Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Evidence for a major route for zinc uptake in human red blood cells: [Zn(HCO3)2Cl]- influx through the [Cl-/HCO3-] anion exchanger

The initial rate of Zn2+ uptake in human red cells was measured by atomic absorption. A very important fraction of Zn2+ uptake was inhibited by DIDS with IC50 = 0.3 microM (and by furosemide and bumetanide with IC50 of 200 and 500 microM, respectively). DIDS-sensitive Zn2+ uptake exhibited the following properties: 1) It required the simultaneous presence of both external HCO3- and Cl-. 2) In Cl- containing media, it was strongly stimulated by external HCO3- following a sigmoidal (S-shaped) and saturable function, which was fitted by a Hanes equation, with n = 2 and an apparent dissociation constant (for external HCO3-) of 5.3 +/- 0.9 mM (mean +/- SD of four experiments). The maximal rate of Zn2+ uptake at saturating HCO3- concentrations was 50.7 +/- 4.8 mmol (liter cells x h)-1. 3) In HCO3- containing media, it was strongly stimulated by external Cl- following a Michaelis-like equation with an apparent dissociation constant (for external Cl-) of 88 +/- 11 mM (mean +/- SD of three experiments). 4) Bicarbonate-stimulated Zn2+ uptake was inhibited by physiological concentrations of phosphate (sulfate was a much less potent inhibitor than phosphate). A kinetic analysis of the data strongly suggested that zinc was transported by the anion carrier in the form of the monovalent anion complex: [Zn(HCO3)2Cl]-.     

Insulin-induced hypoglycemia in fed and fasted exercising rats.

To determine running performance and hormonal and metabolic responses during insulin-induced hypoglycemia, fed and fasted male rats (315 +/- 3 g) were infused with insulin (100 mU/ml, 1.5 ml/h) or saline (1.5 ml/h) for 60 min and then killed at rest or after running on the treadmill (21 m/min, 15% grade). Insulin-infused fed rats ran poorly during the second 10 min of a 20-min exercise test. They were capable of running a total of 43 +/- 5 min, compared with 138 +/- 6 min for saline-infused fed rats. Fasted insulin-infused rats were able to run only 12.8 +/- 0.8 min, compared with 122 +/- 15 min for fasted saline-infused rats. In fasted rats, blood glucose was 1.6 +/- 0.1 mM after 60 min of insulin infusion and 1.2 +/- 0.1 mM after running to exhaustion. Artificial increase of plasma free fatty acids had no effect on performance. Intravenous infusion of glucose at the time of fatigue produced an immediate recovery, allowing the formerly fatigued rats to run 20 min without development of fatigue. These results provide evidence that severe hypoglycemia can be a significant cause of fatigue, even if it occurs early in the course of an exercise bout.     

Efficient syntheses of [3-15N]uracil and [3-15N]thymine.

Regiospecific syntheses of [3-15N]uracil and [3-15N]thymine are described using [15N]ammonium sulfate as a source of labeled nitrogen. The overall yields are excellent, and the reactions are amenable to production of multigram quantities of labeled material.     

Effect of 3-isobutyl-1-methylxanthine on HCO3- transport in turtle bladder. Evidence for electrogenic HCO3- secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO3-/CO2-containing, Cl- -free Na+ media exhibit a short-circuit current (Isc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the Isc and p.d. The IBMX-induced reversal of Isc and p.d. is (1) dependent on the presence of HCO3- (and CO2) in the serosal bathing fluid, (2) independent of Na+ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed Isc elicited by IBMX represents electrogenic secretion of HCO3-.     

Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion

Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.     

Studies on the in vitro metabolism of [3H]cortisol and [3H]oestradiol by sheep skin and wool roots.

The in vitro metabolism of [3H]cortisol, [3H]cortisone and [3H]estradiol-17 beta by adult sheep skin and wool follicle tissue (wool roots) was examined. The main metabolic product of the incubation of [3H]cortisol with sheep skin was [3H]cortisone, and the conversion was reversible. Wool roots were unable to carry out detectable interconversion, nor did this tissue give rise to other significant metabolites. Sheep skin and wool roots both rapidly converted [3H]oestradiol-17 beta to [3H]oestrone and the conversion could be carried out by follicle and non-follicle skin structures. It is suggested that sheep skin contains both 11 beta- and 17 beta-hydroxysteroid dehydrogenases, but that wool follicles contain only the latter enzyme.     

Cholesterol catabolism in the rabbit in fasted and fed states.

Urinary and fecal endogenous steroid excretion of fed or fasted New Zealand white rabbits was determined by the isotopic steady state method after subcutaneous implantation of radioactive cholesterol. While plasma cholesterol was increasing during a 9-day fast, fecal steroid excretion decreased to 10% of the excretion rates in the fed state. Refeeding the fasted rabbits led to a decrease in plasma cholesterol and an increase in fecal endogenous steroid excretion. Urinary steroid excretion, which represented 18% of total endogenous steroid excretion for fed animals, decreased during fasting and increased during refeeding, but these changes were relatively small. The small intestine, cecum, and colon of fed or fasted rabbits had similar endogenous steroid was acidic steroid. During attempts to alter the circulating bile acid concentration by supplying deoxycholate (200 mg/day) to fed rabbits or cholestyramine (2 g/day) to fasted rabbits, plasma cholesterol concentration did not change to the same extent as during fasting or refeeding, respectively. The decreased cholesterol catabolism and the hypercholesterolemia that are seen in the fasting rabbit may result from decreased clearance of plasma cholesterol.     

Fine structure of hepatocytes from fasted and fed rats.

The fine structure of hepatocytes from rats maintained on a controlled feeding schedule are described. Liver samples were processed for electron microscopy, histochemistry and chemical determinations of glycogen at precise time-intervals following a 30-hour fast and a 2-hour meal. Hepatocytes from 30-hour-fasted rats with extremely low hepatic glycogen levels were devoid of glycogen particles. Centrilobular cells showed areas of the cytoplasm rich in vesicles of smooth endoplasmic reticulum (SER) while periportal hepatocytes contained less extensive regions of SER. Soon after feeding the fasted rats, glycogen particles appeared in regions of the cell rich in SER. Centrilobular hepatocytes contained numerous glycogen areas which were infiltrated with tubules of SER, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the masses of glycogen. Throughout glycogen deposition each glycogen particle was closely associated with membranes of SER until maximum glycogen deposition was achieved 12 hours after initiation of feeding. At this point SER was reduced to the lowest amounts of the time-periods studied. During stages of glycogen depletion SER proliferated and reached the highest concentration measured in this study. Tubules of SER were present throughout the glycogen masses of centrilobular hepatocytes, whereas in periportal cells the organelle was restricted to the periphery of the glycogen masses. It is concluded that SER is associated with glycogen particles in rat hepatocytes during both deposition and depletion of glycogen.     

Comparison of glycogen stores in 3- and 7-month-old lean and obese Zucker rats under fed and fasted conditions

Glycogen content (mg/g) and stores (mg) were determined in 3- and 7-month-old obese and lean Zucker rats, under fed and fasted (48 hr) conditions. Hepatic content was higher in fed obese than in lean rats (3 months: 90 vs 70; 7 months: 107 vs 74); it was exhausted after fasting in lean but decreased by 56% in obese rats. Muscle content in fed obese and lean animals did not differ; it decreased comparably after fasting. Myocardial content was higher in fed obese than lean rats (3 months: 7.2 vs 3.6; 7 months: 7.5 vs 6.3); it was enhanced with fasting (10.0 vs 7.5). Total glycogen stores were higher in obese than in lean animals (3 months: 2500 vs 1400; 7 months: 4000 vs 2000) because of the hepatic store. The discussion includes a comparison with available data, taking into account methodological aspects, lipid stores and the FFA/carbohydrate interrelationship.     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport