Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.     

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.     

Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production

The culture levels of glucose and CO(2) have been reported to independently have important influences on mammalian cell processes. In this work the combined effects of glucose limitation and CO(2) partial pressure (pCO(2)) on monoclonal antibody (IgG) producing Chinese Hamster Ovary cells were investigated in a perfusion reactor operated with controlled cell specific medium feed rate, pH and osmolality. Under high glucose conditions (14.3 +/- 0.8 mM), the apparent growth rate decreased (from 0.021 to 0.009 h(-1)) as the pCO(2) increased to approximately 220 mmHg, while the cell specific IgG productivity was almost unchanged. The lactate yield from glucose was not affected by pCO(2) up to approximately 220 mmHg and glucose was mainly converted to lactate. A feed medium modification from high (33 mM) to low (6 mM) glucose resulted in <0.1 mM glucose in the culture. As a result of apparently shifting metabolism towards the conversion of pyruvate to CO(2), both the ratio of lactate to glucose and the alanine production rate were lowered (1.51-1.14 and 17.7-0.56 nmol/10(6) cells h, respectively). Interestingly, when the pCO(2) was increased to approximately 140 mmHg, limiting glucose resulted in 1.7-fold higher growth rates, compared to high glucose conditions. However, at approximately 220 mmHg pCO(2) this beneficial effect of glucose limitation on these CHO cells was lost as the growth rate dropped dramatically to 0.008 h(-1) and the IgG productivity was lowered by 15% (P < 0.01) relative to the high glucose condition. The IgG galactosylation increased under glucose- limited compared to high-glucose conditions.     

Dynamics of protein uptake within the adsorbent particle during packed bed chromatography

Elevated osmolality and pCO(2) have been shown to alter sialylation in a protein-specific manner. In Chinese hamster ovary (CHO)MT2-l-8 cells, tPA sialylation changed only slightly from 40 to 250 mm Hg pCO(2), whereas neural cell adhesion molecule polysialic acid (NCAM PSA) content decreased by up to 70% at 250 mm Hg pCO(2), pH 7.2. NCAM PSA content also decreased with increasing NaCl or NH(4)Cl concentration. This suggests that PSA content is a sensitive indicator of conditions that may alter glycosylation. Amino acids and their derivatives have been used to protect hybridoma and CHO cell growth under hyperosmotic stress. We examined the impact of osmoprotectants on NCAM PSA content in CHO MT2-1-8 cells under hyperosmolality (up to 545 mOsm/kg) and at 195 and 250 mm Hg pCO(2). NCAM PSA content at 545 mOsm/kg was at least two-fold greater in the presence of glycine betaine or L-proline compared to that without osmoprotectant. Surprisingly, in the presence of 20 mM glycine betaine, PSA levels were 50-60% of the control level for osmolalities ranging from 320 to 545 mOsm/kg. Thus, glycine betaine inhibits NCAM polysialylation at osmolalities below 435 mOsm/kg and is beneficial at higher osmolalities. In contrast to glycine betaine, L-proline increased PSA content by 25-120% relative to the unprotected culture at < or =545 mOsm/kg. The decrease in NCAM PSA levels of CHO MT2-1-8 cells cultured at 195 mm Hg pCO(2)-435 mOsm/kg was not mitigated by the presence of 25 mM glycine betaine, glycine, or L-threonine, even though all of these compounds enhanced cell growth. At 250 mm Hg pCO(2), all osmoprotectants tested (20 mM L-threonine, L-proline, glycine, or glycine betaine) increased NCAM polysialylation, with 20 mM glycine betaine restoring NCAM PSA to near control levels. Thus, osmoprotectants may (partially) offset changes in glycosylation, as well as the inhibition of growth, in cells under environmental stress. Supernatant beta-galactosidase levels, which increase upon alkalization of acidic organelles, did not differ significantly under elevated pCO(2) and hyperosmolality from that at control conditions.     

Hyperosmotic stress and elevated pCO2 alter monoclonal antibody charge distribution and monosaccharide content

Medium osmolality increases with pCO2 at constant pH. Elevated pCO2 and osmolality inhibit hybridoma growth to similar extents in both serum-containing and serum-free media. The combination of osmolality and elevated pCO2 synergizes to negatively impact cell growth. IgG2a glycosylation by hybridoma cells was evaluated under elevated pCO2 (to 250 mmHg pCO2) and/or osmolality (to 476 mOsm/kg). IgG2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum-containing and serum-free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum-containing medium decreased by 0.16 pH units at 250 mmHg pCO2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO2 (195 mmHg pCO2-435 mOsm/kg). In serum-free medium, elevated pCO2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO2, elevated pCO2 did not significantly alter the IgG2a charge heterogeneity under any of the conditions studied. The IgG2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO3 or NaCl. However, when osmolality was controlled at elevated pCO2, the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG2a were due to increased organellar pH, which is reflected by increased specific beta-galactosidase activity in the supernatant.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Effects of elevated pCO(2) and/or osmolality on the growth and recombinant tPA production of CHO cells

Carbon dioxide is a by-product of mammalian cell metabolism that will build up in culture if it is not removed from the medium. Increased carbon dioxide levels are generally not a problem in bench-top bioreactors, but inhibitory levels can easily be reached in large-scale vessels, especially if the aeration gas is enriched in oxygen. Due to the equilibrium attained between dissolved CO(2) and bicarbonate, increased pCO(2) is associated with increased osmolality in bioreactors with pH control. While a few preliminary reports indicate that elevated pCO(2) levels can inhibit cell growth and/or recombinant protein production, no comprehensive analysis of the interrelated effects of elevated pCO(2) and osmolality has been published. We have examined the effects of 140, 195, and 250 mm Hg (187, 260, and 333 mbar, respectively) pCO(2) (with and without osmolality control) on the growth of and recombinant tPA production by MT2-1-8 Chinese hamster ovary (CHO) cells. The effects of elevated osmolality were also investigated at the control pCO(2) of 36 mm Hg. Elevated pCO(2) at 310 mOsm/kg osmolality inhibited cell growth in a dose-dependent fashion, with a maximum decrease of 30% in the specific growth rate (mu) at 250 mm Hg. Osmolality alone had no effect on mu, but the combination of elevated pCO(2) and osmolality increased the maximal reduction in mu to 45%. Elevated pCO(2) at 310 mOsm/kg osmolality decreased the specific tPA production rate (q(tPA)) by up to 40% at 250 mm Hg. Interestingly, while increased osmolality decreased q(tPA) significantly at 140 mm Hg pCO(2), it had no effect or even increased q(tPA) at 195 and 250 mm Hg. (c) 1996 John Wiley & Sons, Inc.     

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.     

Effects of CO2 and osmolality on hybridoma cells: growth, metabolism and monoclonal antibody production

CO2 partial pressure (pCO2) in industrial cell culture reactors may reach 150–200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. Due to equilibrium with bicarbonate, increased pCO2 at constant pH results in a proportional increase in osmolality. Hybridoma AB2-143.2 cell growth rate decreased with increasing pCO2 in well-plate culture, with a 45% decrease at 195 mm Hg with partial osmolality compensation (to 361 mOsm kg- 1). Inhibition was more extensive without osmolality compensation, with a 63% decrease in growth rate at 195 mm Hg and 415 mOsm kg-1. Also, the hybridoma death rate increased with increasing pCO2, with 31- and 64-fold increases at 250 mm Hg pCO2 for 401 and 469 mOsm kg- 1, respectively. The specific glucose consumption and lactate production rates were 40–50% lower at 140 mm Hg pCO2. However, there was little further inhibition of glycolysis at higher pCO2. The specific antibody production rate was not significantly affected by pCO2 or osmolality within the range tested. Hybridomas were also exposed to elevated pCO2 in continuous culture. The viable cell density decreased by 25–40% at 140 mm Hg. In contrast to the well-plate cultures, the death rate was lower at the new steady state at 140 mm Hg. This was probably due to higher residual nutrient and lower byproduct levels at the lower cell density (at the same dilution rate), and was associated with increased cell-specific glucose and oxygen uptake. Thus, the apparent effects of pCO2 may vary with the culture system. VMdZ and RK contributed equally to the results in this article. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of hybridoma cell responses to elevated pCO(2) and osmolality: intracellular pH, cell size, apoptosis, and metabolism.

CO(2) partial pressure (pCO(2)) in industrial cell culture reactors may reach 150-200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. The inhibitory effects of elevated pCO(2) at constant pH are due to a combination of the increases in pCO(2) and [HCO(-) (3)], per se, and the associated increase in osmolality. To decouple the effects of pCO(2) and osmolality, low-salt basal media have been used to compensate for this associated increase in osmolality. Under control conditions (40 mm Hg-320 mOsm/kg), hybridoma cell growth and metabolism was similar in DMEM:F12 with 2% fetal bovine serum and serum-free HB GRO. In both media, pCO(2) and osmolality made dose-dependent contributions to the inhibition of hybridoma cell growth and synergized to more extensively inhibit growth when combined. Elevated osmolality was associated with increased apoptosis. In contrast, elevated pCO(2) did not increase apoptotic cell death. Specific antibody production also increased with osmolality although not with pCO(2). In an effort to understand the mechanisms through which elevated pCO(2) and osmolality affect hybridoma cells, glucose metabolism, glutamine metabolism, intracellular pH (pHi), and cell size were monitored in batch cultures. Elevated pCO(2) (with or without osmolality compensation) inhibited glycolysis in a dose-dependent fashion in both media. Osmolality had little effect on glycolysis. On the other hand, elevated pCO(2) alone had no effect on glutamine metabolism, whereas elevated osmolality increased glutamine uptake. Hybridoma mean pHi was approximately 0.2 pH units lower than control at 140 mm Hg pCO(2) (with or without osmolality compensation) but further increases in pCO(2) did not further decrease pHi. Osmolality had little effect on pHi. Cell size was smaller than control at elevated pCO(2) at 320 mOsm/kg, and greater than control in hyperosmotic conditions at 40 mm Hg.     

Identifying inhibitory threshold values of repressing metabolites in CHO cell culture using multivariate analysis methods

Elevation of lactate, ammonia, osmolality, and carbon dioxide to inhibitory levels was reported to have adverse effects on cell growth and protein productivity in mammalian cell culture. Multivariate analysis methods were used to investigate the roles of these repressing metabolites in a fed-batch CHO cell culture for antibody fusion protein B1 (B1) production. Principal Factor Analysis methodology was applied to manufacturing-scale data of 112 cell culture runs, which identified threshold values of four repressing metabolites as follows: (1) ammonium levels above 5.1 mM inhibit cell growth; (2) both lactate and osmolality levels above 58 mM and 382 mOsm/kg affect cell viability; and (3) carbon dioxide levels at or above 111 mmHg reduce protein quality. These threshold values were then verified by simulations using Monod-type equations and Canonical Correlation. These results suggest that adverse effects on cell growth, productivity, and product quality may be minimized under the ideal cell culture condition, in which the peak values of all four repressing metabolites are maintained below the threshold values. This strategy was evaluated in 45 cell culture runs in 50-L bioreactors. Eight out of 45 runs were operated under the ideal condition, while the remaining 37 runs had at least one repressing metabolite with peak value at or above the threshold. In comparison to the remaining runs, the eight cell culture runs under the ideal condition had 17%, 40%, and 11% higher values in peak viable cell density, final B1 titer, and quality attribute, respectively. The unique methodology used in this study may be generally applicable in characterizing cell culture processes.     

HCO3(-) secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3)(-) during Ca(2+)-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl(-) channel, with HCO(3)(-) secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na(+)-dependent pH(i) regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na(+)-free media.     

Contribution of anion transporters to the acidosis-induced swelling and intracellular acidification of glial cells

This study examines the contribution of anion transporters to the swelling and intracellular acidification of glial cells from an extracellular lactacidosis, a condition well-known to accompany cerebral ischemia and traumatic brain injury. Suspended C6 glioma cells were exposed to lactacidosis in physiological or anion-depleted media, and different anion transport inhibitors were applied. Changes in cell volume and intracellular pH (pH(i)) were simultaneously quantified by flow cytometry. Extracellular lactacidosis (pH 6.2) led to an increase in cell volume to 125.1 +/- 2.5% of baseline within 60 min, whereas the pH(i) dropped from the physiological value of 7.13 +/- 0.05 to 6.32 +/- 0.03. Suspension in Cl(-)-free or HCO(3)(-)/CO(2)-free media or application of anion transport inhibitors [0.1 mM bumetanide or 0.5 mM 4, 4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS)] did not affect cell volume during baseline conditions but significantly reduced cell swelling from lactacidosis. In addition, the Cl(-)-free or HCO(3)(-)/CO(2)-free media and DIDS attenuated intracellular acidosis on extracellular acidification. From these findings it is concluded that besides the known activation of the Na(+)/H(+) exchanger, activation of the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and the Na(+)-K(+)-Cl(-) cotransporter contributes to acidosis-induced glial swelling and the intracellular acidification. Inhibition of these processes may be of interest for future strategies in the treatment of cytotoxic brain edema from cerebral ischemia or traumatic brain injury.     

Uroguanylin regulates net fluid secretion via the NHE2 isoform of the Na/H+ exchanger in an intestinal cellular model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na(+)/H(+) exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na(+)/H(+) exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (J(v)) was studied by optic techniques and intracellular pH (pH(i)) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na(+) absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pH(i). In the assayed conditions, cell exposure to apical UGN increases net secretory J(v), without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net J(v) was produced mainly by a reduction in normal Na(+) absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pH(i) is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pH(i) regulation.     

Ammonia excretion via Rhcg1 facilitates Na⁺ uptake in larval zebrafish, Danio rerio, in acidic water

The involvement of a Na(+)/H(+) exchanger (NHE) in mediating Na(+) uptake by freshwater fish is currently debated. Although supported indirectly by empirical molecular and pharmacological data, theoretically its operation should be constrained thermodynamically, owing to unfavorable chemical gradients. Recently, there has been an increasing focus on ammonia channels (Rh proteins) as potentially contributing to Na(+) uptake across the freshwater fish gill. In this study, we tested the hypothesis that Rhcg1, a specific apical isoform of Rh protein, is critically important in facilitating Na(+) uptake in zebrafish larvae via its interaction with NHE. Treating larvae (4 days postfertilization) with 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of NHE, caused a significant reduction in Na(+) uptake in fish reared in acidic water (pH ～ 4.0). A role for NHE in Na(+) uptake was further confirmed by translational knockdown of NHE3b, an isoform of NHE thought to be responsible for Na(+)/H(+) exchange in zebrafish larvae. Exposing the larvae reared in acidic water to 5 mM external ammonium sulfate or increasing the buffering capacity of the water with 10 mM HEPES caused concurrent reductions in ammonia excretion and Na(+) uptake. Furthermore, translational knockdown of Rhcg1 significantly reduced ammonia excretion and Na(+) uptake in larvae chronically (4 days) or acutely (24 h) exposed to acidic water. Unlike in sham-injected larvae, EIPA did not affect Na(+) uptake in fish experiencing Rhcg1 knockdown. Additionally, exposure of larvae to bafilomycin A1 (an inhibitor of H(+)-ATPase) significantly reduced Na(+) uptake in fish reared in acidic water. These observations suggest the existence of multiple mechanisms of Na(+) uptake in larval zebrafish in acidic water: one in which Na(+) uptake via NHE3b is linked to ammonia excretion via Rhcg1, and another facilitated by H(+)-ATPase.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

Indirect regulation of the intestinal H+-coupled amino acid transporter hPAT1 (SLC36A1)

A H(+)-coupled amino acid transporter has been characterised functionally at the brush border membrane of the human intestinal cell line Caco-2. This carrier, hPAT1 (human Proton-coupled Amino acid Transporter 1) or SLC36A1, has been identified recently at the molecular level and hPAT1 protein is localised to the brush border membrane of human small intestine. hPAT1 transports both amino acids (e.g., beta-alanine) and therapeutic agents (e.g., D-cycloserine). In human Caco-2 cells, hPAT1 function (H(+)/amino acid symport) is associated with a decrease in intracellular pH (pH(i)), which selectively activates the Na(+)/H(+) exchanger NHE3, and thus maintains pH(i) and the driving force for hPAT1 function (the H(+) electrochemical gradient). This study provides the first evidence for regulation of hPAT1 function. Activation of the cAMP/protein kinase A pathway in Caco-2 cell monolayers either using pharmacological tools (forskolin, 8-br-cAMP, [(11,22,28)Ala]VIP) or physiological activators (the neuropeptides VIP and PACAP) inhibited hPAT1 function (beta-alanine uptake) at the apical membrane. Under conditions where NHE3 is inactive (the absence of Na(+), apical pH 5.5, the presence of the NHE3 inhibitor S1611) no regulation of beta-alanine uptake is observed. Forskolin and VIP inhibit pH(i) recovery (NHE3 function) from beta-alanine-induced intracellular acidification. Immunocytochemistry localises NHERF1 (NHE3 regulatory factor 1) to the apical portion of Caco-2 cells where it will interact with NHE3 and allow PKA-mediated phosphorylation of NHE3. In conclusion, we have shown that amino acid uptake via hPAT1 is inhibited by activators of the cAMP pathway indirectly through inhibition of NHE3 activity.