Effects of amiloride and diethyl pyrocarbonate on CSF HCO-3 and ventilation in hypercapnia

Amiloride (10(-3) M), a Na+-H+ countertransport inhibitor, infused into the cisterna magna (10 microliter/min for 40 min) of ketamine-xylazine-anesthetized rabbits decreased the cerebrospinal fluid (CSF) HCO3- response to 3 h of hypercapnia [arterial PCO2 (PaCO2) = 60 Torr] by 21.6% (mean delta CSF [HCO3-]/delta PaCO2 0.232 vs. 0.296 mmol.l-1.Torr-1, P less than 0.05). Diethyl pyrocarbonate (DEPC, 10(-3) M), a histidine-blocking agent, infused into the cisterna magna decreased the CSF HCO3- response to hypercapnia by 25.3% (mean delta CSF [HCO3-]/delta PaCO2, 0.230 vs. 0.308 mmol.l-1.Torr-1, P less than 0.02). DEPC is known to inhibit the ventilatory response to hypercapnia (E. Nattie. Respir. Physiol. 64: 161-176, 1986) by a direct effect at the ventrolateral medulla (E. Nattie. J. Appl. Physiol. 61: 843-850, 1986). In this study amiloride had no significant effect on the ventilatory response to hypercapnia. The interpretation is that a Na+-H+ countertransport protein, perhaps with a histidine at a key location, is involved in CSF acid-base regulation and that amiloride appears to have no effects on the chemoreception process. DEPC appears to have effects on chemoreception and on CSF acid-base regulation.     

Effects of SITS, an anion transport blocker, on CSF ionic composition in metabolic alkalosis

Disulfonic stilbenes combine with the carrier protein involved in anion transport and inhibit the exchange of Cl- for HCO3- in a variety of biomembranes. Our aim was to determine whether such a mechanism is operative in the regulation of cerebrospinal fluid (CSF) [HCO3-] in metabolic alkalosis. In anesthetized, curarized, and artificially ventilated dogs either mock CSF (group I, 9 dogs) or mock CSF containing SITS, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (group II, 7 dogs) was periodically injected into both lateral cerebral ventricles. During 6 h of isocapnic metabolic alkalosis, produced by intravenous infusion of Na2CO3 solution, plasma [HCO3-] was increased by approximately 14 meq/l in both groups. In SITS-treated animals the mean cisternal CSF [HCO3-] increased by 7.7 meq/l after 6 h, and this was significantly higher than the respective increment, 3.5 meq/l, noted in the control group. Increments in CSF [HCO3-] in both groups were reciprocated by decrements in CSF [Cl-] with CSF [Na+] remaining unchanged. Cisternal CSF PCO2 and lactate concentrations showed similar increments in both groups. It is hypothesized that in metabolic alkalosis a carrier transports HCO3- out of cerebral fluid in exchange for Cl- and that SITS inhibits this mechanism. The efflux of HCO3- out of CSF in metabolic alkalosis would minimize the rise in CSF [HCO3-] brought about by HCO3-] influx from blood into CSF and therefore contributes to the CSF [H+] homeostasis.     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

CSF acid-base regulation and ventilation in iso- and hypocapnic Hacetate acidosis

Intravenous infusion in conscious rabbits of Hacetate decreases both arterial CO2 partial pressure PaCO2 and cerebrospinal fluid (CSF) HCO3- more than observed with HCl or HNO3 infusion. These acids did not affect CSF HCO3- in isocapnic conditions, and this study asks whether Hacetate infusion will do so. Arterial, central venous, and cisterna magna catheters were implanted in pentobarbital-anesthetized rabbits and all subsequent measurements were performed in the conscious state. Hacetate was infused intravenously over 6 h to decrease plasma HCO3- the same amount in a group allowed to decrease its PaCO2 in response to the acid (hypocapnic) and one in which PaCO2 was maintained at control levels (isocapnic). CSF HCO3- decreased significantly in isocapnia, although the change was less than in hypocapnia. Stoichiometrically by 6 h the measured CSF HCO3- change was balanced by an increase in acetate in hypocapnia and the sum of an increase in acetate and a decrease in chloride in isocapnia. Mechanistically, net acetate entry into CSF appears to involve an exchange for chloride as proposed for NO3-/Cl- and a process that lowers CSF HCO3-. This process could be competitive replacement of HCO3- by acetate in the CSF production mechanism or nonionic diffusive entry of Hacetate into CSF with subsequent titration of HCO3-. The decreases in CSF HCO3- result from the acetate mechanism and the hypocapnic effect on Cl- and HCO3-. The greater ventilatory response results from the greater CSF acidification or a specific effect of acetate per se.     

cAMP activates Cl-/HCO-3 exchange for regulation of intracellular pH in renal epithelial cells.

The role of cAMP in regulation of intracellular pH in the confluent LLC-PK1 cells was investigated. DibutyrylcAMP and forskolin induce intracellular acidification. This acidification is inhibited by DIDS and ethacrynic acid, inhibitors of Na(+)-independent Cl-/HCO3- exchange, and by removal of extracellular Cl-. In addition, Bt2 cAMP causes Cl- entry into LLC-PK1 cells. These results suggest that cAMP activates Cl- transport, namely Na(+)-independent Cl-/HCO3- exchange, which participates in pHi regulation.     

Cl(-)-HCO3- exchange in rat renal basolateral membrane vesicles

Pathways for HCO3- transport across the basolateral membrane were investigated using membrane vesicles isolated from rat renal cortex. The presence of Cl(-)-HCO3- exchange was assessed directly by 36Cl- tracer flux measurements and indirectly by determinations of acridine orange absorbance changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake compared to Cl- uptake under 100% N2 in the presence of a pH gradient alone. Mediated exchange of Cl- for HCO3- was suggested by the HCO3- gradient-induced concentrative accumulation of intravesicular Cl-. Maneuvers designed to offset the development of ion-gradient-induced diffusion potentials had no significant effect on the magnitude of HCO3- gradient-driven Cl- uptake further suggesting chemical as opposed to electrical Cl(-)-HCO3- exchange coupling. Although basolateral membrane vesicle Cl- uptake was observed to be voltage sensitive, the DIDS insensitivity of the Cl- conductive pathway served to distinguish this mode of Cl- translocation from HCO3- gradient-driven Cl- uptake. No evidence for K+/Cl- cotransport was obtained. As determined by acridine orange absorbance measurements in the presence of an imposed pH gradient (pHo 7.5/pHi 6), a HCO3- dependent increase in the rate of intravesicular alkalinization was observed in response to an outwardly directed Cl- concentration gradient. The basolateral membrane vesicle origin of the observed Cl(-)-HCO3- exchange activity was verified by experiments performed with purified brush-border membrane vesicles. In contrast to our previous observations of the effect of Cl- on HCO3- gradient-driven Na+ uptake suggesting a basolateral membrane Na+-HCO3- for Cl- exchange mechanism, no effect of Na+ on Cl-HCO3- exchange was observed in the present study.     

Effects of altered CSF [H+] on ventilatory responses to exercise in the awake goat

We investigated the effects of selective large changes in the acid-base environment of medullary chemoreceptors on the control of exercise hyperpnea in unanesthetized goats. Four intact and two carotid body-denervated goats underwent cisternal perfusion with mock cerebrospinal fluid (CSF) of markedly varying [HCO-3] (CSF [H+] = 21-95 neq/l; pH 7.68-7.02) until a new steady state of alveolar hypo- or hyperventilation was reached [arterial PCO2 (PaCO2) = 31-54 Torr]. Perfusion continued as the goats completed two levels of steady-state treadmill walking [2 to 4-fold increase in CO2 production (VCO2)]. With normal acid-base status in CSF, goats usually hyperventilated slightly from rest through exercise (-3 Torr PaCO2, rest to VCO2 = 1.1 l/min). Changing CSF perfusate [H+] changed the level of resting PaCO2 (+6 and -4 Torr), but with few exceptions, the regulation of PaCO2 during exercise (delta PaCO2/delta VCO2) remained similar regardless of the new ventilatory steady state imposed by changing CSF [H+]. Thus the gain (slope) of the ventilatory response to exercise (ratio of change in alveolar ventilation to change in VCO2) must have increased approximately 15% with decreased resting PaCO2 (acidic CSF) and decreased approximately 9% with increased resting PaCO2 (alkaline CSF). A similar effect of CSF [H+] on resting PaCO2 and on delta PaCO2/VCO2 during exercise also occurred in two carotid body-denervated goats. Our results show that alteration of the gain of the ventilatory response to exercise occurs on acute alterations in resting PaCO2 set point (via changing CSF [H+]) and that the primary stimuli to exercise hyperpnea can operate independently of central or peripheral chemoreception.     

Differential respiratory effects of HCO3- and CO2 applied on ventral medullary surface of rats

To estimate whether H+ is the unique stimulus of the medullary chemosensor, ventilatory effects of HCO3- and/or CO2 applied on the ventral medullary surface using an improved superfusion technique and of CO2 inhalation were compared in halothane-anesthetized spontaneously breathing rats. Superfusion with low [HCO3-]-acid mock cerebrospinal fluid (CSF) (normal Pco2) induced a significant increase in ventilation, with an accompanying reduction in endtidal Pco2 (PETco2). High [HCO3-]-alkaline CSF depressed ventilation. Changes in Pco2 of superfusing CSF, on the other hand, had no significant effect despite the similar changes in pH. Simultaneous decrease in [HCO3-] and Pco2 of mock CSF with normal pH also maintained stimulated respiration. CO2 inhalation during superfusion with various [HCO3-] solutions caused further increase in ventilation as PETco2 increased. The results suggest that the surface area of the rat ventral medulla contains HCO3- (or H+)-sensitive respiratory neural substrates which are, however, little affected by CO2 in the subarachnoid fluid. A CO2 (or CO2-induced H+)-sensitive chemosensor responsible for the increase in ventilation during CO2 inhalation may exist elsewhere functionally apart from the HCO3- (or H+)-sensitive sensor in the examined surface area.     

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla

We examined pH regulation in two chemosensitive areas of the brain, the retrotrapezoid nucleus (RTN) and the nucleus tractus solitarius (NTS), to identify the proton transporters involved in regulation of intracellular pH (pHi) in medullary glia. Transverse brain slices from young rats [postnatal day 8 (P8) to P20] were loaded with the pH-sensitive probe 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein after kainic acid treatment removed neurons. Cells were alkalinized when they were depolarized (extracellular K+ increased from 6.24 to 21.24 mM) in the RTN but not in the NTS. This alkaline shift was inhibited by 0.5 mM DIDS. Removal of CO2/HCO3- or Na+ from the perfusate acidified the glial cells, but the acidification after Na+ removal was greater in the RTN than in the NTS. Treatment of the slice with 5-(N-ethyl-N-isopropyl)amiloride (100 microM) in saline containing CO2/HCO3- acidified the cells in both nuclei, but the acidification was greater in the NTS. Restoration of extracellular Cl- after Cl- depletion during the control condition acidified the cells. Immunohistochemical studies of glial fibrillary acid protein demonstrated much denser staining in the RTN compared with the NTS. We conclude that there is evidence of Na+-HCO3- cotransport and Na+/H+ exchange in glia in the RTN and NTS, but the distribution of glia and the distribution of these pH-regulatory functions are not identical in the NTS and RTN. The differential strength of glial pH regulatory function in the RTN and NTS may also alter CO2 chemosensory neuronal function at these two chemosensitive sites in the brain stem.     

Adaptations to hypercapnic conditions in the nutria (Myocastor coypus)--in vivo and in vitro CO2 titration curves

Arterial blood acid-base status of unanesthetized, unrestrained nutria was studied during exposure to 5, 10 and 14.5% CO2 for 6 hr. Control values, pH = 7.426 +/- 0.037, PaCo2 = 36.5 +/- 3.1 mmHg and [HCO-3] = 24.3 +/- 2.5 mM/1 (n = 24), are within the normal range reported for other mammals. Values after 6 hr of exposure to 10% CO2 were: pH = 7.355 +/- 0.043, PaCO2 = 71.0 +/- 3.6 mmHg and [HCO-3] = 38.0 +/- 4.1 mM/l (n = 5). Arterial blood buffer slopes, obtained from the in vitro titration curve, did not show any pattern of adaptation to hypercapnia. Whole body buffer slopes, calculated from the in vivo CO2 titration curve, showed significantly higher values for the nutria than for the rat, dog and man, under comparable conditions [beta(delta HCO-3/delta pH)] = 57.0 slykes for nutria, 32.6 for rat and 11.8 for man. delta H+/delta PaCO2 = 0.38. mM/l/mmHg for nutria, 0.55 for rat and 0.76 for man. The results suggest that the nutria possesses an efficient metabolic mechanism for regulation of pH level during exposure to hypercapnic conditions.     

The effects of exogenous extracellular carbonic anhydrase on CO2 excretion in rainbow trout (Oncorhynchus mykiss): role of plasma buffering capacity

The buffering capacity (beta) of rainbow trout (Oncorhynchus mykiss) plasma was manipulated prior to intravascular injection of bovine carbonic anhydrase to test the idea that proton (H+) availability limits the catalysed dehydration of HCO3- within the extracellular compartment. An extracorporeal blood shunt was employed to continuously monitor blood gases in vivo in fish exhibiting normal plasma beta (-3.9+/-0.3 mmol 1(-1) pH unit(-1)), and in fish with experimentally (using N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) elevated plasma beta (-12.1+/-1.1 mmol 1(-1) pH unit(-1)). An injection of 5 mg kg(-1) carbonic anhydrase equally reduced (after 90 min) the arterial partial pressure of CO2 in trout with regular (-0.23+/-0.05 Torr) or high (-0.20+/-0.05 Torr) plasma beta; saline injection was without effect. Because ventilation and venous blood gases were unaffected by carbonic anhydrase, the effect of extracellular carbonic anhydrase in lowering arterial partial pressure of CO2 was likely caused solely by a specific enhancement of CO2 excretion owing to acceleration of HCO3- dehydration within the plasma. The lowering of arterial partial pressure of CO2 in trout after injection of exogenous carbonic anhydrase provides the first in vivo evidence that the accessibility of plasma HCO3- to red blood cell carbonic anhydrase constrains CO2 excretion under resting conditions. Because the velocity of red blood cell Cl-/HCO3- exchange governs HCO3- accessibility to red blood cell carbonic anhydrase, the present study also provides evidence that CO2 excretion at rest is limited by the relatively slow rate of Cl-/HCO3- exchange. The effect of carbonic anhydrase in lowering arterial partial pressure of CO2 was unrelated to plasma buffering capacity. While these data could suggest that H+ availability does not limit extracellular HCO3- dehydration in vivo at resting rates of CO2 excretion, it is more likely that the degree to which plasma beta was elevated in the present study was insufficient to drive a substantially increased component of HCO3- dehydration through the plasma.     

Effects of moderate hypoxemia and hypocapnia on CSF [H+] and ventilation in man.

The effects of 26 h of normoxic hypocapnia (PaCO2, 31 MMHg) vs. 26 h of hypocapnia plus hypobaric hypoxia (PaCO2 32, PaO2 57 mmHg) were compared with respect to: a) CSF acid-base status; and b) the spontaneous ventilation (at PIO2 145 mmHg) which followed the imposed (voluntary) hyperventilation. For each condition of prolonged hypocapnia, PaCO2 was held constant throughout and pHa and [HCO3-]a were constant over the final 6-10 h. We assumed that measured changes in lumbar CSF acid-base status paralleled those in cisternal CSF. Spontaneous hyperventilation followed both normoxic and hypoxic hypocapnia but was significantly greater following hypoxic hypocapnia. In the CSF, pH compensation after 26 h of hyperventilation was incomplete (similar to 45-50%), was similar to that in arterial blood, and was unaffected by a superimposed hypoxemia. These data were inconsistent with current theory which proposes the regulation of CSF [HCO2] via local mechanisms and, in turn, the mediation of ventilatory acclimatization to hypoxemia and/or hypocapnia via CSF [H+]. Alternative mediators of ventilatory acclimatization were postulated, including mechanisms both dependent on and independent of "chemoreceptor" stimuli.     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

C1/HCO3 exchange in human placental brush border membrane vesicles

Membrane transport pathways for transplacental transfer of CO2/HCO3 were investigated by assessing the possible presence of a Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. Cl/HCO3 exchange was tested for in preparations of purified brush border membrane vesicles by 36Cl tracer flux measurements and determinations of acridine orange fluorescence changes. Under 10% CO2/90% N2 the imposition of an outwardly directed HCO3- concentration gradient (pHo 6/pHi 7.5) stimulated Cl- uptake to levels approximately 2-fold greater than observed at equilibrium. Maneuvers designed to offset the development of ion gradient-induced diffusion potentials (valinomycin, Ko = Ki) significantly reduced HCO3- gradient-driven Cl- uptake but concentrative accumulation of Cl- persisted. Early time point determinations performed in the presumed absence of membrane potential suggests the reduced level of HCO3- gradient-driven Cl- uptake resulted from a more rapid dissipation of the HCO3- concentration gradient. Concentrative accumulation of Cl- was not observed in the presence of a pH gradient alone under 100% N2, suggesting a preference of HCO3- over OH- as a substrate for transport. As monitored by acridine orange fluorescence the Cl- gradient-dependent collapse of an imposed pH gradient (pHo 8.5/pHi 6) was accelerated in the presence of CO2/HCO3 when compared with its absence, indicating coupling of HCO3- influx to Cl- efflux. Increasing concentrations of the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid were observed to cause a stepwise reduction in HCO3- gradient-driven Cl- uptake (I50 approximately 25 microM) further suggesting the presence of a Cl/HCO3 exchange mechanism. The results of this study provide evidence for a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl/HCO3 exchange mechanism in the maternal-facing membrane of human placental epithelial cells. The identification of an ion-coupled HCO3- transport pathway in placental epithelia may suggest functional roles in mediating transplacental transfer of CO2 as well as maintenance of fetal acid/base balance.     

Intracisternal DIDS enhances ventilatory response to rebreathing CO2 in rats

In 11 anesthetized rats, we tested the hypothesis that carrier-mediated anion transport in part determines the medullary chemoreceptor response to acute hypercapnia by infusing the transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in mock cerebrospinal fluid (CSF) into the cisterna magna. In five additional rats with sham CSF infusion, we found no effect of mock CSF on the response to rebreathing CO2. Dye infused into the cistern stained the putative chemoreceptor areas on the ventral surface of the medulla. DIDS, at 10 to 1,000 nM, increased the respiratory response to CO2 in a dose-related manner but had no effect on arterial pressure or heart rate. At 1,000 nM, the hypercapnic minute ventilation response was almost doubled because of both volume and rate of breathing. We conclude that the net effect of anion transport is to mitigate the stimulus to the medullary chemoreceptors during acute hypercapnia.     

Carbon dioxide storage and nonbicarbonate buffering in the human body before and after an Himalayan expedition

Before and 7-12 days after an Himalayan expedition CO2 equilibration curves were determined in the blood plasma of 12 mountaineers by in vitro and in vivo CO2 titration; in vivo osmolality changes (delta Osm x deltaPCO2(-1), deltaOsm x delta pH(-1), where PCO2 is the partial pressure of CO2) during the latter experiments yielded estimates of whole body CO2 storage. In vitro -delta[HCO3-] x delta pH(-1) [nonbicarbonate buffer capacity (beta) of blood] was increased 7 days after descent [before 31.3 (SEM 0.4) mmol x kgH2O(-1), after 38.3 (SEM 3.9) mmol x kgH2O(-1); P<0.05] resulting from an increased proportion of young erythrocytes; in additional experiments an augmented beta was found in young (low density cells) compared to old cells [<1.097 g x ml(-1): 0.216 (SEM 0.028) mmol x gHb(-1), >1.100 g x ml(-1): 0.145 (SEM 0.013) mmol x gHb(-1), where Hb is haemoglobin; P < 0.02]. In spite of increased Hb mass in vivo delta[CO2total] x deltaPCO2(-1) [0.192 (SEM 0.010) mmol x kgH2O(-1) x mmHg(-1)] and -delta[HCO3-] x delta pH(-1) [17.9 (SEM 1.0) mmol x kgH2O(-1)] as indicators of extracellular beta rose only slightly after altitude (7 days +16%, P<0.02; +7%, NS) because of haemodilution. The deltaOsm x deltaPCO2(-1) [0.230 (SEM 0.015) mosmol x kgH2O(-1) x mmHg(-1)] remained unchanged. Prealtitude differences in deltaOsm x delta pH(-1) between hypercapnia [-41 (SEM 5) mosmol x kgH2O(-1)] and hypocapnia [-20 (SEM 3) mosmol x kgH2O(-1); P<0.01] disappeared temporarily after return since the former slope was reduced. The high value during hypercapnia before ascent probably resulted from mechanisms stabilizing intracellular pH during moderate hypercapnia which were attenuated after descent.     

Na(+)-dependent Cl-HCO3 exchange in the squid axon. Dependence on extracellular pH.

Intracellular pH (pHi) in squid giant axons recovers from acid loads by means of a Na(+)-dependent Cl-HCO3 exchanger, the actual mechanism of which might be exchange of: (i) external Na+ and HCO3- for internal Cl- and H+, (ii) Na+ plus two HCO3- for Cl-, (iii) Na+ and CO3= for Cl-, or (iv) the NaCO3- ion pair for Cl-. Here we examine sensitivity of transport to changes of extracellular pH (pHo) in the range 7.1-8.6. We altered pHo in four ways, using: (i) classical "metabolic" disturbances in which we varied [HCO3-]o, [NaCO3-]o, and [CO3=]o at a fixed [CO2]o; (ii) classical "respiratory" disturbances in which we varied [CO2]o, [NaCO3-]o, and [CO3=]o at a fixed [HCO3-]o; (iii) novel mixed-type acid-base disturbances in which we varied [HCO3-]o and [CO2]o at a fixed [CO3=]o and [NaCO3-]o; and (iv) a second series of novel mixed-type disturbances in which we varied [CO2]o, [CO3=]o, and [Na+]o at a fixed [HCO3-]o and [NaCO3-]o. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. The equivalent acid extrusion rate (JH) was computed from the rate of pHi recovery (i.e., increase) in the presence of Na+ and HCO3-. When pHo was varied by method (i), which produced the greatest range of [CO3=]o and [NaCO3-]o values, JH increased with pHo in a sigmoidal fashion; the relation was fitted by a pH titration curve with a pK of approximately 7.7 and a Hill coefficient of approximately 3.0. With method (ii), which produced smaller changes in [CO3=]o and [NaCO3-]o, JH also increased with pHo, though less steeply. With method (iii), which involved changes in neither [CO3=]o nor [NaCO3-]o, JH was insensitive to pHo changes. Finally, with method (iv), which involved changes in neither [HCO3-] nor [NaCO3-]o, but reciprocal changes in [CO3=]o and [Na+]o, JH also was insensitive to pHo changes. We found that decreasing pHo from 8.6 to 7.1 caused the apparent Km for external HCO3- ([Na+]o = 425 mM) to increase from 1.0 to 26.7 mM, whereas Jmax was relatively stable. Decreasing pHo from 8.6 to 7.4 caused the apparent Km values for external Na+ ([HCO3-]o = 48 mM) to increase from 8.6 to 81 mM, whereas Jmax was relatively stable.(ABSTRACT TRUNCATED AT 400 WORDS)