Biomedical subjects

W F Boron

Publications and source records attributed to W F Boron.

At least 55 records · Page 3Linked to original sources

PubMed1995

An apical permeability barrier to NH3/NH4+ in isolated, perfused colonic crypts.

Fermentation of nonabsorbed nutrients in the colon generates high concentrations of NH3/NH4+ in the colonic lumen. NH3 is a small, lipophilic neutral weak base that readily permeates almost all cell membranes, whereas its conjugate weak acid NH4+ generally crosses membranes much more slowly. It is not known how colonocytes maintain intracellular pH in the unusual acid-base environment of the colon, where permeant acid-base products of fermentation exist in high concentration. To address this issue, we hand dissected and perfused single, isolated crypts from rabbit proximal colon, adapting techniques from renal-tubule microperfusion. Crypt perfusion permits control of solutions at the apical (luminal) and basolateral (serosal) surfaces of crypt cells. We assessed apical- vs. basolateral-membrane transport of NH3/NH4+ by using fluorescent dyes and digital imaging to monitor intracellular pH of microvacuolated crypt cells as well as luminal pH. We found that, although the basolateral membranes have normal NH3/NH4+ permeability properties, there is no evidence for transport of either NH3 or NH4+ across the apical borders of these crypt cells. Disaggregating luminal mucus did not increase the transport of NH3/NH4+ across the apical border. We conclude that, compared to the basolateral membrane, the apical border of crypt colonocytes has a very low permeability-area product for NH3/NH4+. This barrier may represent an important adaptation for the survival of crypt cells in the environment of the colon.

Absorption↗

PubMed1995

Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter.

Active ion-coupled glutamate transport is of critical importance for excitatory synaptic transmission, normal cellular function, and epithelial amino acid metabolism. We previously reported the cloning of the rabbit intestinal high affinity glutamate transporter EAAC1 (Kanai, Y., and Hediger, M. A. (1992) Nature 360, 467-471), which is expressed in numerous tissues including intestine, kidney, liver, heart, and brain. Here, we report a detailed stoichiometric and kinetic analysis of EAAC1 expressed in Xenopus laevis oocytes. Uptake studies of 22Na+ and [14C]glutamate, in combination with measurements of intracellular pH with pH microelectrodes gave a glutamate to charge ratio of 1:1, a glutamate to Na+ ratio of 1:2, and a OH-/H+ to charge ratio of 1:1. Since transport is K+ dependent it can be concluded that EAAC1-mediated glutamate transport is coupled to the cotransport of 2 Na+ ions, the countertransport of one K+ ion and either the countertransport of one OH- ion or the cotransport of 1 H+ ion. We further demonstrate that under conditions where the electrochemical gradients for these ions are disrupted, EAAC1 runs in reverse, a transport mode which is of pathologic importance. 22Na+ uptake studies revealed that there is a low level of Na+ uptake in the absence of extracellular glutamate which appears to be analogous to the Na+ leak observed for the intestinal Na+/glucose cotransporter SGLT1. In voltage clamp studies, reducing extracellular Na+ from 100 to 10 mM strongly increased K0.5L-glutamate and decreased I(max). The data indicate that Na+ binding at the extracellular transporter surface becomes rate-limiting. Studies addressing the cooperativity of the substrate-binding sites indicate that there are two distinct Na(+)-binding sites with different affinities and that Na+ binding is modulated by extracellular glutamate. A hypothetical ordered kinetic transport model for EAAC1 is discussed.

ATP-Binding Cassette Transporters↗

PubMed1995

Extracellular and intracellular alkalinization and the constriction of rat cerebral arterioles.

1. Direct observations of perfused cerebral arterioles in vivo and in vitro have demonstrated that alkalinization of blood or cerebrospinal fluid (CSF) causes arteriolar constriction. Inasmuch as such alkalinizations lead to increases in intracellular pH (pHi) as well as interstitial pH (pHo), it is possible that increases in either pHi or pHo (or both) underlie alkalinization-induced cerebral vasoconstriction. In order to test the hypothesis that changes in pHi alone underline alkalinization-induced cerebral vasoconstriction, we simultaneously measured vessel diameter and pHi (using the pH-sensitive dye, SNAFL) in isolated cerebral arterioles from adult rats during imposed alterations in pHo and pHi. 2. Penetrating cerebral arterioles from the distribution of the middle cerebral artery were hand dissected, cannulated on one end and occluded distally. Vessels were inflated hydrostatically to 60 cmH2O under no-flow conditions. Confocal microscopy verified specific pH-sensitivity dye staining of the vascular smooth muscle cells within the vessel wall. 3. Extracellular alkalinization from pH 7.3 to 7.8 caused pHi to increase by 0.06 +/- 0.01 of a pH unit, and vessel diameter to decrease by 21.8 +/- 1.8% (mean +/- S.E.M.). 4. Intracellular alkalinization at constant pHo was produced by exposure to weak bases, including NH3 and trimethylamine, or by exposure to, followed by withdrawal of, weak acids, including CO2 and acetic acid. None of these treatments evoked vasoconstriction even though each of them caused increases in pHi greater than those observed in the same vessels during exposure to the pHo 7.8 solution. 5. We conclude that, at least in cerebral arterioles, alkalinization-induced vasoconstriction is mediated by an increase in pHo, not pHi [corrected].

Alkalies↗

PubMed1995

Out-of-equilibrium CO2/HCO3- solutions and their use in characterizing a new K/HCO3 cotransporter.

In typical physiological solutions, CO2 is in equilibrium with HCO3- and H+ (CO2 + H2O<==>HCO3- +H+). Because one cannot independently alter CO2 and HCO3- concentrations and pH, it is impossible to distinguish between the effects of CO2 and HCO3- on physiological processes. Here we describe a continuous-flow, rapid-mixing approach for generating out-of-equilibrium CO2/HCO3- solutions with a physiological pH and CO2 (but little HCO3-), or pH and HCO3- (but little CO2). We have exploited these out-of-equilibrium solutions to introduce HCO3- exclusively to either the outside or inside of a squid giant axon, and verify the presence of a new K/HCO3 cotransporter. The out-of-equilibrium approach could be useful in a variety of applications for independently controlling CO2 and HCO3- concentrations and pH.

Animals↗

PubMed1995

Use of BCECF and propidium iodide to assess membrane integrity of acutely isolated CA1 neurons from rat hippocampus.

We used 2 fluorescent dyes, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and propidium iodide (PI), to assess the membrane integrity of neurons acutely isolated from the CA1 region of the rat hippocampus. Exciting BCECF at a relatively pH-insensitive wavelength (440 nm), or exciting PI at 490 nm, we quantitatively recorded, in real time and in single cells, the rate constants for BCECF loss (-k440) and PI uptake (k490). We found that approximately 98% of intracellular BCECF is rapidly released by applying 0.01% saponin. In neurons not treated with saponin, rate constants for BCECF loss and PI uptake typically were 1% min-1 or less under control conditions, in the presence of NH3/NH4+ and in the absence of Na+. However, in a small number of neurons, the rate constant for BCECF loss increased markedly (-k440 > 5% min-1), while pHi approached pHo, suggesting that the plasma membrane spontaneously became leaky. When neurons were progressively swollen in hypotonic solutions, rates constants for BCECF loss and PI uptake generally were affected minimally unless osmolality was decreased to approximately 75 mOsmol/kg. Treating neurons with 0.001% saponin caused an increase in PI uptake rate only in a minority of neurons, whereas in most experiments a similar treatment caused -k440 for BCECF to exceed 5% min-1, and led to a rapid deterioration of the pH gradient across the cell membrane. At even lower saponin levels (0.0005-0.0007%), we observed a much slower deterioration of pHi, which occurred at low rates of BCECF loss (-k440 = approximately 3% min-1). We conclude that computing rate constants for BCECF loss and PI uptake may be useful for assessing neuronal health, and that BCECF loss may be more sensitive to cell damage than PI uptake.

Ammonia↗

PubMed1995

Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts.

Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H+ ATPase, be complemented by other acid-base transporters. We investigated acid-extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO3-, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta 1) decreased curvilinearly from approximately 25 mM at pHi = 6.4 to approximately 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and approximately 60% of round osteoclasts (approximately 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 microM ethylisopropylamiloride (EIPA), or 50 microM hexamethyleneamiloride (HMA). The apparent K1/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent K1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 microM/s. In the remaining approximately 40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na(+)-independent pHi recovery was blocked by 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), a vacuolar-type H+ pump inhibitor. Corrected for NBD-Cl insensitive fluxes, H+ pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 microM/s at pHi 7.45. In approximately 45% of pattern-2 cells, Na+ readdition elicited a further pHi recovery, suggesting that H+ pumps and Na-H exchangers can exist simultaneously. We conclude that, under the conditions of our study, most neonatal rat osteoclasts express Na-H exchangers that are probably of the ubiquitous basolateral subtype. Some cells express vacuolar-type H+ pumps in their plasma membrane, as do active osteoclasts in situ.

4-Chloro-7-nitrobenzofurazan↗

PubMed1995

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux.

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons II. Base influx.

We used microelectrodes to determine whether the K/HCO3 cotransporter tentatively identified in the accompanying paper (Hogan, E. M., M. A. Cohen, and W. F. Boron. 1995. Journal of General Physiology. 106:821-844) can mediate an increase in the intracellular pH (pHi) of squid giant axons. An 80-min period of internal dialysis increased pHi to 7.7, 8.0, or 8.3; the dialysis fluid was free of K+, Na+, and Cl-. Our standard artificial seawater (ASW), which also lacked Na+, K+, and Cl-, had a pH of 8.0. Halting dialysis unmasked a slow pHi decrease. Subsequently introducing an ASW containing 437 mM K+ and 0.5% CO2/12 mM HCO3- had two effects: (a) it caused membrane potential (Vm) to become very positive, and (b) it caused a rapid pHi decrease, because of CO2 influx, followed by a slower plateau-phase pHi increase, presumably because of inward cotransport of K+ and HCO3- ("base influx"). Only extracellular Rb+ substituted for K+ in producing the plateau-phase pHi increase in the presence of CO2/HCO3-. Mean fluxes with Na+, Li+, and Cs+ were not significantly different from zero, even though Vm shifts were comparable for all monovalent cations tested. Thus, unless K+ or Rb+ (but not Na+, Li+, or Cs+) somehow activates a conductive pathway for H+, HCO3-, or both, it is unlikely that passive transport of H+, HCO3-, or both makes the major contribution to the pHi increase in the presence of K+ (or Rb+) and CO2/HCO3-. Because exposing axons to an ASW containing 437 mM K+, but no CO2/HCO3-, produced at most a slow pHi increase, K-H exchange could not make a major contribution to base influx. Introducing an ASW containing CO2/HCO3-, but no K+ also failed to elicit base influx. Because we observed base influx when the ASW and DF were free of Na+ and Cl-, and because the disulfonic stilbene derivatives SITS and DIDS failed to block base influx, Na(+)-dependent Cl-HCO3 exchange also cannot account for the results. Rather, we suggest that the most straightforward explanation for the pHi increase we observed in the simultaneous presence of K+ and CO2/HCO3- is the coupled uptake of K+ and HCO3-.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO3-.

Monitoring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein, we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of rabbit proximal tubule. To explain a previous observation, that steady-state pHi is higher in the presence than in the absence of CO2/HCO3- (N. L. Nakhoul, L. K. Chen, and W. F. Boron. J. Gen. Physiol. 102: 1171-1205, 1993), we examined the effect of bilateral (i.e., luminal and basolateral) CO2/HCO3- on the acid extrusion processes responsible for recovery of pHi from acid loads. To compute fluxes from rates of pHi change, we determined the pHi dependence of intrinsic intracellular buffering power, which was approximately 50 mM/pH at pHi 6.5 and fell linearly to approximately 20 mM at pHi 7.4. In one series of experiments, we monitored the rate of pHi recovery from an acid load imposed by an NH4+/NH3 prepulse. Over a broad range of pHi values, total net acid extrusion was approximately four times higher in bilateral presence of CO2/HCO3- than in its absence. In a second group of experiments, which were designed to determine the effect of CO2/HCO3- on luminal Na+/H+ exchange, we monitored the rate of pHi recovery elicited by adding Na+ back to only the lumen, after first removing Na+ bilaterally. Initial rate of luminal Na(+)-dependent net acid extrusion in presence of CO2/HCO3- was approximately 229 microM/s (pHi 6.92), approximately 1.8 times higher than the flux of approximately 127 microM/s (P < 0.005) obtained in absence of CO2/HCO3- (pHi 6.66). CO2/HCO3- alkali-shifted the flux vs. pHi relationship by 0.3-0.4 pH units. In a final series of experiments, we examined the effect of CO2/HCO3- on the Na(+)-independent alkalinization that follows the rapid, initial acidification elicited by bilateral Na+ removal. In the presence of CO2/HCO3-, lag time for initiation of the Na(+)-independent alkalinization was only approximately 36 vs. approximately 211 s (P < 0.002) in absence of CO2/HCO3-. Also, Na(+)-independent net acid extrusion rate was approximately two to three times higher in presence than in absence of CO2/HCO3- at comparable pHi. This Na(+)-independent acid extrusion was insensitive to N-ethylmaleimide (2 mM), but was inhibited approximately 94% by efforts to deplete intracellular ATP (i.e., removal of glucose and amino acids, plus addition of 2 mM cyanide and 10 mM iodoacetic acid). Stimulation of luminal Na+/H+ exchange and Na(+)-independent acid extrusion appears to be the major, if not the entire, explanation for the higher steady-state pHi caused by bilateral addition of CO2/HCO3-.

Acids↗

PubMed1995

Acid extrusion in S3 segment of rabbit proximal tubule. II. Effect of basolateral CO2/HCO3-.

Monitoring intracellular absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of the rabbit proximal tubule. In the preceding study [Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F179-F192, 1995.], we demonstrated that simultaneously adding CO2/HCO3- to both lumen and bath stimulated two acid-extruding mechanisms, one dependent on luminal Na+ (a Na+/H+ exchanger) and one independent of Na+ (presumably a H+ pump). Here, we examine the effects of adding CO2/HCO3- to the lumen only or to the bath only. Over a broad pHi range, the total rate of pHi recovery from an acid load in the presence of Na+ was not increased by luminal CO2/HCO3-. On the other hand, basolateral CO2/HCO3- increased the pHi recovery rate to an even greater extent than had bilateral CO2/HCO3-. Regarding the Na(+)-independent pHi recovery mechanism, we found that luminal CO2/HCO3- failed to increase the pHi recovery rate compared with controls also studied in the absence of Na+. Neither did luminal CO2/HCO3- significantly affect the lag time between the maximal acid load and the initiation of the Na(+)-independent pHi recovery (approximately 147 vs. approximately 212 s in controls). On the other hand, adding CO2/HCO3- to only the bath substantially increased the rate of Na(+)-independent pHi recovery, which generally was greater than that observed with bilateral CO2/HCO3-. CO2/HCO3- only in the bath also reduced the lag time to approximately 51 s, which is not significantly different from the value of approximately 36 s observed with bilateral CO2/HCO3-. Our data are consistent with the hypothesis that basolateral (but not luminal) CO2/HCO3- stimulates both a luminal Na+/H+ exchanger and a luminal H+ pump.

Acids↗

PubMed1995

Fluid absorption in isolated perfused colonic crypts.

A spatial segregation of ion transport processes between crypt and surface epithelial cells is well-accepted and integrated into physiological and pathophysiological paradigms of small and large intestinal function: Absorptive processes are believed to be located in surface (and villous) cells, whereas secretory processes are believed to be present in crypt cells. Validation of this model requires direct determination of fluid movement in intestinal crypts. This study describes the adaptation of techniques from renal tubule microperfusion to hand-dissect and perfuse single, isolated crypts from rat distal colon to measure directly fluid movement. Morphologic analyses of the isolated crypt preparation revealed no extraepithelial cellular elements derived from the lamina propria, including myofibroblasts. In the basal state, crypts exhibited net fluid absorption (mean net fluid movement = 0.34 +/- 0.01 nl.mm-1.min-1), which was Na+ and partially HCO3- dependent. Addition of 1 mM dibutyryl-cyclic AMP, 60 nM vasoactive intestinal peptide, or 0.1 mM acetylcholine to the bath (serosal) solution reversibly induced net fluid secretion (net fluid movement approximately -0.35 +/- 0.01 nl.mm-1.min-1). These observations permit speculation that absorption is a constitutive transport function in crypt cells and that secretion by crypt cells is regulated by one or more neurohumoral agonists that are released in situ from lamina propria cells. The functional, intact polarized crypt described here that both absorbs and secretes will permit future studies that dissect the mechanisms that govern fluid and electrolyte movement in the colonic crypt.

Animals↗

PubMed1994

Expression cloning of a mammalian proton-coupled oligopeptide transporter.

In mammals, active transport of organic solutes across plasma membranes was thought to be primarily driven by the Na+ gradient. Here we report the cloning and functional characterization of a H(+)-coupled transporter of oligopeptides and peptide-derived antibiotics from rabbit small intestine. This new protein, named PepT1, displays an unusually broad substrate specificity. PepT1-mediated uptake is electrogenic, independent of extracellular Na+, K+ and Cl-, and of membrane potential. PepT1 messenger RNA was found in intestine, kidney and liver and in small amounts in brain. In the intestine, the PepT1 pathway constitutes a major mechanism for absorption of the products of protein digestion. To our knowledge, the PepT1 primary structure is the first reported for a proton-coupled organic solute transporter in vertebrates and represents an interesting evolutionary link between prokaryotic H(+)-coupled and vertebrate Na(+)-coupled transporters of organic solutes.

Amino Acid Sequence↗

PubMed1994

Unusual permeability properties of gastric gland cells.

Physiologists have long pondered the riddle of why the stomach is itself not digested by the very juice it secretes. One explanation is that a mucus-bicarbonate barrier, coating the stomach lumen as well as superficial portions of gastric glands, prevents autodigestion. However, this leaves unanswered the question of what protects cells deeper in the glands, which seem to lack a mucus barrier. These are the parietal and chief cells, which secrete acid and pepsin. Using perfused single gastric glands from rabbit, we recently found that intracellular pH is uniquely resistant to extreme degrees of luminal acidification, suggesting that the apical (luminal) barrier might also exclude ammonia and carbon dioxide, to which cell membranes are generally highly permeable. We now show that this is indeed the case. There are three reports of membranes with very low permeabilities to NH3 (refs 5-7), and none of membranes impermeable to CO2.

Ammonia↗

PubMed1994

Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na(+)-dependent Cl(-)-HCO3- exchange.

1. We have measured intracellular pH (pHi) in freshly isolated pyramidal neurones from the CA1 region of the rat hippocampus using the fluorescent indicator 2',7'-bis(carboxy-ethyl)-5-(and-6)-carboxyfluorescein (BCECF). 2. The neurones selected by our isolation procedure, when studied in the nominal absence of CO2-HCO3-, had a mean steady-state pHi of 6.81 +/- 0.02 (n = 163). The recovery of pHi from acid loads was very slow. The rate of recovery from acid loads was reduced by Na+ removal, but only very slightly inhibited by 1 mM amiloride. 3. The addition of 5% CO2-25 mM HCO3- caused steady-state pHi to increase from 6.74 +/- 0.05 to 7.03 +/- 0.03 (n = 28). In the presence of 5% CO2-25 mM HCO3-, the rate of pHi recovery from acid loads was much faster than in its absence. 4. The HCO(3-)-induced alkalinization was reversible, and did not occur in the absence of extracellular Na+ or in the presence of DIDS (4,4'-diisothiocyanatostilbene- 2,2'-disulphonic acid). 5. In the absence of external Cl-, successive exposures to CO2-HCO3- elicited alkalinizations that were progressively reduced in rate and amplitude. This effect, presumably due to gradual depletion of internal Cl-, was rapidly reversed by returning Cl- to the external medium. 6. We conclude that the major acid-extrusion mechanism in pyramidal CA1 neurones is the Na(+)-dependent Cl(-)-HCO3- exchanger. The Na(+)-dependent mechanism that operates in the nominal absence of HCO3- is far less active.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Long-term expression of c-H-ras stimulates Na-H and Na(+)-dependent Cl-HCO3 exchange in NIH-3T3 fibroblasts.

In a study of intracellular pH (pHi) regulation, we compared c-H-ras-transformed fibroblasts (FT9s) with parental NIH 3T3 cells. Cells were grown on a coverslip and placed in a flow-through cuvette; pHi was monitored using a fluorescent dye. Whether cells were in a CO2-free or CO2-containing solution, steady-state pHi was approximately 0.5 higher in FT9s than in 3T3s. Rates of pHi recovery from acid loads in the presence of CO2 indicate that total net acid extrusion (Jtotal) was far greater in FT9s than 3T3s in the pHi range 6.3-7.4. In both FT9s and 3T3s, pHi recovery was blocked by removing Na+. In FT9s, most of Jtotal was blocked by ethyl isopropyl amiloride (EIPA) and was probably due to Na-H exchange. A smaller component was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and required Cl- and was probably due to Na(+)-dependent Cl-HCO3 exchange. In 3T3s, the DIDS-sensitive and Cl(-)-dependent components of Jtotal were very small. The dominant acid-extruder in 3T3s appears to be a Na-H exchanger insensitive to 50 microM EIPA. We determined the flux-pHi relationships for both the Na-H and Na(+)-dependent Cl-HCO3 exchangers in both FT9s and 3T3s. p21ras alkaline shifts the pHi profile of each transporter by approximately 0.7, without affecting the Vmax. These shifts in the flux-pHi relationships provide a mechanism for the large increase in steady-state pHi produced by long-term expression of p21ras.

3T3 Cells↗

PubMed1994

Luminal perfusion of isolated gastric glands.

We have extended to rabbit gastric glands the technique for perfusing single isolated renal tubules. We isolated glands by hand dissection and used concentric glass pipettes to hold them and perfuse their lumina. Parietal cells (PCs), which tended to be located toward the gland opening, were identified by their pyramidal shape, large size, and autofluorescence. Chief cells (CCs) were identified by their round shape and smaller size. In some experiments, we perfused the lumen with hydroxypyrenetrisulfonate, a pH-sensitive fluorophore, at pH 7.4 and used digital image processing to monitor luminal pH (pH1). Solutions were buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to pH 7.4 at 37 degrees C. With fast perfusion, we found no evidence of decreased pH1, even with stimulation by 10 microM carbachol. With slow perfusion, pH1 often fell below the dye's sensitive range (pH < 5), especially at low perfusate buffering power. In other experiments, we loaded cells with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and monitored intracellular pH (pHi) in multiple individual PCs and CCs in a single gland. Mean pHi was 7.21 +/- 0.02 (n = 136 cells) for PCs and 7.27 +/- 0.03 (n = 103) for CCs. To examine the response to decreased pH1 and basolateral pH (pHb), we lowered pHb to 6.4 or lowered pH1 to 3.4 or 1.4. Lowering pHb to 6.4 for approximately 1 min caused pHi to fall reversibly by 0.39 +/- 0.05 (n = 53) in PCs and 0.58 +/- 0.03 (n = 50) in CCs. Lowering pH1 to 3.4 or 1.4 caused no significant pHi changes in PCs (n = 38 and 82) or in CCs (n = 44 and 77). Carbachol did not affect the response to changes in pH1 or pHb. We conclude that the apical surfaces of PCs and CCs are unusually resistant to extreme pH gradients.

Ammonia↗

PubMed1994

Effects of CGRP, forskolin, PMA, and ionomycin on pHi dependence of Na-H exchange in UMR-106 cells.

We examined the effects of calcitonin gene-related peptide (CGRP), forskolin, phorbol 12-myristate 13-acetate (PMA), and ionomycin on the intracellular pH (pHi) dependence of Na-H exchange in UMR-106 cells. In the nominal absence of CO2-HCO3-, each agent increased pHi, measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). From the rate of pHi recovery (dpHi/dt) from an acid load, and intracellular buffering power, we computed the pHi dependence of the total acid-extruding flux (JTotal). All four agents increased JTotal. From dpHi/dt data obtained in the presence of ethylisopropyl amiloride (EIPA, a blocker of Na-H exchange), we determined the EIPA-resistant component of JTotal (JEIPA/R). We estimated the Na-H exchange flux (JNa-H) as the difference JTotal-JEIPA/R-CGRP, forskolin, and PMA produced similar increases in the slope of the JNa-H vs. pHi-relationship. The net effect of these agents, as well as ionomycin, was to increase JNa-H over a broad pHi range. Ionomycin alkaline shifted the JEIPA/R vs. pHi relationship; the other agents had no effect. Our results indicate that CGRP increased JTotal by stimulating Na-H exchange, with little effect on EIPA-resistant processes. A signaling pathway involving only adenosine 3',5'-cyclic monophosphate, only protein kinase C, or only Ca2+ cannot account for the effects of CGRP on both pHi and pHi dependence of JNa-H. Thus, CGRP probably affects UMR-106 pHi physiology via more than one pathway.

Animals↗

PubMed1994

Shrinkage-induced activation of Na(+)-H+ exchange in barnacle muscle fibers.

We examined the effect of shrinkage on Na(+)-H+ exchange in single muscle fibers at intracellular pH (pHi) values of 6.8, 7.2, and 7.6 using pH microelectrodes and internal dialysis. Under normotonic conditions (975 mosmol/kgH2O) at pHi 6.8, the amiloride-sensitive acid-extrusion rate (JAmil/s) averaged 17 microM/min. Exposure to hypertonic solutions (1,600 mosmol/kgH2O) increased JAmil/s to 304 microM/min at pHi 6.8. At pHi approximately 7.2 and 7.6, hypertonicity increased JAmil/s from approximately 0 to approximately 172 microM/min (pHi 7.2) and approximately 0 to approximately 90 microM/min (pHi 7.6). Thus, under normotonic conditions, Na(+)-H+ exchange activity is slight at pHi approximately 6.8 and virtually nil at higher pHi values. Shrinkage stimulated Na(+)-H+ exchange, more at low pHi values. We also examined the Cl- dependence of the Na(+)-H+ exchanger's response to shrinkage. Our results indicate that shrinkage-induced activation of Na(+)-H+ exchange requires Cl-, specifically intracellular Cl-. These results establish that shrinkage is both pHi dependent and requires intracellular Cl-.

Animals↗