Effects of acetazolamide on cerebrospinal fluid ions in metabolic alkalosis in dogs

We hypothesized that inhibition of carbonic anhydrase in the central nervous system by acetazolamide should limit the rise in cisternal cerebrospinal fluid (CSF) [HCO3-] observed in metabolic alkalosis. To test this hypothesis, isosmotic isonatremic metabolic alkalosis was produced in two groups of anesthetized, paralyzed, and mechanically ventilated dogs (8 in each group). Group II animals received 50 mg/kg of acetazolamide intravenously 1 h before induction of metabolic alkalosis of 5-h duration. Renal effects of acetazolamide were eliminated by ligation of renal pedicles. In both groups cisternal CSF [Na+] remained relatively constant during metabolic alkalosis. In group I CSF [Cl-] decreased 3.6 and 8.2 meq/l, respectively, 2.5 and 5 h after induction of metabolic alkalosis. Respective increments in CSF [HCO3-] were 3.4 and 6.0 meq/l. In acetazolamide-treated dogs, during metabolic alkalosis, increments in CSF [HCO3-] (4.8 and 7.2 meq/l, respectively, at 2.5 and 5 h) and decrements in CSF [Cl-] (9.1 and 13.3 meq/l) were greater than those observed in group I. We conclude that, in dogs with metabolic alkalosis and bilateral ligation of renal pedicles, acetazolamide impairs CSF regulation of HCO3- and Cl- ions; acetazolamide not only failed to impede HCO3- rise but actually appeared to increase it. The mechanisms for these observations are discussed.     

Cerebrospinal fluid ions in metabolic acidosis in dogs: effects of acetazolamide

We hypothesized that, during isosmotic isonatremic HCl acidosis with maintained isocapnia in cisternal cerebrospinal fluid (CSF), acetazolamide, by inhibiting carbonic anhydrase (CA) in the central nervous system (CNS), should produce an isonatric hyperchloric metabolic acidosis in CSF. Blood and CSF ions and acid-base variables were measured in two groups of anesthetized and paralyzed dogs with bilateral ligation of renal pedicles during 5 h of HCl acidosis (plasma [HCO3-] = 11 meq/l). Mechanical ventilation was regulated such that arterial PCO2 dropped and CSF Pco2 remained relatively constant. In group I (control group, n = 6), CSF [Na+] remained unchanged, [HCO3-] and strong ions difference (SID) fell, respectively, 6.1 and 5 meq/l, and [Cl-] rose 3.5 meq/l after 5 h of acidosis. In acetazolamide-treated animals, (group II, n = 7), CSF [Na+] remained unchanged, [HCO3-], and SID fell 11 and 7.1 meq/l, respectively, and [Cl-] rose 7.1 meq/l. We conclude that during HCl acidosis inhibition of CNS CA by acetazolamide induces an isonatric hyperchloric metabolic acidosis in CSF, which is more severe than that observed in controls.     

Dual contribution theory of regulation of CSF HCO3 in respiratory acidosis.

Regulation of CSF HCO3-in respiratory acidosis was studied in light of the "dual contribution theory," which proposed that there were two sources for the CSF HCO3-increase: 1) HCO3-by diffusion from plasma and 2) HCO3-generated in the CNS and catalyzed by the local carbonic anhydrase (J. Appl. Physiol. 38: 504-512, 1975). In anesthetized dogs with an increase in Paco2 of 30 mmHg for 4 h the plasma HCO3 increased 2 meq/1 and CSF 6 meq/1. In combined respiratory and metabolic acidosis, plasma HCO3-did not increase but CSF HCO3-increased 6 meq/1. In combined acidosis and intraventricular injections of acetazolamide no increase in plasma or CSF HCO3-occurred. In combined respiratory acidosis and metabolic alkalosis and intraventricular acetazolamide, plasma HCO3-increased 15 meq/1 but CSF HCO3-increased 6 meq/1. Brain and CSF ammonia increased linearly and selectively with the increase in the relative contribution of CNS HCO3-increase. Therefore regulation of CSF HCO3-in respiratory acidosis depends on both components of the dual contribution theory, where each component can provide the total CSF HCO3-increase under appropriate experimental conditions. The control mechanism may be sensitive to changes in [H+] on the brain side of the blood-brain barrier.     

Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4- isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.     

Furosemide and cerebrospinal fluid ions during acute respiratory acidosis

The purpose of this study was to investigate the effects of furosemide, an inhibitor of NaCl cotransport, on cisternal cerebrospinal fluid (CSF) acid-base balance during acute respiratory acidosis (ARA). We measured blood and CSF acid-base variables in two groups (n = 7 in each) of anesthetized, paralyzed, and mechanically ventilated dogs with bilateral ligation of renal pedicles (to eliminate saluresis). After base-line samples were obtained (-1 h), furosemide (50 mg/kg) was administered intravenously within 15 min (group II); group I received an equal volume of half-normal saline. ARA was induced 1 h later (0 h) and arterial CO2 tension was maintained between 55 and 60 Torr for 5 h. Mean cisternal CSF PCO2 was 42.8 +/- 2.6 and 39.5 +/- 1.7 Torr, respectively in groups I and II and rose approximately 20 Torr during ARA. In group I, CSF [HCO3-] was 22.0 +/- 1.0, 24.8 +/- 0.6, and 25.4 +/- 1.6 meq/l, respectively at 0, 2.5, and 5 h. Respective values for group II were 22.2 +/- 1.3, 24.3 +/- 1.8, and 24.6 +/- 1.0 meq/l. These values were not significantly different from each other. In each group, CSF [Na+-Cl-] increased significantly during ARA, but the changes were not significantly different when the two groups were compared. We conclude that furosemide at the dose used in the present study does not change ionic composition and acid-base balance of cisternal CSF compared with control. Because changes in CSF [Na+-Cl-] during ARA were similar in both groups, any inhibition of Cl- influx into CSF by furosemide should have been proportional to that of Na+.     

Effect of plasma [K+] on the DC potential and on ion distributions between CSF and blood.

Keeping the arterial pH at 7.4 and PaCO2 at 40 mmHg in eight anesthetized dogs, we acutely raised plasma potassium concentration from 3.4 to 8.2 meq/1, then allowed it to decay back to control levels. The cerebrospinal fluid (CSF)-blood electrical potential difference (pd) increased 13.2 mV per 10-fold increase in plasma [K+]. Again keeping arterial pH at 7.4 and PaCO2 at 40 mmHg, we elevated plasma [K+] in four dogs from 3.3 to 8.0 meq/1 and maintained this level for 6 h. We found 1) that the PD increased from a control value of +1.3 to +8.9mV, showing no tendency to decay over the 6 h; and 2) that the change in PD did not affect the distribution of Na+, K+, H+, Cl-, or HCO3- between blood and CSF over the 6 h. These results suggest that under these conditions the PD between CSF and blood may play no effective role in determining the distributions of these charged species by 6 h. These results are contrasted with recent findings which suggest that H+ and HCO3- are distributed according to passive forces between CSF and blood.     

Changes in smooth muscle cell pH during hypoxic pulmonary vasoconstriction: a possible role for ion transporters

Hypoxic pulmonary vasoconstriction (HPV) occurs in smooth muscle cells (SMC) from small pulmonary arteries (SPA) and is accompanied by increases in free cytoplasmic calcium ([Ca2+]i) and cytoplasmic pH (pHi). SMC from large pulmonary arteries (LPA) relax during hypoxia, and [Ca2+]i and pHi decrease. Increases in pHi and [Ca2+]i in cat SPA SMC during hypoxia and the augmentation of hypoxic pulmonary vasoconstriction by alkalosis seen in isolated arteries and lungs suggest that cellular mechanisms, which regulate inward and outward movement of Ca2+ and H+, may participate in the generation of HPV. SMC transport systems that regulate pHi include the Na+ - H+ transporter which regulates intracellular Na+ and H+ and aids in recovery from acid loads, and the Na+ -dependent and Na+ -independent Cl-/HCO3- transporters which regulate intracellular chloride. The Na+ -dependent Cl-/HCO3- transporter also aids in recovery from acidosis in the presence of CO2 and HCO3-. The Na+ -independent Cl-/HCO3- transporter aids in recovery from cellular alkalosis. The Na+ - H+ transporter was present in SMC from SPA and LPA of the cat, but it seemed to have little if any role in regulating pHi in the presence of CO2 and HCO3-. Inhibiting the Cl-/HCO3- transporters reversed the normal direction of pHi change during hypoxia, suggesting a role for these transporters in the hypoxic response. Future studies to determine the interaction between pHi, [Ca2+]i and HPV should ascertain whether pHi and [Ca2+]i changes are linked and how they may interact to promote or inhibit SMC contraction.     

DIDS decreases CSF HCO3- and increases breathing in response to CO2 in awake rabbits

An inhibitor of the HCO3-/Cl- exchange carrier protein, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or vehicle was infused in mock cerebrospinal fluid (CSF) via the cisterna magna in conscious rabbits at 10 mumol/l for 40 min at 10 microliter/min. Neither treatment had any effect over 2-5 h on the non-CO2-stimulated CSF ion values or blood gases. With CO2 stimulation such that arterial PCO2 (PaCO2) was increased 25 Torr over 3 h, DIDS treatment significantly decreased the stoichiometrically opposite changes in CSF [HCO3-] and [Cl-] that normally accompany hypercapnia and reflect ionic mechanisms of CSF pH regulation. Expressed as delta CSF [HCO3-]/delta PaCO2, DIDS treatment decreased the CSF ionic response by 35%. In a separate paired study design DIDS administration via the same protocol had no effect on resting ventilation but significantly increased the ventilation and tidal volume responses to a 28-Torr increase in PaCO2. Expressed as change in minute ventilation divided by delta PaCO2, DIDS treatment produced a 39.6% increase. The results support the concept of a DIDS-inhibitable anion exchange carrier being involved in CSF pH regulation in hypercapnia and suggest a DIDS-related effect on the ventilatory response to CO2.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

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)     

Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.     

Roles of CO2, O2, and acid in arteriovenous [H+] difference during muscle contractions

To determine the origins of the arteriovenous [H+] difference of muscle during contractions, arterial and muscle venous blood sample pairs were taken before and after 0.5, 5.0, and 30.0 min of 4/s isometric twitches of the gastrocnemius-plantaris muscle group of anesthetized dogs. These samples were analyzed for PO2, PCO2, and pH, the concentrations of O2, CO2, K+, Na+, La-, and Cl- in whole blood, and La-, K+, Na+, and Cl- in plasma. Whole blood was hemolyzed and analyzed for PO2, PCO2, and pH. Net O2 uptake, CO2 output, L, K+, Na+, and Cl- were calculated in addition to net output of non-CO2 acid (HA) and strong ion difference ([SID]) and common ion [SID] ([K+] + [Na+] - [Cl-] - [La-]). From these data we partitioned the origins of the arteriovenous [H+] difference via the common PCO2-pH diagram and via a [H+]-PCO2 diagram and determined whether true plasma arteriovenous [H+] differences reflect plasma and cell arteriovenous [H+] differences. The arteriovenous [H+] differences of plasma and hemolyzed blood were the same, showing that true plasma does reflect plasma and cells. K+ showed a small significant but transient output. Na+ was not significant, whereas Cl- showed a significant transient uptake. Lactate output and HA, calculated for dog blood acid-base, showed transient outputs and were the same. At 5.0 min when the arteriovenous difference was largest, CO2 alone would have increased [H+] 15.9 nmol/l whereas desaturation of Hb would have decreased [H+] 4.2 nmol/l and lactate could have raised [H+] 1.0 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water and electrolyte balance in the vascular space during graded exercise in humans

We analyzed the changes in water content and electrolyte concentrations in the vascular space during graded exercise of short duration. Six male volunteers exercised on a cycle ergometer at 20 degrees C (relative humidity = 30%) as exercise intensity was increased stepwise until voluntary exhaustion. Blood samples were collected at exercise intensities of 29, 56, 70, and 95% of maximum aerobic power (VO2max). A curvilinear relationship between exercise intensity and Na+ concentration in plasma ([Na+]p) was observed. [Na+]p significantly increased at 70% VO2max and at 95% VO2max was approximately 8 meq/kgH2O higher than control. The change in lactate concentration in plasma ([Lac-]p) was closely correlated with the change in [Na+]p (delta[Na+]p = 0.687 delta[Lac-]p + 1.79, r = 0.99). The change in [Lac-]p was also inversely correlated with the change in HCO3- concentration in plasma (delta[HCO3-]p = -0.761 delta[Lac-]p + 0.22, r = -1.00). At an exercise intensity of 95% VO2max, 60% of the increase in plasma osmolality (Posmol) was accounted for by an increase in [Na+]p. These results suggest that lactic acid released into the vascular space from active skeletal muscles reacts with [HCO3-]p to produce CO2 gas and Lac-. The data raise the intriguing notion that increase in [Na+]p during exercise may be caused by elevated Lac-.     

Red cell hemoglobin, hydrogen ion and electrolyte concentrations during exercise in trained and untrained subjects

Red cell concentrations of hemoglobin (MCHC), H+, Na+, K+, Mg++, cl- were measured in femoral venous blood of six untrained (UT), six endurance trained (TR) and three semitrained (ST) subjects during graded increasing work (4, 8, 12, 18 and 24 mkp/s, 10-15 min on each step) on a bicycle ergometer. Before exercise no significant differences were detected for the measured variables when comparing UT and TR. During exercise MCHC, [Na+], [K+] and [Mg++] remained constant indicating lack of water shift into the erythrocytes in spite of a marked acidosis (lowest pH Blood value 7.225). This lack resulted from an elevated extracellular osmolality. [H+]Ery and [Cl-]Ery maximally increased by 2.0 X 10(-8) eq/kg H2O and 10 meq/l, respectively. The change was markedly greater in UT than in TR at equal load. However, if [H+] Ery and [Cl-] Ery were related to pH of whole blood, differences between groups, almost disappeared and the ions were distributed as predictable from in vitro experiments (Fitzsimmons and Sendroy, 1961). Behaviour of H+ and Cl- may be of importance for oxygen dissociation under in vivo conditions.     

Intracellular pH-regulating mechanism of the squid axon. Relation between the external Na+ and HCO-3 dependences

The intracellular pH-regulating mechanism of the squid axon was examined for its dependence on the concentrations of external Na+ and HCO3-, always at an external pH (pHo) of 8.0. Axons having an initial intracellular pH (pHi) of approximately 7.4 were internally dialyzed with a solution of pH 6.5 that contained 400 mM Cl- and no Na+. After pHi had fallen to approximately 6.6, dialysis was halted, thereby returning control of pHi to the axon. With external Na+ and HCO-3 present, intracellular pH (pHi) increased because of the activity of the pHi-regulating system. The acid extrusion rate (i.e., equivalent efflux of H+, JH) is the product of the pHi recovery rate, intracellular buffering power, and the volume-to-surface ratio. The [HCO3-]o dependence of JH was examined at three fixed levels of [Na+]o: 425, 212, and 106 mM. In all three cases, the apparent Jmax was approximately 19 pmol X cm-2 X s-1. However, the apparent Km (HCO3-) was approximately inversely proportional to [Na+]o, rising from 2.6 to 5.4 to 9.7 mM as [Na+]o was lowered from 425 to 212 to 106 mM, respectively. The [Na+]o dependence of JH was similarly examined at three fixed levels of [HCO3-]o: 12, 6, and 3 mM. The Jmax values did not vary significantly from those in the first series of experiments. The apparent Km (Na+), however, was approximately inversely related to [HCO3-]o, rising from 71 to 174 to 261 mM as [HCO3-]o was lowered from 12 to 6 to 3 mM, respectively. These results agree with the predictions of the ion-pair model of acid extrusion, which has external Na+ and CO3= combining to form the ion pair NaCO3-, which then exchanges for internal Cl-. When the JH data are replotted as a function of [NaCO3-]o, data from all six groups of experiments fall along the same Michaelis-Menten curve, with an apparent Km (NaCO3-) of 80 microM. The ordered and random binding of Na+ and CO3= cannot be ruled out as possible models, but are restricted in allowable combinations of rate constants.     

Bioelectric properties and ion transport across excised canine fetal and neonatal airways

Knowledge of liquid secretion by fetal lung stems from studies of sheep. We extended these studies to dogs and examined the persistence of the fetal pattern of airway epithelial permeability and ion transport in the neonatal animal. Plasma and lung liquid from fetal dogs were analyzed for Na+, K+, Cl-, and HCO3-. Only the Cl- concentration of fetal lung liquid (129 meq/l) was significantly different from that of fetal plasma (111 meq/l). Segments of trachea from fetal and neonatal (less than 1, 7-10, and 21-46 days after birth) dogs were excised and mounted in flux chambers. The transepithelial potential difference (PD) of all tissues was oriented lumen negative (9.8-14.8 mV). Under short-circuit conditions, unidirectional Na+ flows were symmetrical. Cl- was secreted, and the secretion was equivalent to short-circuit current (Isc). Cl- secretion persisted under open-circuit conditions. Lobar bronchi from 21- to 46-day neonates absorbed Na+ (1.9 mueq.cm-2.h-1), but unidirectional flows of Cl- were symmetrical. Amiloride (10(-4) M) reduced Isc of neonatal bronchi by 47% but did not affect fetal bronchi. Isoproterenol increased Isc of both fetal (33%) and neonatal (40%) bronchi. These responses suggest that fetal bronchi do not absorb Na+ but can be stimulated to secrete Cl-. We conclude that Cl- secretion by epithelium of large airways may contribute to fetal lung liquid production, but it is unlikely that the tracheal epithelium is involved in fluid absorption at birth. Whereas fetal bronchi appear to secrete Cl-, neonatal bronchi absorb Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bicarbonate transport mechanisms in the Ambystoma kidney proximal tubule: transepithelial potential measurements

Modes of bicarbonate entry from tubule lumen to cell were examined in isolated Ambystoma proximal tubules, using determinations of transepithelial potential differences (V3). (1) Upon removal of luminal substrate, tubules first equilibrated in bilateral (lumen and bath) 94.72 mM Cl- and 10 mM HCO3- yielded a change in V3 between the experimental and control circumstances of +1.8 mV (delta V3). (2) The identical experiment conducted under the condition of symmetrical 4.72 mM Cl- produced a delta V3 of +7.6 mV. This reduction of luminal and bath Cl- generates an amplification of delta V3 by a factor of 4.4 and reflects a substantial increase in the paracellular Cl- shunt resistance. Ensuing experiments were conducted in bilateral nominally Cl(-)-free solutions and in the absence of luminal substrate. The experimental protocols are divided into several situations where HCO3- is removed from the lumen, bath, or lumen and bath; the HCO3- removal sequences are repeated in the presence of luminal SITS and then after SITS washout. 0.5 mM SITS (4-acetoamido-4-isothiocyanostilbene-2,2'-disulfonate) was applied exclusively to the luminal perfusate. (1) Removal of luminal HCO3- in the absence of SITS produces a delta V3 of -1.9 mV, whereas, in the presence of SITS, the delta V3 measures -1.3 mV. Subsequent removal of luminal HCO3- in the presence of bath HCO3- (in the presence of luminal SITS) yields a delta V3 of -1.0 mV. All of these measurements reflect a decrease in HCO3- current across the basolateral membrane Na+ (HCO3-)n co-transporter; the role of a possible Cl-/Anion- antiport cannot be assessed. (2) Removal of bath HCO3- in the absence of SITS yields a delta V3 of +1.5 mV, whereas, in the presence of SITS, the delta V3 value measures +1.2 mV. Subsequent removal of bath HCO3- in the absence of luminal HCO3- (in the presence of SITS) yields a delta V3 of +0.8 mV. These experiments are consistent with an increase in HCO3- current across the basolateral Na+(HCO3-)n co-transporter, do not rule out the possibility of an apical HCO3- conductance pathway, and diminish the likelihood of an apical Cl-/HCO3- antiport system.     

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.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Effect of changing [K+], [Na+] and [Cl-] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO3(-) but with 95% O2/5% CO2 in both cases. In both cases, changing from 4 to 40 mM K+ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO3(-). Changing from 102 to 8 mM Na+ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO3(-) was insignificant but with 25 mM HCO3(-) the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K+ and Na+ were largely related to (Na+ + K+)-ATPase pump. Changes in nutrient Cl- from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of (Na+ + K+)-ATPase pump and Na+/Cl- symport. Removal of HCO3(-) and concurrent reduction of pH modify resistance of these pathways.     

Acid-base changes in the running greyhound: contributing variables.

To determine the factors responsible for changes in [H+] during and after sprint exercise in the racing greyhound, Stewart's quantitative acid-base analysis was applied to arterial blood plasma samples taken at rest, at 8-s intervals during exercise, and at various intervals up to 30 min after a 402-m spring (approximately 30 s) on the track. [Na+], [K+], [Cl-], [total Ca], [lactate], [albumin], [Pi], PCO2, and pH were measured, and the [H+] was calculated from Stewart's equations. This short sprint caused all measured variables to change significantly. Maximal changes were strong ion difference decreased from 36.7 meq/l at rest to 16.1 meq/l; [albumin] increased from 3.1 g/dl at rest to 3.7 g/dl; PCO2, after decreasing from 39.6 Torr at rest to 27.9 Torr immediately prerace, increased during exercise to 42.8 Torr and then again decreased to near 20 Torr during most of recovery; and [H+] rose from 36.6 neq/l at rest to a peak of 76.6 neq/l. The [H+] calculated using Stewart's analysis was not significantly different from that directly measured. In addition to the increase in lactate and the change in PCO2, changes in [albumin], [Na+], and [Cl-] also influenced [H+] during and after sprint exercise in the running greyhound.