its contribution from 48% at +100 mV to 87% at -100 mV, and the medium open state reduced its length to one half, resulting in gating dominated by fast events. Similarly, the fast Lorentzian increased its amplitude, and its corner frequency increased from 44 Hz at +100 mV to 91 Hz at -100 mV, while the slow Lorentzian was voltage independent. In multi-channel recordings N.Po (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased Po of CFTR to approximately 0.5, which resulted from a decrease of the longest closed state while the faster open and closed states were unaffected. Neither corner frequency was affected during stimulation. Recordings from multichannel patches revealed in addition, unique, very long channel openings (high Po mode, average 13 s). Channels exhibiting high Po (i.e., Po approximately 1.0) or low Po (i.e., Po approximately 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high Po mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 microM) favored transition into the high Po mode, suggesting a cellularly mediated regulation of model switching due to a fundamental change in configuration of the CFTR. Thus, during stimulation the CFTR increased its activity through two distinct effects: the reduction of the long closed state and modal switching to the high Po mode.

Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. Measurements in the open-circuit state.

A modified version of a capacitance probe technique has been used to measure fluid transport across the isolated retinal pigment epithelium (RPE)-choroid of the bullfrog. The accuracy of this measurement is 0.5-1.0 nl/min. Experiments carried out in the absence of external osmotic or hydrostatic gradients show that the RPE-choroid transports fluid from the retinal to the choroid side of the tissue at a rate of approximately 10 nl/min (4-6 microliters/cm2 X h). Net fluid absorption (Jv) was abolished within 10 min by the mitochondrial uncoupler 2,4-dinitrophenol. It was also inhibited (70%) by the removal of bicarbonate from the bulk solutions bathing the tissue. Ouabain caused a slow decrease in Jv (no effect at 10 min, 70% at 3 h), which indicates that RPE fluid transport is not directly coupled to the activity of the Na-K pump located at the apical membrane of this epithelium. In contrast to ouabain, cyclic AMP (cAMP) produced a quick decrease in Jv (84% within 5 min). Radioisotope experiments in the open circuit show that cAMP stimulated secretory fluxes of Na and Cl, which accounted for the observed cAMP-induced decrease in Jv. The direction of net fluid absorption, the magnitudes of the net ionic fluxes in the open circuit, and the dependence of Jv on external bicarbonate concentration strongly suggest that fluid absorption is generated primarily by the active absorption of bicarbonate.

Organelle pH studies using targeted avidin and fluorescein-biotin.

BACKGROUND: Mammalian organelles of the secretory pathway are of differing pH. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still ( approximately pH 5). The mechanisms that regulate pH in these organelles are still unknown. RESULTS: Using a novel method, we tested whether differences in H(+) 'leak' and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. A pH-sensitive, membrane-permeable fluorescein-biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. In live, intact cells, ER pH (pH(ER)) was 7.2 +/- 0.2 and Golgi pH (pH(G)) was 6.4 +/- 0.3 and was dissipated by bafilomycin. Buffer capacities of the cytosol, ER and Golgi were all similar (6-10 mM/pH). ER membranes had an apparent H(+) permeability three times greater than that of Golgi membranes. Removal of either K(+) or Cl(-) did not affect ER and Golgi H(+) leak rates, or steady-state pH(G) and pH(ER). CONCLUSIONS: The Golgi is more acidic than the ER because it has an active H(+) pump and fewer or smaller H(+) leaks. Neither buffer capacity nor counterion permeabilities were key determinants of pH(G), pH(ER) or ER/Golgi H(+) leak rates.

Does nitric oxide regulate capacitative Ca influx in HEK 293 cells?

It has been proposed that capacitative Ca influx into both pancreatic acinar cells and HT-29 colonic cells is regulated by stimulation of nitric oxide synthase (NOS). NO, in turn, controls cGMP levels through effects on guanylate cyclase. We tested this possibility by measuring Ca (and Ba) entry into human embryonic kidney 293 cells and into 293 cells that had been transfected with the neuronal NOS gene (293/NOS). 293 cells had undetectable levels of NOS, while 293/NOS cells exhibited very high levels [Bredt D.S., Ferris C.D., Snyder S.H. Nitric oxide synthase regulatory sites. J Biol Chem 1992; 267: 10976-10981]. Ca (or Ba) entry into single cells was measured as the rate of increase of the Fura-2 fluorescence ratio (digital imaging microscopy) during rapid changes from Ca-free (or Ba-free) to Ca- (or Ba-) containing solutions (using high K to depolarize the membrane potential). cGMP levels (EIA method) were measured to correlate to rates of Ca entry. 100 microM ATP caused release of Ca from internal stores, but no sustained plateau due to Ca entry in either 293 or 293/NOS cells. Cyclopiazonic acid (CPA, which inhibits the Ca pump of the internal store, allowing Ca to leak from the store) caused apparent Ca entry to increase 5-10-fold from similar, low levels in both 293 and 293/NOS cells. CPA-stimulated Ca entry was unaffected by the NOS inhibitor N-nitro-L-arginine (L-NA) in either 293 or 293/NOS cells. In 293 cells [cGMP] was low; ATP and CPA both increased [cGMP] by 2-fold, and the guanylate cyclase inhibitor LY83583 and L-NA decreased [cGMP] by 50-75%. [cGMP] was 20-fold higher in 293/NOS cells than in 293 cells; these [cGMP] were not affected by ATP and CPA, but were effectively decreased by 80-90% by L-NA and by LY83583. Thus, [cGMP] and Ca or Ba entry showed no relationship to each other: Ca entry was small into cells in which [cGMP] was either low (resting 293, CPA + L-NA or CPA + LY83583), intermediate (ATP-treated 293) or high (resting 293/NOS). Similarly, Ca entry was high into cells in which [cGMP] was low (CPA + L-NA- or CPA + LY83583-treated 293), intermediate (CPA-treated 293 and CPA + L-NA-treated 293/NOS) or high (CPA- or ATP-treated 293/NOS). We conclude that, as in most other non-excitable cells, Ca entry into 293 cells is stimulated by loss of Ca from the store but, unlike pancreatic and colonic cells, this capacitative Ca entry does not appear to be regulated by NO and cGMP. Therefore, although capacitative entry across the plasma membrane may be regulated by NO and cGMP in Gl epithelial cells, this regulation does not occur in all cells.

Possible regulation of capacitative Ca2+ entry into colonic epithelial cells by NO and cGMP.

A possible role of the nitric oxide (NO)/cGMP pathway in the regulation of Ca2+ entry into HT29/B6 human colonic epithelial cells was investigated using digital image processing of Fura-2 fluorescence and immunoblotting for nitric oxide synthase (NOS). We tested the hypothesis that Ca2+ store depletion causes increased NOS activity and [NO], which is stimulatory to Ca2+ entry by increasing guanylate cyclase (GC) and [cGMP]. Cells were incubated in 95 mM K(+)-containing solutions to depolarize the cell membrane potential and thereby exclude effects of NO and CGMP on K+ or Cl- channels, which might affect Ca2+ entry. Sodium nitroprusside (SNP, 0.5 microM and 30 microM), a NO donor, only slightly raised intracellular [Ca2+] ([Ca2+]i) in resting cells, but in 100 microM carbachol-stimulated cells the sustained, elevated Ca2+ plateau (reflecting Ca2+ entry) as well as Ba2+ entry were increased by 0.5 microM SNP, while 5, 10 or 30 microM SNP either had no effect or were inhibitory. Pretreatment of cells with the NOS inhibitor N-nitro-L-arginine (1 mM) reduced carbachol-stimulated Ca2+ entry, and simultaneous treatment with 0.5 microM (but not 30 microM) SNP restored Ca2+ influx. 8-Br-cGMP (1 mM) had little effect on [Ca2+]i or on rates of Ca2+ or Ba2+ influx into resting cells, but there were large effects on cells in which capacitative Ca2+ entry was activated by carbachol or cyclopiazonic acid (10 microM). The GC inhibitor LY83583 (10 microM) reduced carbachol-stimulated Ca2+ entry, and this entry was restored with 8-Br-cGMP. Western blotting revealed that endothelial-type NOS was present in the particulate fraction of cells. The data are consistent with the notion that Ca2+ entry into HT29/B6 cells is regulated by endothelial NOS/NO and GC/cGMP, but effects are most pronounced in store-depleted cells. Thus, NO and cGMP appear to potentiate the action of messengers released from the store during the emptying process, but NO and cGMP have only small effects of their own to open the Ca2+ channel in the plasma membrane. High [SNP] appeared to be inhibitory while low [SNP] was stimulatory, indicating that a precise range of [NO] may be required for effective stimulation of Ca2+ entry.

Cystic fibrosis transmembrane conductance regulator and H+ permeability in regulation of Golgi pH.

This paper reviews experiments from this lab that have tested the hypothesis that pH of the Golgi (pH(G)) of cystic fibrosis (CF) airway epithelial cells is alkaline compared to normal, that this altered pH affects sialyltransferase and other Golgi enzymes controlling biochemical composition of the plasma membrane and that altered surface biochemistry increases bacterial binding. We generated a plasmid encoding a modified green fluorescence protein-sialyltransferase (GFP-ST) chimera protein that was pH-sensitive and localized to the Golgi when transfected into HeLa cells and also CF and normal or cystic fibrosis transmembrane conductance regulator- (CFTR)-corrected airway epithelial cells. Digital imaging microscopy of these Golgi-localized probes showed that there was no correlation between pH(G) (6.4-7.0) and the presence of CFTR, whether cells were in HCO(3)(-)/CO(2)-containing or in HCO(3)(-)/CO(2)-free solutions. Activation of CFTR by raising cell [cAMP] had no effect on pH(G). Thus, CFTR seemed not to be involved in controlling pH(G). Experiments on HeLa cells using an avidin-sialyltransferase chimera in combination with a pH-sensitive fluorescent biotin indicated that even in cells that do not express CFTR, Cl(-) and K(+) conductances of the Golgi and other organelle membranes were large and that pH(G) was controlled solely by the H(+) v-ATPase countered by a H(+) leak. A mathematical model was applied to these and other published data to calculate passive H(+) permeability (P(H+)) of the Golgi, endoplasmic reticulum, trans-Golgi network, recycling endosomes and secrety granules from a variety of cells. An organelle's acidity was inversely correlated to its calculated P(H+). We conclude that the CFTR plays a minor role in organelle pH regulation because other (Cl(-) and K(+)) channels are present in sufficient numbers to shunt voltages generated during H(+) pumping. Acidity of the Golgi (and perhaps other organelles) appears to be determined by the activity of H(+) pumps countered by H(+) leaks.

Biosynthesis and secretion of pituitary hormones: dynamics and regulation.

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to

Regulation of intracellular pH in the stomach.

The following picture emerges from the relatively small literature concerned with pHi regulation in the stomach. Oxyntic cells have a H,K-ATPase at the luminal membrane and both Na+-H+ and Cl(-)-HCO3 exchangers at the serosal membrane. The intrinsic buffer capacity is 40-50 mM/pH. In the resting state, when the H, K-ATPase is inactive, pHi is 7.1. The Na+-H+ exchanger prevents acidic shifts of pHi, and the Cl(-)-HCO3 exchanger prevents alkaline shifts. The combined operation of the two appears to contribute directly to the net Cl- secretion (and short-circuit current) generated by resting OC. In the stimulated state, H+ secretion into the gland lumen via the H,K-ATPase increases, and Cl- movement across the serosal membrane via exchange for HCO3 doubles or triples; however, pHi remains roughly constant (delta pHi less than or equal to 0.1 units). The large increase in Cl- and HCO3 movement across the serosal membrane requires one (or all) of the following events: The anion exchanger changes its characteristics (Km or Vmax); [Cl-]i decreases; or other membrane mechanisms must be activated. Chief cells also have a pHi of 7.1 and a Bi of 40-50 mM/pH. They exhibit Na+-dependent and Cl(-)-dependent changes of pHi that are consistent with the presence of both Na+-H+ and Cl(-)-HCO3 exchangers. The Na+-dependent changes of pHi are larger and the Cl(-)-dependent changes are smaller in CC than in OC. Surface cells exhibit changes of AO fluorescence that indicate that there is a Na+-H+ exchanger and a HCO3 conductance. The H+ permeability of the luminal membranes of all cells is very low, while that of the serosal membrane (via the cation and anion exchangers) is high.

Intracellular pH dependence of buffer capacity and anion exchange in the parietal cell.

When parietal cells (PC) are stimulated with histamine, the anion exchanger rate increases three to five times to compensate for alkaline loading induced by H+-K+ adenosinetriphosphatase (ATPase) and to provide Cl for acid secretion. It has been hypothesized that this increased activity is caused by the increase in intracellular pH (pHi) that often occurs in stimulated PC (from 7.1 to a maximum of 7.3). The dependence of the anion exchanger on pHi was studied using microspectrofluorimetry of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions were used because the anion exchanger can transport OH- (or HCO3) in exchange for Cl- even with [HCO3]o = 200 microM. It was found that when solutions were changed either from NaCl to Cl- free or Cl- free to NaCl, rates of change of pHi (delta pH/delta t) were strongly dependent on pHi: nearly 0 at pHi 6.6 and 1.25 pH/min at pHi 8.0. To convert these pHi changes into anion flux rates, the intrinsic buffer capacity (beta i) was determined over the same pHi range by making small changes of [NH4]o to determine the resulting changes of [NH4]i and pHi (i.e., beta i = delta[NH4]i/delta pHi) in PC that had been pretreated with 1 mM amiloride and 200 microM [H2]4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) [to block Na+-H+ and Cl- -OH-(HCO3-) exchange]. beta i was also strongly dependent on pHi: at pHi 6.5 beta i = 48 mM/pH, and this decreased as pHi increased; at pHi 7.75 beta i = 8 mM/pH. The derived anion fluxes (i.e., JOH = beta i x delta pH/delta t) were roughly linearly related to pHi between 6.6 (JOH near 0) and 8.1 (JOH = 13 mM/min). Between pHi 7.1 and 7.3, the range normally observed during stimulation of PC, rates of anion exchange increased by 75%. This pHi sensitivity cannot explain the 300-500% increase in anion exchanger activity observed during secretagogue stimulation of PC.

Intracellular pH regulation in basal corneal epithelial cells measured in corneal explants: characterization of Na/H exchange.

Intracellular pH (pHi) was measured in basal corneal epithelial cells from fresh corneal explants using the pH sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The overlying superficial and wing cells were removed by mechanical scraping to expose basal cells attached to their basal lamina. Tissue pieces with attached, dye-loaded basal cells were mounted in a microscope-stage-perfusion chamber which allowed rapid changes of Ringer's bathing solutions while measuring BCECF fluorescence. In NaCl-Ringer's (bicarbonate free). pHo 7.40, resting cell pHi was 7.34 +/- 0.03 (+/- S.E.M., n = 31). Buffering capacity measured by NH4Cl treatment was 31 mM pH at pHi 7.34 and increased with decreasing pHi. Recovery from 20 mM NH4Cl-induced acid loads was dependent on the presence of Na and inhibited by 1 mM amiloride. Adding amiloride to resting cells caused a slow, reversible acidification (0.04 pH units min-1). These results indicate the presence of Na:H exchange, its role in responding to acid loads and in maintaining resting cell pHi. Activation of Na:H by Nao showed simple saturation kinetics, with Km = 44 mM. Net proton efflux via Na:H exchange increased with decreasing pHi and was enhanced by depleting cells of Nai, suggesting roles for both pHi and Nai in control of Na:H activation.

pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport.

Regulation of intracellular pH (pHi) was studied in Fu5, a rat hepatoma cell line that maintains a variety of differentiated functions. Microspectrofluorimetry of the pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was used to measure pHi in 10-15 cells growing on cover glasses that were mounted in a flow-through chamber on the stage of a microscope. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, pHi was 7.14, and intrinsic buffer capacity was inversely related to pHi. Amiloride (0.1 mM) caused pHi to decrease by 0.33 pH units in 4 min. Recovery from an acid load (using either NH4 prepulse technique or Na-free solutions) was completely blocked by amiloride. In HCO3-CO2-buffered solutions, pHi was 7.15, and buffer capacity was relatively insensitive to pHi between pHi of 6.6 and 7.2. Amiloride caused pHi to decrease by only 0.09 units. Recovery from an acid load was Na dependent, occurred in Cl-free solutions, and was totally blocked by the combination of amiloride plus 0.5 mM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS); recovery occurred when either amiloride or H2DIDS was removed. Removal of external Cl caused a rapid, H2DIDS-blockable alkalinization that was faster in HCO3-CO2 than in HEPES. The apparent Km for Clout for relaxation of Cl-free alkalinization was 4.5 mM. Rate of HCO3 transport during Cl-free treatment increased at alkaline resting pHi. It is concluded that Fu5 cells have two Na-dependent base-loading mechanisms and an acid-loading Cl-HCO3 exchanger. In solutions containing HCO3-CO2, the Na-H exchanger accounts for approximately 40% of recovery from an acid load, and a Na-HCO3 cotransporter accounts for the remainder. Recovery from an alkaline load appears to occur through the activity of the Cl-HCO3 exchanger.

Release and reloading of intracellular Ca stores after cholinergic stimulation of the parietal cell.

Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of intact rabbit gastric glands loaded with the Ca-sensitive fluorescent dye, fura-2. Cells were repeatedly stimulated with the cholinergic agonist carbachol to gain insights into the membrane mechanisms involved in hormonally stimulated Ca metabolism. In either Ca-containing or Ca-free solutions, carbachol (100 microM) caused a rapid (within 30 s) elevation of Cai from a resting level of 100 nM to greater than 600 nM. After the spike, Cai decreased within 3 min to a lower level that was somewhat elevated (greater than 200 nM) over base line. This plateau was dependent on both carbachol and extracellular Ca (Cao) and could be blocked by the addition of atropine (1 microM) or lanthanum ion (La, 50 microM). The spike is due to the release of Ca from internal stores, whereas the plateau is due to Ca entry across the plasma membrane through agonist-controlled, La-inhibitable channels. After a carbachol stimulation of 3 min or longer, reloading of the internal store was absolutely dependent on Cao. Under these conditions, reloading occurred through a La-sensitive (but nifedipine- and verapamil-insensitive) pathway in the plasma membrane. No significant change in Cai was detectable during the reloading. In contrast to the longer treatments, if carbachol stimulation was terminated with atropine while Cai was still elevated, significant reloading occurred from the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)