Extracellular and intracellular pH with changes of temperature in the dogfish Scyliorhinus stellaris.

Larger Spotted Dogfish, Scyliorhinus stellaris, were exposed to varied ambient temperature (t) in order to determine the behavior of extracellular pH (pHe), Pco2 and bicarbonate concentration as well as intracellular pH (pHi) in three muscle types. pHe was found to vary with temperature slightly less than expected on the basis of the rule of constant relative alkalinity in juvenile (deltapH/deltat= -0.0148 per degree centigrade) as well as in adult (deltapH/deltat= -0.0136) fish. The absolute pHe values of adult fish were about 0.08 pH units higher than in juvenile fish. Arterial pco2 increased with rising temperature, the increase being much more marked in adult than in juvenile fish. Extracellular bicarbonate concentration (calculated from the pH and Pco2 values measured in arterial blood) was not maintained constant, but diminished in juvenile and increased in adult fish with increasing temperature, indicating that extracellular pH in dogfish is regulated by variations of both Pco2 and bicarbonate concentration. Variations of intracellular pH with temperature (deltapHi/deltat), -0.0178 for white muscle, -0.0334 for red muscle, and -0.0098 for heart muscle, were significantly different from the values of the extracellular compartment and, except for white muscle, significantly different from the condition for constant relative alkalinity (deltapH/deltat= -0.0183). These results are in agreement with the rule of constant relative alkalinity with respect to extracellular pH and possibly also with respect to an overall mean intracellular pH, but the rule is not quantitatively followed by the individual body compartments and tissues.     

Effect of hypocapnia on intracellular pH during metabolic acidosis.

Separate and combined effects of acute metabolic acidosis and hypocapnia were determined in skeletal and cardiac muscles of intact rats. Normocapnic metabolic acidosis, imposed by intraperitoneal injection of hydrochloric acid (6 mEq/kg), did not change skeletal muscle intracellular acid--base parameters. Hypocapnia, induced by mechanical hyperventilation, resulted in intracellular alkalosis within skeletal muscle during both respiratory alkalosis and compensated metabolic acidosis; changes of skeletal muscle intracellular bicarbonate concentration per unit change in carbon dioxide tension were identical during these two experimental procedures. These data suggest that processes other than physicochemical buffering neutralize protons taken into skeletal muscle cells during acute metabolic acidosis. The acid--base state of the heart was quite stable during these experimental manipulations; thus, it appears that cardiac muscle has an extraordinary buffering ability. Moreover, our data suggest that processes other than physicochemical buffering maintain cardiac intracellular pH normal during hypocapnia.     

Bicarbonate exchange between body compartments after changes of temperature in the larger spotted dogfish (Soyliorhinus stellaris)

Intracellular/extracellular and extracellular/sea-water bicarbonate exchanges were measured in Larger Spotted Dogfish (Sycliorhinus stellaris) exposed to 10 degrees C temperature step changes in a closed sea-water recirculation system. Changes of the bicarbonate concentration in blood plasma (= extracellular space) and in the recirculating sea-water were monitored for 36 h after the temperature change. Intracellular/extracellular transfer of bicarbonate was computed from bicarbonate changes in the extracellular space and sea-water. When the temperature was changed from 10 to 20 degrees C a signigicant transfer of bicarbonate was observed from the intracellular to the extracellular compartment and from the extracellular compartment to the sea-water. These transfers were reversed when the temperature was lowered from 20 to 10 degrees C. The exchange processes were practically completed after 18 h. The amount of bicarbonate exchanged between intracellular and extracellular compartments and sea-water was larger than predicted on the basis of in vitro buffer values of white, red and heart muscle, suggesting that additional tissues exchange significant amounts of bicarbonate with the extracellular space. It is concluded that physicochemical buffering is not sufficient to account for the observed adjustment of intracellular and extracellular pH and that bicarbonate exchange between body compartments and environment may be the most important regulatory mechamism, responsible for the final adjustment of acid-base balance in dogfish.     

Effect of systemic pH, PCO2 and bicarbonate concentration on biliary bicarbonate secretion in the rat

The effect of acute metabolic or respiratory acid-base disturbances on biliary bicarbonate secretion was examined in bile fistula rats. Animals were infused with ursodeoxycholate at a rate that stimulates bicarbonate secretion (1 mumole . min-1 X 100 gm-1), in control conditions and during acute acid-base disturbances. Metabolic acidosis or alkalosis were induced by HCl or NaHCO3 infusions, and respiratory acidosis or alkalosis were created respectively by adding CO2 to the inspired gas or by hyperventilation in artificially ventilated animals. Biliary bicarbonate concentration was always higher than plasma bicarbonate concentration. During metabolic disturbances, changing the plasma bicarbonate concentration from 9.2 to 30.2 mM stimulated biliary bicarbonate secretion by 113%. During respiratory disturbances, changing the plasma PCO2 from 25.5 to 59.8 mm Hg also increased biliary bicarbonate secretion by 89%. Biliary bicarbonate output was thus independent of plasma pH. When all animals were considered, bile flow was positively correlated with biliary bicarbonate concentration (r = 0.71, p less than 0.001). Acetazolamide significantly decreased ursodeoxycholate-induced bile flow and bicarbonate secretion by 20 and 22%, respectively. These results support the hypothesis that there is a relationship between ursodeoxycholate-induced bicarbonate secretion and bile flow. They are also consistent with the view that ursodeoxycholate-stimulated biliary bicarbonate secretion in the rat is strongly affected by plasma bicarbonate and PCO2, but not by plasma pH, and involves carbonic anhydrase.     

Intracellular buffering of heart and skeletal muscles during the onset of hypercapnia.

Changes in the total CO2 content of tissues were determined in order to characterize variations in intracellular acid-base parameters during the onset of hypercapnia. Within two minutes after an increasement in the CO2 tension of the inspired air of rats, there were large increases in the intracellular bicarbonate concentrations of both cardiac and skeletal muscles. Greater changes occurred in the heart, and its intracellular pH remained near normal during the first hour of hypercapnia; whereas there was an intracellular acidosis in skeletal muscle. This greater capacity of the heart to buffer excess CO2 has been linked to an increased movement of bicarbonate ions into and/or hydrogen ions out of cardiac cells during hypercapnia (Lai et al., 1973c). Yet, the buffer capacity of the heart was not compromised by metabolic acidosis during which there was a greatly reduced extracellular bicarbonate ion concentration and a greatly increased extracellular hydrogen ion concentration. The intracellular pH of the cardiac ventricle was stable following the imposition of a noncarbonic acid load on normocapnic rats.     

Brain cellular and mitochondrial respiration in media of altered pH

This study was designed to investigate the effects of altered pH on cellular aerobic energy metabolism in the immature and adult rat cerebral cortex. Cerebral cortical slice respiration was measured polarographically in acid and alkaline media. In separate experiments, the extracellular pH was changed by altering the HCO ₃ ^– concentration or the intracellular pH and extracellular pH were changed by altering the CO₂. Respiratory rates and oxidative phosphorylation in adult rat cerebral mitochindria also were measured in media with an altered pH. Increased intracellular pH inhibited respiratory rates in cortical slices from immature rats more than in tissue from adults. Decreasing the pH to 6.7 produced no changes in respiration in mature cortical slices and moderate inhibition of immature tissue respiration. In cerebral mitochondria, altered pH caused inhibition of State 3 respiration, respiratory control ratios, and ADP/O ratios. These changes were greater and occurred with smaller pH changes in the alkaline compared to the acid direction. From the results of these studies, we conclude that brain cellular respiration is not affected by moderate decreases in intracellular pH. With increased pH, there is inhibition of cellular and mitochondrial respiration, which may be the mechanism for the rise in lactic acid previously observed to result from hypocarbiain vivo.Abbreviations used BSA bovine serum albumin - DNP dinitrophenol - RCR respiratory control ratio     

Acid-base regulation in response to environmental hypercapnia in two aquatic salamanders, Siren lacertina and Amphiuma means

The partial pressure of CO2 (PCO2) in certain areas of the aquatic habitat of the salamanders Siren lacertina and Amphiuma means frequently rises to values of up to 60 mm Hg. This ambient hypercapnia occurs due to hindrance of gas exchange between water and air caused by dense water-surface vegetation. In order to investigate the acid-base regulation in response to the respiratory acidosis, which wound be expected to result from the high CO2 conductance of the amphibian skin, specimens of both species were subjected to water PCO2 of 47 mm Hg while having free access to normocapnic air in a closed water recirculation system. Arterial PCO2 rose considerably from 12 to 35 mm Hg in Siren and from 17 to 36 mm Hg in Amphiuma. The resultant fall in plasma pH remained uncompensated, whereas intracellular pH of white muscle and heart muscle of Siren were little affected owing to elevated intracellular bicarbonate concentrations. The bicarbonate accumulated in the intracellular compartments was in part produced by intracellular and extracellular nonbicarbonate buffering, and in part gained from the environment in exchange for Cl- ions. Elevated water bicarbonate concentration or bicarbonate infusion into Siren had no effect on the acid-base regulation. These data suggest that the availability of bicarbonate is not a limiting factor for extracellular compensation of increased PCO2, but that the threshold of the bicarbonate-regulating structures is simply not readjusted in hypercapnia. This type of regulation may have evolved as a result of the specific environmental conditions of these animals and may be considered as an energetically efficient way of maintaining a constant milieu for the pH-sensitive intracellular structures.     

Effects of PCO2, pH and extracellular calcium on contraction of airway smooth muscle from rats

The effect of changes in PCO2 on airway smooth muscle was studied in acetylcholine-induced contractions of isolated rat trachea. Elevation of superfusate PCO2 from control PCO2, 38 mm Hg (pH 7.49), to 168 mm Hg (pH 6.74) decreased tension to 68% of control tension; reduction of PCO2 to 19 mm Hg (pH 7.84) increased tension to 104%. Similar effects on tension occurred when pH was altered by varying superfusate bicarbonate concentration at constant PCO2. Modification of the response to changes in PCO2 by varying extracellular calcium (Ca2+) concentration and also by verapamil indicated that changes in PCO2 and pH may alter Ca2+ uptake by the smooth muscle. Calcium uptake was measured by 45Ca2+ and the lanthanum method. At control pH 7.49, net Ca2+ uptake was 5.34 mmol Ca2+/kg trachea 60 min after the onset of contraction; this decreased to 4.26 at pH 6.88, and increased to 6.58 at pH 7.85. The results suggest that the mechanism whereby changes in PCO2 affect airway smooth muscle contraction is a pH-dependent alteration of Ca2+ uptake.     

Bicarbonate and the regulation of ventilation

The regulation of ventilation involves a multifactorial control system with several feedback loops transmitting deviations from normal in pH, carbon dioxide tension (pCO₂) and oxygen tension (pO₂) to the control area. Variations in the size of the bicarbonate pool, caused by ventilatory or metabolic disturbances, can be expected to modify resting ventilation if hydrogen ion activity is the ultimate stimulus of the regulation of ventilation. A relationship between serum bicarbonate and resting ventilation can be identified in patients with stable acid-base disturbances including those in whom correction of the arterial blood pH was not achieved by respiratory adaptation. Why the pH in arterial blood is rarely returned to the normal range is not well understood. It may be an inadequacy of the control system, a “compromise” solution avoiding hypoxia in metabolic alkalosis or increasing work of breathing in metabolic acidosis, or a consequence of discrepancies in hydrogen ion activity in body fluids adjacent to and remote from the control site.Additional information about the role of bicarbonate in the control of ventilation may be obtained by measuring the response to carbon dioxide inhalation at varying extracellular bicarbonate concentrations. The increments in ventilation during inhalation of carbon dioxide are within individual limitations, inversely and exponentially related to the bicarbonate concentrations in blood.These observations are in accord with the concept that the extracellular bicarbonate concentration modulates resting ventilation and the ventilatory response to inhalation of fixed concentrations of carbon dioxide by acting as a determinant for the hydrogen ion activity within or adjacent to the central chemosensitive control area.     

Metabolic energy production during adrenergic pH regulation in red cells of the Atlantic salmon, Salmo salar

Whole blood from Atlantic salmon was incubated anaerobically at 10 degrees C so as to measure the metabolic activity of the nucleated erythrocytes. An acute extracellular acidosis was produced by adding either an acid solution (sham) or an acid solution with adrenaline (final concentration, 5 x 10(-4) M). The extracellular acidosis produced by the sham solution was transferred to the erythrocytes, whereas with adrenaline, intracellular pH actually increased in the face of a plasma acidosis. Indeed, the extracellular acidosis in the adrenaline-treated blood was significantly higher than that of the sham as a result of net H+ excretion from the erythrocyte. This pH response of the erythrocyte was accompanied by a proportional increase in the O2 consumption of the blood, with no change in lactate production. In comparison to sham-treated cells, the content of erythrocytic nucleotide triphosphates initially decreased upon addition of adrenaline but was thereafter maintained at a constant NTP/Hb ratio presumably due to an increased ATP turnover. In conclusion, it appears that the aerobic rather than anaerobic metabolism of erythrocytes is accelerated upon addition of adrenaline to blood, and that this increased metabolism is involved in fueling the membrane transport processes involved in adrenergic pH regulation of salmonid red cells.     

The effect of hypocapnia on intracellular pH and bicarbonate

The intracellular pH (pHi) and bicarbonate concentration ([HCO₃⁻]icw) of cardiac and skeletal muscles were monitored during respiratory alkalosis in order to further elucidate the homeostatic processes which operate in these tissues to ameliorate deviations from normal acid-base status. Rats were mechanically hyperventilated to induce hypocapnia, pHi was determined by the DMO method, and [HCO₃⁻]icw was calculated from the Henderson-Hasselbach equation using pHi and the partial pressure of carbon dioxide of vena caval blood. A significant intracellular alkalosis occurred in both cardiac and skeletal muscles during hypocapnia, but the changes in pHi were less in the heart than in skeletal muscle. The decreases in cardiac [HCO₃⁻]icw were greater than those attributable to the physicochemical buffering of the heart. These data are consistent with an intramyocardial source of protons other than physicochemical buffering respiratory alkalosis. The decreases in skeletal muscle [HCO₃⁻]icw were less than those due to physicochemical buffering. These data are consistent with a net extrusion from skeletal muscles cells of the protons derived from physicochemical buffering during respiratory alkalosis.     

Intracellular and extracellular acid-base changes in hemorrhagic shock.

Each of 21 dogs was bled until mean arterial blood pressure fell to 50 torr; this hemorrhagic shock state was then maintained for two hours. During hemorrhagic shock, the blood lactate concentration increased sixfold. The severe metabolic acidosis in arterial blood was partially compensated by a decreased PCO2 caused by increased ventilation. However, in mixed venous blood, the metabolic acidosis was combined with a respiratory acidosis. This hypercapnia in venous blood was indicative of the increased PCO2 in tissues poorly perfused following hemorrhage. The increase in the PCO2 of the femoral venous blood was greater than that in mixed venous blood, suggesting that some tissue beds were better perfused than those of the hind limb during shock. The intracellular lactate concentration of hind limb skeletal muscle was greatly increased in the shock state, and tissue PCO2 rose. Intracellular pH of skeletal muscle was only slightly decreased and bicarbonate concentration was unchanged during this combined metabolic and respiratory acidosis. This capacity of skeletal muscle to maintain a high HCO-3 concentration in intracellular fluid during metabolic acidosis may be an enhanced response of the mechanism responsible for maintaining (HCO-3)i normally at a level approximately ten times that which would be expected if HCO-3 were distributed passively.     

Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid–base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H⁺-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH_e and pH_i) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO₂ conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO₂. Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH_e whenever seawater pH changes. However, measurements of pH_i demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na⁺ and HCO₃⁻, suggesting a bicarbonate buffer mechanism involving secondary active Na⁺-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH_i enables calcification to proceed despite decreased pH_e. However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.     

Gastrointestinale Ursachen von metabolischer Alkalose

Buffer systems tightly regulate the extracellular H⁺-ion concentration to achieve a pH of 7.4 (40?nmol/l). Decreases in H⁺-ions result in alkalemia and the corresponding disease conditions are either metabolic or respiratory alkalosis. Metabolic alkalosis results from loss of acid, gain of bicarbonate or a combination of both. Acid loss is frequently caused by vomiting of acidic gastric fluid. Bicarbonate can be obtained from exogenous sources but is most often generated by the kidneys via aldosterone (mineralocorticoid effect). Diarrhea usually causes metabolic acidosis because of the bicarbonate loss; however, congenital or acquired enteropathy with chloridorrhea may result in bicarbonate excess and metabolic alkalosis. Metabolic alkaloses caused by gastrointestinal conditions will be discussed. The relationship of an acid-base disorder on the one hand and volume contraction, chloride and potassium deficit on the other hand will be highlighted. The alveolar-arterial pO₂ gradient will be introduced to estimate the consequences of the respiratory compensation in patients with primary metabolic alkalosis.     

The effect of prolonged pancreatic secretion on blood acid-base balance in the conscious dog

Prolonged near maximal pancreatic secretion in conscious dogs has been found to result in a metabolic acidosis. This is mild and is accompanied by respiratory and other forms of compensation. Measurements of blood bicarbonate or base-excess changes cannot be used to estimate pancreatic bicarbonate output. The acidosis caused by pancreatic secretion cannot explain the changes in bicarbonate concentration seen in pancreatic juice during prolonged secretion.     

Urinary ammonia content as a determinant of urinary pH during chronic metabolic acidosis

During the course of metabolic acidosis, the urine pH initially falls but then rises to levels that seem inappropriate to the degree of systemic acidosis. We investigated the factors responsible for the reduced urinary acidity during chronic metabolic acidosis, using fasting as the model of acidosis and varying the serum bicarbonate concentration by acid or alkali supplements. The rise in urine pH was found to be greatest in those with the most profound acidosis and least in those with milder degrees. This paradoxical relation-ship could not be related to improvement of systemic acidosis, external potassium deficits, acidification defects, or increases of nonvolatile buffer excretion. The factor which correlated with the rise of urine pH was the urinary content of ammonia. This observation suggests that the urinary ammonia concentration may determine, rather than be determined by, the urinary acidity during chronic metabolic acidosis. Addition of ammonia to segments of the nephron with large secretory reserves for H⁺ should not elevate urine pH. Since the collecting duct is the only segment of the nephron shown to have limited H⁺ secretory capacity, we propose that the alkalinizing effect of ammonia on urine pH during prolonged acidosis is due to augmented addition of basic NH₃ to the collecting duct by diffusion from the loop of Henle. This view is compatible with the concept that ammonia entering the proximal tubule may be excreted by this route.     

Urinary ammonia content as a determinant or urinary pH during chronic metabolic acidosis.

During the course of metabolic acidosis, the urine pH initially falls but then rises to levels that seem inappropriate to the degree of systemic acidosis. We investigated the factors responsible for the reduced urinary acidity during chronic metabolic acidosis, using fasting as the model of acidosis and varying the serum bicarbonate concentration by acid or alkali supplements. The rise in urine pH was found to be greatest in those with the most profound acidosis and least in those with milder degrees. This paradoxical relation-ship could not be related to improvement of systemic acidosis, external potassium deficits, acidification defects, or increases of nonvolatile buffer excretion. The factor which correlated with the rise of urine pH was the urinary content of ammonia. This observation suggests that the urinary ammonia concentration may determine, rather than be determined by, the urinary acidity during chronic metabolic acidosis. Addition of ammonia to segments of the nephron with large secretory reserves for H⁺ should not elevate urine pH. Since the collecting duct is the only segment of the nephron shown to have limited H⁺ secretory capacity, we propose that the alkalinizing effect of ammonia on urine pH during prolonged acidosis is due to augmented addition of basic NH₃ to the collecting duct by diffusion from the loop of Henle. This view is compatible with the concept that ammonia entering the proximal tubule may be excreted by this route.     

Constant set points for pH and P(CO2) in cold-submerged skin-breathing frogs

The low temperatures encountered by overwintering frogs result in a large downregulation of metabolism and behaviour. However, little is known about acid-base regulation in the extreme cold, especially when frogs become exclusive skin-breathers during their winter submergence. Blood and muscle tissue acid-base parameters (pH, P(CO2), bicarbonate and lactic acid concentrations) were determined in submerged frogs exposed to a range of low temperatures (0.2-7 degrees C). At overwintering temperatures between T = 0.2 and 4 degrees C plasma pH and P(CO2) were maintained constant, whereas intracellular pH regulation resulted in larger pH-temperature slopes occurring in the presumably more active heart muscle (deltapH/deltaT = -0.0313) than in the gastrocnemius muscle (deltapH/deltaT = -0.00799). Although blood pH was not significantly affected by submergence between 0.2 and 4 degrees C (pH = 8.220-8.253), it declined in the 7 degrees C frogs (pH = 8.086), a decrease not linked to the recruitment of anaerobiosis. Plasma P(CO2) and pH in the cold appear to be regulated at constant levels, implying that cutaneous CO2 conductance in submerged frogs is adjusted within the range of overwintering temperatures. This is likely geared toward facilitating the uptake of oxygen under conditions of greater metabolic demand, however there remains the possibility that acid-base balance itself is maintained at a constant set point at the frog's natural overwintering temperatures.     

pH dependency of potassium efflux from sickled red cells

Potassium efflux from deoxygenated, hemoglobin S-containing red cells is often used as an "objective" in vitro measure of aed cell sickling, particularly during tests with antisickling agents. Since varying pH is known to affect both the extent of sickling and passive K+-flux across the red cell membrane, in opposite directions, we measured the sickling-related K+-efflux in sickle cell anemia (SS) and sickle cell trait (AS) red cells as a function of extracellular and intracellular pH. The sickling-related K+-efflux was found to show the same direction of pH dependence as normal red cells, so that as the extracellular pH was reduced below 7.6, sickling and K+-efflux were increasingly dissociated. A similar dissociation was observed between sickling and K+-efflux when the intracellular pH was lowered by increasing red cell organic phosphate levels. The sickling-related K+-efflux from osmotically shrunken AS cells (whose sickling tendency resembles that of SS cells) was similar in magnitude and pH dependency to that of the SS cells. The findings suggest that measurement of K+-efflux may be an accurate estimate of the extent of intracellular polymerization in sickled red cells, provided that both the intracellular and extracellular pH levels are carefully controlled, and the experimental conditions produce no independent effects on K+ permeability.     

Regulation of intracellular pH by human peripheral blood lymphocytes as measured by 19F NMR.

We have measured the intracellular pH of human peripheral blood lymphocytes by means of high-resolution 19F NMR spectroscopy using D,L-2-amino-3,3-difluoro-2-methylpropanoic acid (F2MeAla) as a probe. Lymphocytes readily took up the methyl ester of F2MeAla, and endogenous esterase hydrolyzed the ester to the free amino acid inside the cell. This alpha-methyl amino acid is not metabolized by the cell, and its 19F NMR spectrum exhibits large pH-dependent shifts as the alpha-amino group is protonated. The size of the 19F shifts, the high sensitivity of 19F NMR, and the favorable pKa of the alpha-amino group of F2MeAla (pKa = 7.3) allowed us to measure intracellular pH of lymphocytes at 25-30 degrees C with approximately 5-min acquisition times. Measurements at various external pH values demonstrated that human peripheral blood lymphocytes regulate their internal pH, a process requiring expenditure of metabolic energy. In the pH range between 6.8 and 7.4, lymphocytes maintain a constant internal pH of 7.17 +/- 0.06 pH unit. Outside this range, intracellular pH changes with extracellular pH. The accuracy of this 19F pH probe has been confirmed by independent measurements of intracellular pH using equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione.