Demonstration of an osteoblast defect in two cases of human malignant osteopetrosis. Correction of the phenotype after bone marrow transplant.

Osteopetrosis is an inherited disorder characterized by bone sclerosis due to reduced bone resorption. Here we report that human osteopetrotic osteoblast-like (Ob) cells express a defective phenotype in primary cultures in vitro, and that bone marrow transplant (BMT) corrects osteoblast function. DNA analysis at polymorphic short-tandem repeat loci from donor, recipient, and primary Ob-like cells pre-BMT and 2 yr post-BMT revealed that Ob were still of recipient origin post-BMT. Osteopetrotic Ob-like cells obtained pre-BMT showed normal and abnormal 1,25(OH)2D3-induced alkaline phosphatase (ALPase) and osteocalcin production, respectively, and failed to produce macrophage colony-stimulating factor (M-CSF) in response to IL-1a and TNF-alpha. These parameters were all normalized in primary Ob-like cells prepared 2 yr post-BMT. X-linked clonality analysis at the human androgen receptor (HUMARA) locus revealed that osteoblasts showed a polyclonal and an oligoclonal derivation pre- and post-BMT respectively, indicating that a limited number of progenitor reconstituted this population. Because osteoblasts were still of recipient origin post-BMT, this suggests that functional osteoclasts, due to the replacement of hematopoeitic cells, provided a local microenvironment in vivo triggering the differentiation and/or recruitment of a limited number of functional osteoblasts.

Evidence from incorporation experiments for an anionic channel of small conductance at the apical membrane of the rabbit distal tubule.

Many of the hormone-regulated ion transport processes in distal nephron involve transcellular pathways which require a passive entry of ions at the apical membrane of the distal tubule cells. To investigate molecular mechanisms underlying the ionic permeability of the distal tubule apical membrane, a study was undertaken in which vesicles prepared from apical membranes from isolated rabbit distal tubules were fused onto a planar lipid bilayer. These experiments led to the identification of several ionic channels including a Cl(-)-permeable channel of 14 pS with a Na+ over Cl- permeability ratio, PNa/PCl < 0.09. The open channel probability (Po) showed a weak voltage dependency with Po increasing slightly at negative potential values (intracellular (trans) relative to extracellular (cis) for right-side-out vesicles). Channel activity was inhibited by NPPB at high concentrations (> 100 microM) and by DIDS (300 microM). A small inhibitory effect was also observed in the presence of DPC at concentrations ranging from 200 microM to 500 microM. The presence of SO4(2-) (32 mmol/l) in the trans solution caused a complete inhibition of channel activity, but no modification of channel behaviour was observed with the non-selective channel blocking agent gadolinium (Gd3+) at 100 microM. Finally, addition of the catalytic subunit of protein kinase A into the trans chamber (60 U/ml to 80 U/ml) led to an increase in channel activity characterized by a greater number of active channels coupled to an increase of the individual channel open probability. The action of the protein kinase A could be cancelled by the addition of a non specific protein phosphatase, such as alkaline phosphatase. Our results suggest that the apical membrane of the rabbit distal tubule contains a Cl- permeable channel of small conductance the activity of which may be modulated by hormones linked to the adenylate cyclase pathway.

The mechanism of parathyroid hormone action on calcium reabsorption by the distal tubule.

PTH increases calcium reabsorption exclusively in the distal nephron. Two mechanisms of Ca++ transport through the basolateral membrane (BLM) have been described: the ATP-dependent and the sodium gradient-dependent transport. In the present study, we investigated the effect of PTH and (Bu)2cAMP on these two mechanisms. We recently reported, using 100 microM Ca++ as the substrate, that whereas the ATP-dependent system was present in the proximal and distal tubule (DCT), the Na+/Ca++ exchanger was located only in the DCT. Using 2 microM Ca++ as the substrate, the Na+/Ca++ exchanger was again found to be present only in the DCT. Incubation of DCT suspension with 10(-8) M bovine PTH (1-34) resulted in a significant increase in the Na(+)-dependent Ca++ uptake by the corresponding BLM vesicles. This effect was dose dependent. The half-maximal stimulation was obtained with 1 X 10(-8) M PTH. At this concentration, PTH increased the maximum velocity (Vmax) from 0.34 +/- 0.06 t 0.54 +/- 0.02 nmol/mg/10 s (P less than 0.05) without influencing the Michaelis-Menten constant (Kms). Incubation of DCT suspensions with (Bu)2cAMP mimicked this effect. The dose-response curve showed a peak action at 1 mM (Bu)2cAMP. In contrast, neither (Bu)2cAMP nor PTH influenced the ATP-dependent Ca++ transport through BLM from proximal tubule nor DCT. It is proposed that PTH influences Ca++ reabsorption by the DCT because the target molecule, the Na+/Ca++ exchanger, is located exclusively at this site.

Parathyroid hormone and hydrochlorothiazide increase calcium transport by the luminal membrane of rabbit distal nephron segments through different pathways.

The present study was designed to investigate the effect of PTH on calcium (Ca2+) transport through the luminal membrane of proximal and distal rabbit tubule segments. Proximal tubule and distal tubule segment suspensions were incubated with the hormone, and the luminal membranes were subsequently purified. Incubation with 10(-8) M human PTH(1-34) strongly increased initial Ca2+ uptake by the distal membranes. The effect of PTH was dose dependent, with an apparent ED50 of 8.2 +/- 1.0 nM. We recently reported the presence of two kinetics of Ca2+ uptake by the distal luminal membranes. PTH affected exclusively the high affinity component, increasing the maximum velocity from 0.31 +/- 0.02 to 0.76 +/- 0.07 pmol/micrograms.10 sec (P < 0.001), and leaving the Michaelis-Menten constant Ca2+ unchanged. The addition of 500 microM hydrochlorothiazide (HCTZ) to the luminal membranes of distal tubules incubated with PTH further enhanced Ca2+ uptake. The effect of HCTZ was on the low affinity system. HCTZ (100 microM) enhanced the maximum velocity from 2.5 +/- 0.3 to 3.7 +/- 0.6 pmol/micrograms protein.10 sec (P < 0.01) without affecting the Michaelis-Menten constant. Whereas 1 microM nitrendipine alone did not affect Ca2+ transport by the distal tubule luminal membranes, the Ca2+ channel inhibitor completely abolished the effect of PTH. Conversely, 1 microM Bay K 8644 increased Ca2+ uptake by membranes from PTH-treated distal tubules but was ineffective in membranes from control tubules. Neither PTH nor nitrendipine nor Bay K 8644 had any effect on the luminal membranes from proximal tubules. These results suggest that: 1) the high affinity, low velocity Ca2+ transport system in the luminal membrane from distal cortical segments is sensitive to PTH; 2) the effects of nitrendipine and Bay K 8644 on Ca2+ uptake were observed only in membranes from tubules incubated with PTH; 3) this uptake is distinct from the thiazide-sensitive Ca2+ transport system; and 4) PTH does not influence Ca2+ transport by the luminal membrane of proximal tubules.

Effect of vitamin D depletion on calcium transport by the luminal and basolateral membranes of the proximal and distal nephrons.

To study the effect of vitamin D on calcium (Ca2+) reabsorption by the kidney, we measured Ca2+ uptake by the basolateral and luminal membranes of proximal and distal tubules obtained from rabbits fed a vitamin D-deficient diet for 3 weeks. Results were compared to those obtained with a group of control animals fed a normal diet. Serum Ca2+ concentrations were comparable in both groups. In the control group, serum PTH, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 remained relatively stable. In the vitamin D-deficient animals, serum PTH levels slightly, but not significantly, increased, and the levels of vitamin D metabolites abruptly fell. Vitamin D depletion produced a 40% decrease in ATP-dependent Ca2+ uptake by the basolateral membrane of the distal tubule. There was no change in the activity of the Na+/Ca2+ exchanger. A very significant effect was also observed in the luminal membrane of the distal tubule, where a 50% decrease in Ca2+ uptake was observed after the third week of vitamin depletion. Administration of 0.1 microgram 1,25-dihydroxyvitamin D3 16 and 2 h before death partially reestablished normal uptake. In contrast, no change in Ca2+ uptake could be detected in the basolateral or luminal membranes of the proximal tubule. These observations provide the first evidence of an effect of vitamin D on Ca2+ transport at both the basolateral and luminal membranes of the distal segment of the nephron.

Localization of immunoreactive vitamin D-dependent calcium binding protein in chick nephron.

Immunoreactive vitamin D-dependent calcium binding protein (iCaBP) has been localized in the chicken nephron, using microdissection and subsequent radioimmunoassay (RIA). Six to ten weeks old vitamin D-replete chicks were sacrificed and the kidneys removed. The various segments of the nephron, i.e. the proximal convoluted tubule (PCT), the thick part of the hairpin loops (thick loops), the thin part of the hairpin loops (thin loops), the distal convoluted tubules (DCT) and the cortical collecting tubules (CCT) were microdissected and grouped according to their morphological characteristics. Because the transition between DCT and CCT was not evident, these two segments were gathered together. The samples were assayed for iCaBP by a microradioimmunoassay, using antisera against the vitamin D-dependent chicken intestinal CaBP. iCaBP was localized in the thin loops and in the DCT-CCT segments (24.7 +/- 5 and 20.8 +/- 3 ng/mg protein, respectively). No iCaBP was detected either in the PCT using up to 32 micrograms protein, or in fragments of medullary papilla, assaying up to 4 micrograms protein.

Parathyroid hormone receptor in human placental syncytiotrophoblast brush border and basal plasma membranes.

The syncytiotrophoblast of the placenta is the site of exchange of nutrients and minerals between the mother and fetus. We have recently demonstrated that PTH influences, in vitro, phosphate transport through the placenta brush border membranes (BBM) and increases cAMP accumulation in placental tissue. To demonstrate the site of binding of PTH in the cytoplasmic membrane, we have purified two polar membranes: the first located on the apical side, the BBM, and the second, on the fetal side, the basal plasma membrane (BPM). BBM were enriched 24-fold in alkaline phosphatase (marker for BBM), and the BPM was enriched 37-fold in binding of [3H] dihydroalprenolol (marker for BPM) compared to homogenate. Both placental membranes contain binding sites (maximum binding = 0.550 +/- 0.032 and 0.298 +/- 0.065 pmol/mg protein for BBM and BPM, respectively) with similar affinities (Kd = 2.05 +/- 0.23 and 1.78 +/- 0.19 nM, respectively) for 125I-[Nle8,Nle18,Tyr34] bovine (b) PTH-(1-34) amide. The three bovine preparations [bPTH-(1-34), its analog [Nle8,Nle18,Try34]bPTH-(1-34) amide, and the antagonist bPTH-(3-34)] were equipotent in binding to both placental membranes. In contrast, human PTH-(1-84) was more effective in displacing the bovine radioligand in BBM. Thyrocalcitonin and insulin, two non-PTH peptides, did not significantly displace the radioligand in BBM and BPM. Adenylate cyclase activity, located exclusively in BPM, was stimulated by PTH. Since the enzyme is absent from BBM, it is probable that the binding of the hormone to this membrane activates another system of messengers.

Childhood asthma: prevention of attacks with short-term corticosteroid treatment of upper respiratory tract infection.

In this study, the potential for short courses of glucocorticoids to prevent or reduce the severity of asthma induced by viral respiratory infections in preschool children was investigated. Two groups of children with a mean age of 36.4 +/- 3.9 months and 40.4 +/- 4.9 months were monitored during a 2-year period. Group 1, considered as the control group, received theophylline preparations and orciprenaline either on a continuous basis or during attacks. During severe attacks, albuterol was administered by nebulization, with corticosteroids occasionally added for seven to 14 days in cases of poor response to albuterol. Group 2 received the same treatment during the first year. During the second year, however, a short-term course of prednisone (1 mg/kg) was given as soon as the first symptoms of an upper respiratory tract infection appeared, prior to any signs of wheezing. Results indicate that, whereas morbidity remained constant in the control group during the 2-year observation period, a significant decrease in the number of wheezing days (65%), attacks (56%), visits to the emergency room (61%), and hospitalizations (90%) occurred in group 2. It was concluded that preschool children who suffer from repeated asthma attacks related to upper respiratory tract infections may benefit greatly from the preventive administration of corticosteroids.

Effect of estrogen on calcium and sodium transport by the nephron luminal membranes.

Estrogens are widely used for contraception and osteoporosis prevention. The aim of the present study was to investigate the effect of 17 beta-estradiol on calcium (Ca(2+)) transport by the nephron luminal membranes, independently of any other Ca(2+)-regulating hormones. Proximal and distal tubules of rabbit kidneys were incubated with 17 beta-estradiol or the carrier for various periods of time, and the luminal membranes of these tubules were purified and vesiculated. Ca(2+) uptake by membrane vesicles was measured using the Millipore filtration technique. Incubation of proximal tubules with the hormone did not influence Ca(2+) uptake by the luminal membranes. In contrast, incubation of distal tubules with 10(-8) M 17 beta-estradiol for 30 min decreased the initial uptake of 0.5 mM Ca(2+) from 0.34+/-0.04 (s.e.m. ) to 0.17+/-0.04 pmol/microg per 5 s (P<0.05). In the presence of 100 mM Na(+), 0.5 mM Ca(2+) uptake was strongly diminished and the effect of 17 beta-estradiol disappeared (0.17+/-0.01 and 0.21+/-0.07 pmol/microg per 5 s in vesicles from the control and treated tubules). Direct incubation of the membranes with 17 beta-estradiol, however, failed to show any influence of the hormone on Ca(2+) transport. The action of 17 beta-estradiol was dose-dependent, with a half-maximal effect at approximately 10(-9) M. Ca(2+) uptake by the distal tubule membranes presents dual kinetics. 17 beta-Estradiol decreased the V(max) value of the high-affinity component from 0.42+/-0.02 to 0.31+/-0.03 pmol/microg per 10 s (P<0.02). In contrast with the effect of the hormone on Ca(2+) transport, estradiol increased Na(+) uptake by both the proximal and distal tubule luminal membranes. In conclusion, incubation of proximal and distal tubules with estrogen decreases Ca(2+) reabsorption by the high-affinity Ca(2+) channels of the distal luminal membranes, and enhances Na(+) transport by the membranes from proximal and distal nephrons.

Hyperglycemia induces insulin resistance on angiotensinogen gene expression in diabetic rat kidney proximal tubular cells.

Clinical and animal studies have shown that treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II (Ang II) receptor antagonists slows the progression of nephropathy in diabetes, indicating that Ang II plays an important role in its development. We have reported previously that insulin inhibits the stimulatory effect of high glucose levels on angiotensinogen (ANG) gene expression in rat immortalized renal proximal tubular cells (IRPTCs) via the mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We hypothesize that the suppressive action of insulin on ANG gene expression might be attenuated in renal proximal tubular cells (RPTCs) of rats with established diabetes. Two groups of male adult Wistar rats were studied: controls and streptozotocin (STZ)-induced diabetic rats at 2, 4, 8 and 12 weeks post-STZ administration. Kidney proximal tubules were isolated and cultured in either normal glucose (i.e. 5 mM) or high glucose (i.e. 25 mM) medium to determine the inhibitory effect of insulin on ANG gene expression. Immunoreactive rat ANG (IR-rANG) in culture media and cellular ANG mRNA were measured by a specific radioimmunoassay and reverse transcription-polymerase chain reaction assay respectively. Activation of the p44/42 MAPK signal transduction pathway in rat RPTCs was evaluated by p44/42 MAPK phosphorylation employing a PhosphoPlus p44/42 MAPK antibody kit. Insulin (10(-7) M) inhibited the stimulatory effect of high glucose levels on IR-rANG secretion and ANG gene expression and increased p44/42 MAPK phosphorylation in normal rat RPTCs. In contrast, it failed to affect these parameters in diabetic rat RPTCs. In conclusion, our studies demonstrate that hyperglycaemia induces insulin resistance on ANG gene expression in diabetic rat RPTCs by altering the MAPK signal transduction pathway.

Sodium gradient-dependent phosphate transport in placental brush border membrane vesicles.

We recently described a sodium gradient-dependent transport of phosphate through the brush border membrane vesicles from human placenta. In order to characterize this transport carrier further, we studied the influence of temperature and membrane potential on the transport of this electrolyte, the stoichiometry of the sodium-phosphate interaction, and the interrelationship between phosphate uptake and other sodium-dependent systems. Temperature influenced phosphate uptake by changing the maximal velocity and the affinity of the carrier for the substrate. The Arrhenius plot for uptake velocity exhibited an abrupt breakpoint at 28.6 degrees C, suggesting that membrane fluidity is a factor in phosphate uptake. Increasing the sodium concentration in the incubation medium augmented the phosphate uptake according to a sigmoid curve, and the Hill plot analysis of these data indicates that at least two sodium ions are transported with each phosphate radical. The effect of membrane potential on phosphate uptake was studied by inducing potassium diffusion with valinomycin and by using various sodium salts with different anion conductance in the incubation medium. In both series of experiments, the inside-negative potential significantly enhanced phosphate uptake. We concluded that the phosphate-sodium cotransport is an electrogenic process, a conclusion which is compatible with the observation that at least two sodium ions accompany each phosphate radical. Glycine, alanine and proline all inhibited phosphate uptake according to an uncompetitive type of inhibition. In contrast, the addition of glucose to the incubation medium had no effect.

The paradoxical effect of adrenomedullin on Na+ transport by the renal distal tubule luminal membrane.

Adrenomedullin (ADM) is a potent hypotensive and natriuretic peptide which is synthetized in several mammalian tissues including the kidney. The purpose of this study was to investigate whether the natriuresis was due to a change in Na+ transport by either the proximal (PT) or the distal tubule (DT) luminal membrane, and to characterize this effect, if present. PT and DT suspensions were incubated with human ADM for 20 min at 37 degrees C and luminal membranes of these tubules were purified using the Mg2+ precipitation technique. Na+ uptake was measured by the Millipore filtration technique. A volume of 10(-8) M ADM had no effect on Na+ uptake by the PT luminal membranes. In contrast and unexpectedly, the hormone increased Na+ transport by the DT membranes from 0.28 +/- 0.03 to 0.68 +/- 0.06 pmol/microg per 5 s (P < 0.01). The dose-response curve of this effect showed a maximal response with 10(-7) M ADM. The hormone influenced exclusively the Na+/H+ exchanger, leaving the N-ethyl-N-isopropyl-amiloride (EIPA) insensitive transport intact. The addition of Rp cAMPs to the preparations completely abolished the effect of the hormone on Na+ transport suggesting that cAMP was the messenger involved in this action. Finally, incubation of the DT suspensions with aldosterone also stimulated 1 mM Na+ uptake by the luminal membrane and the two hormone actions were not additive. We conclude that, although ADM is a natriuretic hormone probably through its vasodilating action, it increases distal Na+ reabsorption by the stimulation of the Na+/H+ exchanger activity, as does aldosterone at the same site.

Effect of (1-34) parathyroid hormone-related peptide on the composition and turnover of phospholipids in syncytiotrophoblast brush border and basal plasma membranes of human placenta.

The effect of parathyroid hormone-related peptide on the lipid composition and the turnover of phosphoinositides was studied in brush border and basal plasma membranes of human placenta syncytiotrophoblasts. Lipid composition of the two polar membranes differed markedly with respect to the cholesterol/phospholipid ratio (0.57 +/- 0.04 and 0.91 +/- 0.05 in basal plasma membranes and brush border membranes, respectively). Sphingomyelin was the major phospholipid in both membranes. Except for the phosphoinositide-phosphatidylserine complex which was higher in basal plasma membranes, the phospholipid composition was comparable in the brush border membrane and basal plasma membranes. Incubation of the tissue with 10(-8) M parathyroid hormone-related peptide (1-34) resulted in a significant increase in the phosphatidylinositol phosphate content of the two membranes and in the phosphatidylinositol biphosphate concentration in the basal plasma membranes. Finally, when the tissue was preincubated with [3H]myo-inositol in the presence of 10(-8) M parathyroid hormone-related peptide (1-34), the hormone significantly stimulated the inositol phosphate release by the two membranes. These results demonstrate that: (1) in the placental syncytiotrophoblast, as found in other transport epithelia, the lipid composition of the polar membranes is different; (2) parathyroid hormone-related peptide stimulates the phosphoinositide turnover in both membranes.

Effect of insulin on D-glucose transport by human placental brush border membranes.

We studied the effect of insulin on the uptake of D-glucose by human placental brush border membranes (BBM) in vitro. D-glucose transport through placental BBM is a Na(+)-independent transport, inhibited by 0.5 mM phloretin. Increasing the substrate concentration from 1 to 50 mM resulted in an increase in glucose uptake according to an S-shaped relationship. Hill plot analysis suggests that at least two molecules of D-glucose are transported at the same time by the carrier. Preincubation of the placental tissue with insulin for 45 min at 22 degrees C significantly enhanced the D-glucose influx into the membrane vesicles, without influencing the slope of the Hill plot. A dose-response curve of the effect of insulin revealed that although the effect was already significant at 10(-9) M, the maximal activity was reached at 10(-8) M. The influence of insulin on D-glucose uptake was present only when preincubation of the placental tissue with the hormone was performed in the presence of Mn2+. Incubation of placental tissue with 10(-8) M insulin did not influence D-glucose efflux from the BBM vesicles. Finally, direct incubation of the membranes with insulin had no effect on the glucose influx into these membrane vesicles. We conclude that insulin, at physiological concentrations, enhances glucose uptake by the BBM, and that such a regulation might contribute to the glucose homeostasis in the fetal circulation, independent of the maternal variations in glycemia.

Influence of insulin on phosphate uptake by brush border membranes from human placenta.

Regulation of phosphate transport by insulin was investigated in brush border membranes from human placenta at term. At 22 degrees C, a 45 min incubation of the total tissue with 10(-6) M insulin significantly decreased both the initial rate and the peak of sodium-dependent phosphate uptake by the corresponding brush border membranes. In contrast, Na+ transport was not influenced by the hormone. Increasing the insulin concentration from 0 to 10(-5) M resulted in a dose-dependent inhibition of phosphate uptake with half-maximal effect at 1.1 x 10(-9) M. The hormone decreased PO4 transport by decreasing the affinity of the carrier for the substrate (Km = 0.180 +/- 0.010 mM and 0.215 +/- 0.015 mM in absence and presence of 10(-6) M insulin respectively, P less than 0.05). The inhibitory effect of insulin required the presence of Mn2+ whereas neither Mn2+ nor insulin alone had any influence on PO4 uptake. It is therefore assumed that receptor phosphorylation, which needs the presence of Mn2+, is an intermediate step of insulin action on PO4 uptake by the subsequently isolated brush border membranes. In contrast, insulin had no effect on PO4 uptake when the membranes were directly incubated with the hormone prior to the transport measurement, suggesting that an intracellular messenger is needed for the inhibitory effect. This messenger is not cAMP since insulin at 10(-6) M concentration has no effect on cAMP content of the total placental tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calbindin D 28K on sodium transport by the luminal membrane of the rabbit nephron.

We previously reported that in the rabbit, the vitamin D-dependent calcium binding protein 28K (CaBP 28K) increases calcium (Ca2+) transport in the distal tubule by opening a high affinity Ca2+ channel in the luminal membrane. Since Na+ and Ca2+ transports are interdependent in this membrane, we questioned whether the calbindin has any influence on Na+ transport. Luminal membranes from rabbit proximal and distal tubules were purified and 22Na uptake by the membrane vesicles was measured using the rapid filtration technique. The vesicles were loaded with 280 mM mannitol and 20 mM Tris-Hepes pH 7.4, with either 3 microM CaBP or the carrier. Incubation medium contained 1 mM 22NaCl, 278 mM mannitol, and 20 mM Tris-Hepes pH 7.4. The presence of 3 microM CaBP 28K in the distal luminal membrane vesicles increased the 0.5 mM Ca2+ uptake from 0.91 +/- 0.21 to 1.84 +/- 0.33 pmol/microg/10 s (P < 0.01) and decreased 1 mM Na+ uptake from 0.62 +/- 0.15 to 0.27 +/- 0.08 pmol/microg/10 s (P < 0.05). A similar decrease of Na+ uptake was observed in proximal luminal membrane experiments. The effect on Na+ uptake by the distal membrane was dose-dependent with a IC50 of 4.5 microM. Addition of 2 mM Ca2+ to the incubation medium decreased 1 mM Na + uptake from 0.62 +/- 0.15 to 0.49 +/- 0.12 pmol/microg/10 s (P < 0.05), but did not influence the effect of CaBP 28K on Na+ uptake. Experiments performed in the presence and absence of ethyl isopropyl amiloride (EIPA) suggest that the effect of calbindin involves the Na+/H+ exchanger activity.