Stimulation of HCO3− secretion by the prostaglandin E2 analog enprostil: Studies in human stomach and rat duodenum

This study investigated the action of enprostil, a synthetic analog of PGE₂, on gastric HCO₃⁻ secretion in humans and on duodenal HCO₃⁻ secretion in the anesthetized rat. A previously validated 2-component model was used to calculate gastric HCO₃⁻ and H⁺ secretion in 10 human subjects. Compared to placebo, a single 70 μg oral dose of enprostil increased basal gastric HCO₃⁻ secretion from 1810 +- 340 to 3190 ± 890 μmol/hr (P < 0.05). In addition, enprostil reduced basal gastric H⁺ secretion from 5240 ± 1140 to 1680 ± 530 μmol/hr (P < 0.02). Enprostil also increased HCO₃⁻ and reduced H⁺ secretion during intravenous pentagastrin infusion. In the rat, duodenal HCO₃⁻ secretion was measured by direct titration in situ using perfused segments of duodenum just distal to the Brunner gland area dn devoid of pancreatic and biliary secretions. Addition of enprostil(10 μg/ml) to the duodenal bathing solution increased duodenal HOC₃⁻ secretion from 6.3 ± 1.3 to 15.1 ± 2.0 μmol/cm·hr (P < 0.01, n = 6). The stimulatory action of enprostil on duodenal HCO₃⁻ secretion at 10 μg/ml was comparable in magnitude and duration to that of 10 μg/ml natural PGE₂. In summary, the PGE₂ analog enprostil stimulated gastroduodenal HCO₃⁻ secretion, effects which may be beneficial in protection of the gastroduodenal mucosa against luminal acid.     

Impaired duodenal bicarbonate secretion in diabetic rats. Salutary effect of nitric oxide synthase inhibitor

We previously reported the impaired HCO₃⁻ secretion and the increased mucosal susceptibility to acid in the duodenum of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the salutary effect of the NO synthase inhibitor L-NAME (N^G-nitro-L-arginine methyl ester) on these changes and compared it with those of insulin. Animals were injected streptozotocin (STZ: 70 mg/kg, ip) and used after 1, 3–4, and 5–6 weeks of diabetes with blood glucose levels of > 300 mg/dL. Under urethane anesthesia the HCO₃⁻ secretion was measured in the proximal duodenal loop using a pH-stat method and by adding 10 mM HCl. L-NAME (20 mg/kg × 2) or insulin (4 units/rat) was administered sc for 4–5 weeks, starting 1 week after STZ treatment. The duodenal HCO₃⁻ secretory responses to various stimuli such as mucosal acidification (10 mM HCl for 10 min), 16,16-dimethyl prostaglandin E₂ (dmPGE₂: 10 μg/kg, iv), and vagal stimulation (0.5 mA, 2 ms, 3 Hz) were significantly decreased in STZ-treated rats, depending on the duration of diabetes. Repeated administration of L-NAME, starting from 1 week after STZ treatment, significantly reduced blood glucose levels toward normal values and restored the HCO₃⁻ responses to various stimuli in STZ rats, the effects being similar to those observed after supplementation of insulin. Diabetic rats developed duodenal lesions after perfusion of the duodenum with 150 mM HCl for 4 h, but this ulcerogenic response was significantly inhibited by the repeated treatment with L-NAME as well as insulin. We conclude that L-NAME is effective in ameliorating hyperglycemic conditions in STZ-diabetic rats, similar to insulin, and restores the impaired HCO₃⁻ secretion and the increased mucosal susceptibility to acid in diabetic rat duodenums.     

Stimulatory effect of PACAP on gastroduodenal bicarbonate secretion in rats

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO₃⁻ secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO₃⁻ secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO₃⁻ secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO₃⁻ secretion after intracisternal administration (1 nmol/rat). PGE₂ (300 μg/kg, iv) significantly increased HCO₃⁻ secretion in both the stomach and the duodenum. The potency of duodenal HCO₃⁻ secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE₂. The duodenal HCO₃⁻ secretory action of PACAP-27 as well as PGE₂ was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 μg/kg, iv), the stimulator of adenylate cyclase, also increased HCO₃⁻ secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO₃⁻ secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO₃⁻ secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO₃⁻ secretion.     

Fish red blood cell carbon dioxide transport in vitro: A comparative study

Red blood cell (rbc) carbon dioxide transport was examined in vitro in three teleosts (Oncorhynchus mykiss, Anguilla anguilla, Scophthalmus maximus) and an elasmobranch (Scyliorhinus canicula) using a radioisotopic assay that measures the net conversion of plasma HCO₃⁻ to CO₂. The experiments were designed to compare the intrinsic rates of rbc CO₂ excretion and the impact of haemoglobin oxygenation/deoxygenation among the species.Under conditions simulating in vivo levels of plasma HCO₃⁻ and natural haematocrits, the rate of whole blood CO₂ excretion varied between 14.0 μmol ml⁻¹ h⁻¹ (S. canicula) and 17.6 μmol ml⁻¹ h⁻¹ (O. mykiss). The rate of CO₂ excretion in separated plasma was significantly greater in the dogfish, S. canicula. The contribution of the rbc to overall whole blood CO₂ excretion was low in the dogfish (46 ± 6%) compared to the teleosts (trout, 71 ± 4%; turbot, 64 ± 5%; eel, 55 ± 3%).To eliminate the naturally occurring differences in haematocrit and plasma [HCO₃⁻] as inter-specific variables, the rates of whole blood CO₂ excretion were determined in blood that had been resuspended to constant [HCO₃⁻] (5 mmol⁻¹) and haematocrit (20%) in appropriate teleost and elasmobranch Ringer solutions. Under such normalized conditions, the rate of whole blood CO₂ excretion was significantly higher in the turbot (22.4 ± 1.3 μmol ml⁻¹ h⁻¹) in comparison to the other species (16.4–18.4 μmol ml⁻¹ h⁻¹) and thus revealed a greater intrinsic rate of rbc CO₂ excretion in the turbot.To study the contribution of Bohr protons, the rates of whole blood CO₂ excretion were assessed in blood subjected to rapid oxygenation during the initial phase of the 3 min assay period. Rapid oxygenation significantly enhanced the rate of CO₂ excretion in the teleosts but not in the elasmobranch. The extent of the increase provided by the rapid oxygenation of haemoglobin was a linear function of the extent of the Haldane effect, as quantified in each species from in vitro CO₂ dissociation (combining) curves. Under steady-state conditions, deoxygenated blood exhibited greater rates of CO₂ excretion than oxygenated blood in the teleosts but not in the elasmobranch. As a consequence of the Haldane effect, rbc intracellular pH was increased in the teleosts by deoxygenation but was unaltered in the elasmobranch.The results, by extrapolation, suggest that the rates of CO₂ excretion in vivo are influenced by the magnitude of the Haldane effect and the extent of haemoglobin oxygenation during gill transit in addition to the intrinsic rate at which the rbc converts plasma HCO₃⁻ to CO₂.     

The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

The ability of the seagrass Zostera muelleri Irmisch ex Aschers. to use HCO₃⁻ as well as CO₂ for photosynthesis was investigated by measuring photosynthetic O₂ evolution over a range of pH values. It was found that the apparent K_m CO₂ fell from 0.128 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO₃⁻ as well as CO₂ may act as a substrate for photosynthesis.The true K_m CO₂ could not be determined due to inhibition of photosynthesis at pHs less than 7.8 K_m CO₂ must be at least 0.128 mM, the apparent K_m at pH 7.9, and is probably of the order of 0.200 mM CO₂, the same as that reported for other marine plants. K_m HCO₃⁻¹ is about 20 mM when CO₂-dependent photosynthesis is minimal. Such a high K_m HCO₃⁻ resembles values reported for freshwater, rather than marine plants.Photosynthetic O₂ evolution is not saturated with respect to total inorganic carbon in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pH 8.1 to 8.5 in the pH profile of photosynthetic O₂ evolution at a constant concentration of inorganic carbon is caused by an effect of pH on HCO₃⁻ uptake. The effect of pH on HCO₃⁻ uptake was determined by constructing a pH profile of photosynthesis at constant HCO₃⁻ concentration, and subtracting the estimated contribution of CO₂ to photosynthesis from this rate. The resultant curve has a maximum at pH 8.4 and declines sharply at pHs less than 8.     

Mucus glycoprotein secretion by duodenal mucosa in response to luminal arachidonic acid

The effect of luminal application of arachidonic acid on the alkaline secretion, prostaglandin generation, and mucus glycoprotein output and composition was studied in proximal and distal duodenum of conscious dogs. Surgically prepared duodenal loops were instilled in vivo for up to 2 h with saline (control) followed by various concentrations (12.5–100 μg/ml) of arachidonic acid. The experiments were conducted with and without intravenous pretreatment with indomethacin. The recovered instillates were assayed for the content of prostaglandin and HCO₃⁻, and used for the isolation of mucus glycoprotein. Exposure of duodenal mucosa to arachidonic acid led to concentration-dependent increase in the output of HCO₃⁻ and prostaglandin generation. In both cases this response was greater in the proximal duodenum. Pretreatment with indomethacin caused reduction in the basal HCO₃⁻ and prostaglandin output, and prevented the increments evoked by arachidonic acid. The proximal and distal duodenum displayed similar basal output and composition of mucus glycoprotein. Comparable increases in these glycoproteins were also obtained with arachidonic acid, the effect of which was abolished by indomethacin. Compared to basal conditions, mucus glycoproteins elaborated in response to arachidonic acid exhibited higher contents of associated lipids and covalently bound fatty acids, and contained less protein. The associated lipids of mucus glycoproteins elaborated in the presence of arachidonic acid showed enrichment in phospholipids and decrease in neutral lipids. The carbohydrate components in these glycoproteins also exhibited higher proportions of sialic acid and sulfate. The changes brought about by arachidonic acid were prevented by indomethacin pretreatment, and in both cases the glycoprotein composition returned to that obtained under basal conditions. The enrichment of mucus glycoprotein in lipids, sialic acid and sulfate in response to endogenous prostaglandin may be of significance to the function of this glycoprotein in the hostile environment of the duodenum.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Influence of the hormone analogue 13-NLE-molitin and of 1-methyl-3-isobutylxanthine on tone and cyclic 3',5'-AMP content of antral and duodenal muscles in the rabbit.

1. By the action of 1-methyl-3-isobutylxanthine (isobutyltheophylline, 2 - 3 × 10⁻⁴ M), the content of cyclic 3', 5'-AMP in the antral and duodenal muscles of the rabbit is increased by 72 % and 126 %, respectively; by 1.8 × 10⁻⁷ M 13-norleucine-motilin and 1.8 × 10⁻⁶ M acetylcholine it is not changed. 13-norleucine-motilin is an analogue of the recently discovered duodenal tissue hormone motilin and has identical effects. 1-methyl-3-isobutylxanthine has a more powerful inhibiting effect on phosphodiesterase than has theophylline.2. 3 × 10⁻⁴ M isobutyltheophylline reduces the tone of the duodenal muscle while simultaneously increasing the content of cyclic AMP and negates the tone-enhancing effect of nle-motilin on the duodenal muscle, while nle-motilin increases the muscle tone lowered by isobutyltheophylline.3. The basic tone of the antral muscle is not reduced by isobutyltheophylline. However, the contraction-promoting effect of nle-motilin after an increase in cyclic AMP due to isobutyltheophylline is significantly lower.4. It is assumed that the changes in the tone or in the response of the antral and duodenal muscles to nle-motilin observed after the administration of isobutyltheophylline, are due to the increase of cyclic AMP in the tissue.5. The antagonistic effects of cyclic AMP and motilin on the gastro-intestinal muscles might be of physiological importance for the regulation of the gastro-intestinal motor activity.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Evidence for HCO3− conductance pathways in nutrient membrane of bullfrog antrum

The effect of changing the nutrient-side HCO₃⁻ concentration on potential difference (PD) and resistance in bullfrog antrum bathing in Cl⁻ media was determined. Changes in HCO₃⁻ concentration were from 25 mM to several lower concentrations and back to 25mM. A plot of 6ΔPD6 versus log [HCO₃⁻] gave a linear relation for changes of HCO₃ concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO₃⁻ concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part the same changes with a simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO₃⁻ conductance pathways rather than OH⁻ conductance pathways predominated. There was no evidence of HCO₃⁻, OH⁻ and H⁺ conductance pathways in secretory membrane.     

24-Hour plasma secretin level and pancreatic secretion in dog

Plasma secretin concentrations were determined and duodenal pH was recorded continuously for a period of 24 hours after ingestion of a meal in 3 dogs with gastric cannula and duodenal cannula and in 4 dogs with pancreatic fistulae. The mean plasma secretin concentration increased significantly after a meal and it remained elevated for the first 12-hour period (peak at 30 min). Duodenal pH frequently decreased below 4.5 during the first 12-hour postprandial period, but it remained above 5.0 during the second 12 hours. Pancreatic secretion peaked during the first hour of meal ingestion and remained elevated until the end of 12 hours. The increased plasma secretin level in pancreatic fistula dog during the postprandial period was significantly greater than that of duodenal cannula dog, but the trends of increase in the secretin levels were quite identical. The present study indicates that: (1) plasma secretin concentration increases significantly within 30 min after a meal and remains increased during the first 12-hour period, (2) duodenal pH frequently decreased below 4.5 during the same 12 hours but more frequently during the first 6 hours, and (3) a significant increase in pancreatic water, HCO₃^? and protein occurred during the same time period.     

Complete ammonia oxidization in agricultural soils: High ammonia fertilizer loss but low N2O production

The contribution of agriculture to the sustainable development goals requires climate-smart and profitable farm innovations. Increasing the ammonia fertilizer applications to meet the global food demands results in high agricultural costs, environmental quality deterioration, and global warming, without a significant increase in crop yield. Here, we reported that a third microbial ammonia oxidation process, complete ammonia oxidation (comammox), is contributing to a significant ammonia fertilizer loss (41.9 ± 4.8%) at the rate of 3.53 ± 0.55 mg N kg⁻¹ day⁻¹ in agricultural soils around the world. The contribution of comammox to ammonia fertilizer loss, occurring mainly in surface agricultural soil profiles (0–0.2 m), was equivalent to that of bacterial ammonia oxidation (48.6 ± 4.5%); both processes were significantly more important than archaeal ammonia oxidation (9.5 ± 3.6%). In contrast, comammox produced less N₂O (0.98 ± 0.44 μg N kg⁻¹ day⁻¹, 11.7 ± 3.1%), comparable to that produced by archaeal ammonia oxidation (16.4 ± 4.4%) but significantly lower than that of bacterial ammonia oxidation (72.0 ± 5.1%). The efficiency of ammonia conversion to N₂O by comammox (0.02 ± 0.01%) was evidently lower than that of bacterial (0.24 ± 0.06%) and archaeal (0.16 ± 0.04%) ammonia oxidation. The comammox rate increased with increasing soil pH values, which is the only physicochemical characteristic that significantly influenced both comammox bacterial abundance and rates. Ammonia fertilizer loss, dominated by comammox and bacterial ammonia oxidation, was more intense in soils with pH >6.5 than in soils with pH <6.5. Our results revealed that comammox plays a vital role in ammonia fertilizer loss and sustainable development in agroecosystems that have been previously overlooked for a long term.     

Effects of hypercapnia on blood-gas and acid-base status in the white sturgeon, Acipenser transmontanus

The effect of environmental hypercapnia on respiratory and acid-base variables was studied in white sturgeon, Acipenser transmontanus. Blood PCO₂, PO₂, pH, hemoglobin concentration, and plasma lactate, glucose, catecholamines and cortisol were measured first under normocapnia (water PCO₂ < 0.5 Torr, 1 Torr = 133.32 Pa), then under hypercapnia (25–35 Torr) and a final return to normocapnia at 19 ± 0.5 °C. Acute (≤ 2h) hypercapnia significantly increased arterial PCO₂ (8-fold increase), ventilation frequency (2-fold increase), plasma HCO₃ ⁻ (2.3-fold) and decreased arterial pH (to 7.15 ± 0.02). After 24 h, norepinephrine, epinephrine and cortisol, were significantly increased, and arterial pH reached its nadir (7.10 ± 0.03). During the 72- and 96-h-periods, arterial PCO₂ (24 ± 4.4 Torr) and ventilatory frequency (105 ± 5 breaths min⁻¹) stabilized, HCO₃ ⁻ reached its apparent maximum (23.6 ± 0.0 mmol⁻¹), glucose decreased by 32%, and pH increased significantly to 7.31 + 0.03. The return to normocapnia completely restored arterial PCO₂ (2.5 ± 0.14 Torr), HCO₃ ⁻ (7.4 ± 0.59 mmol · l⁻¹), ventilation frequency (71 ± 7 breaths · min⁻¹), and pH (7.75 ± 0.04). Overall, hypercapnia produced a respiratory acidosis, hyperventilation, a transient norepinephrine “spike”, and increased plasma catecholamines, cortisol, and arterial PO₂. The respiratory acidosis was only partially compensated (35% pH restoration) 96 h after the onset of hypercapnia and resulted in a significantly decreased blood-O₂ affinity (Bohr effect), as determined by construction of in vitro blood O₂ equilibrium curves at 15 °C and 20 °C. Prolonged exposure to hypercapnia may lead to acid-base disturbances and negatively affect growth of white sturgeon. Accepted: 17 August 1997     

Functional expression of basolateral Cl−/HCO3− exchange from rat jejunum in Xenopus laevis oocytes

Poly(A)⁺ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl⁻/HCO₃⁻ antiport was investigated by means of ³⁶Cl⁻ uptake. Two days after injection of 50 ng of poly(A)⁺ RNA, Cl⁻ uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0·2 mM DIDS, whereas endogenous Cl⁻ uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl⁻ uptake was detected with mRNA size fraction of about 2–4 kb in length. The expressed Cl⁻ uptake can occur against a Cl⁻ concentration gradient and is unaffected by the known Cl⁻ channel blocker anthracene-9-carboxylic acid. Cl⁻ transport mechanism has properties similar to jejunal basolateral Cl⁻/HCO₃⁻ exchange with regard to Na⁺ dependence. © 1998 John Wiley & Sons, Ltd.     

Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production

The contribution of metabolic bicarbonate to cytosolic pH (pH_cyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO^?₃ decreased proton efflux from 18.6±5.0 to 1.64±0.29 μmol/min per g liver wet weight (w.w.) and pH_cyto from 7.17±0.06 to 6.87±0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69±0.26 μmol/min per g liver w.w. reflecting a reduction in metabolic CO₂ hydration, and hence a decrease of H⁺ and HCO^?₃ supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pH_cyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and amiloride since pH_cyto decreased by 0.16±0.05 and proton efflux by 0.60±0.14 μmol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pH_cyto regulation. The involvement of two mechanisms, most likely Na⁺/H⁺ antiport and Na⁺:HCO^?₃ symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO^?₃ in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pH_cyto regulation.     

Role of peptide YY in regulation of duodenal motility during the interdigestive period in sheep

Temporal coordination between duodenal migrating myoelectric complexes (MMC) and pancreatic exocrine secretion, and the effects of porcine peptide YY (PYY) on gastroduodenal motility and pancreatic exocrine secretion were examined during the interdigestive period in conscious mature sheep. Fluid and enzyme secretions from the exocrine pancreas showed a periodic pattern corresponding to the phases of duodenal MMC, although these secretion rates were maintained at a high level during phase II in sheep. Intravenous continuous infusion of PYY at doses ranging from 50 to 200 pmol · kg⁻¹ · h⁻¹ or intravenous bolus infusion of PYY at doses ranging from 50 to 200 pmol · kg⁻¹ showed a tendency to prolong the first cycle of the duodenal MMC and significantly shorten the second cycle. However, there was almost no effect on ruminal contractions from the PYY administration. In the pancreatic exocrine secretion, PYY could inhibit only bicarbonate secretion at only the highest dose of 200 pmol · kg⁻¹. These results imply that endogenous PYY may play a physiological role in the regulation of the duodenal MMC cycles in sheep but not in ruminal contractions. PYY seems unlikely to regulate the pancreatic exocrine secretion in normal sheep, because a supraphysiological dose of PYY was required to inhibit the pancreatic exocrine secretion. Accepted: 3 March 1997     

The metabolic cost of bicarbonate use in the submerged plant Elodea nuttallii

The aquatic plant Elodea nuttallii (Planch.) St. John has been shown to express plasticity in the source of inorganic carbon it uses for photosynthesis. An investigation was undertaken to determine what effect the switch from CO₂ to HCO₃⁻ use had on the growth of E. nuttallii. Plants were grown under reduced CO₂ availability that favoured the switch, together with control plants (CO₂ at equilibrium with air) that continued to use CO₂ only. The extent to which both sets of plants could utilise HCO₃⁻ was determined (as the ratio of oxygen evolution at pH 9 and 6.5), and several measures of growth were made. Although reduced CO₂ availability produced an increase in HCO₃⁻ utilisation, no differences were found in the measured growth of the plants. Therefore, it was possible to estimate, from the difference between the estimated rate of photosynthesis of the plants utilising HCO₃⁻ and those using CO₂ only, the approximate cost of constructing, maintaining and running the bicarbonate utilisation mechanism in this species as 69 μmol photons m⁻² s⁻¹. This value can be used to estimate an irradiance of circa 80 μmol m⁻² s⁻¹ below which HCO₃⁻ use would not be expected in this species, an irradiance commonly experienced by submerged macrophytes in the field.     

Acid–base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid–base regulation during respiratory acidosis either by facilitating or constraining HCO₃ ⁻ accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid–base status during exposure to increasing levels of hypercapnia (nominally 1–5% CO₂). Fish exhibited a well-developed ability to increase plasma HCO₃ ⁻ levels with values of 39.8 ± 2.8 mmol l⁻¹ being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO₂ = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO₃ ⁻ during hypercapnia was associated with marked increases in branchial net acid excretion (J _NETH⁺) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J _NETH⁺ was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na⁺/K⁺-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO₃ ⁻ concentrations of 92.2 ± 4.8 mmol l⁻¹. At the highest level of hypercapnia, rectal fluid HCO₃ ⁻ levels were increased significantly to 141.8 ± 7.4 mmol l⁻¹ but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l⁻¹). Thus, the impressive accumulation of plasma HCO₃ ⁻ to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO₃ ⁻ excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO₃ ⁻ concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO₃ ⁻. The apparent inability of P. notatus to effectively excrete HCO₃ ⁻ following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.     

The metabolic responses and acid–base status after feeding, exhaustive exercise, and both feeding and exhaustive exercise in Chinese catfish (Silurus asotus Linnaeus)

Feeding and exhaustive exercise are known to elevate metabolism. However, acid–base status may be oppositely affected by the two processes. In this study, we first investigated the acid–base response of Chinese catfish to feeding (the meal size was about 8% of body mass) to test whether an alkaline tide (a metabolic alkalosis created by gastric HCl secretion after feeding) would occur. We then determined the combined effects of feeding and exhaustive exercise on excess post-exercise oxygen consumption and acid–base status to determine whether the alkaline tide induced by feeding protects against acid–base disturbance during exhaustive exercise and affects subsequent recovery. Arterial blood pH increased from 7.74 ± 0.02 before feeding to 7.88 ± 0.02 and plasma [HCO₃ ⁻]_pl increased from 5.42 ± 0.29 to 7.83 ± 0.37 mmol L⁻¹ 6 h after feeding, while feeding had no significant effect on P_\textCO2 P_{{{\text{CO}}_{2} }} . Exhaustive exercise led to a significant reduction in pH by 0.46 units and a reduction of [HCO₃ ⁻]_pl by ~3 mmol L⁻¹. Lactate concentrations in white muscle and plasma increased by 2.4 mmol L⁻¹ and 13.4 μmol g⁻¹, respectively. Fed fish had a higher pH and [HCO₃ ⁻]_pl than fasting fish at rest, and the reductions in pH (0.36 units) and [HCO₃ ⁻]_pl (~2 mmol L⁻¹) were thus lower after exhaustive exercise. However, the recovery of acid–base status and metabolites were similar in digesting and fasting fish. Overall, a significant alkaline tide was found in Chinese catfish after feeding. The alkaline tide elicited by feeding significantly prevented the decreases in pH and [HCO₃ ⁻]_pl immediately after exhaustive exercise, but recovery from exhaustive exercise was not affected by digestion.     

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·10⁵ M⁻¹·s⁻¹) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H₂O₂ concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·10⁴ M⁻¹·s⁻¹ to 6.8·10⁶ M⁻¹·s⁻¹ when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·10³ M⁻¹·s⁻¹ at pH 6.0 to 1.0·10⁵ M⁻¹·s⁻¹ at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.