Unidirectional Calcium Fluxes Research Articles

Individual Amino Acids Differentially Regulate Paracellular Calcium Flux across an Intestinal Cell Model

Acute high protein intake results in increased intestinalcalcium absorption in humans and animals. Calcium is absorbed through both passive paracellular and facilitated trans cellular pathways. In rats, the increase in intestinal calcium absorption observed during a high protein dietis mediated at least in part through increased apical intestinal calcium uptake suggesting involvement of the transcellular pathway. However, we previously found that the expression of a paracellular calcium‐permissive tight junction protein, Claudin‐2, is higher in the duodenum of rats consuming a high compared to lowprotein diet. Whether dietary protein affects paracellular calcium flux has not been evaluated. Furthermore, the bioactive amino acids that might underlie this effect have not been identified. The purpose of this study was to evaluate the direct effects of mixed and individual amino acids on paracellular calcium flux across a small intestinal absorptive cell model.MethodsCaco‐2 Bbe cells were used as a model of small intestinal absorptive epithelial cells. Confluent monolayers were grown on snap well inserts and mounted in Ussing chambers for flux studies under a mucosal to serosal calcium gradient to simulatepost‐prandial conditions. Unidirectionalcalcium flux over 90 min was calculated using 44Ca as a tracer with serosal samples collected every 30 min. Treatments were applied acutely to bothmucosal and serosal chambers and included vehicle (no treatment control), 80mMraffinose as a positive control, mixed amino acids, and 2, 5 and 10mMleucine, phenylalanine and lysine. RNA expression of calcium permissive tight junction proteins was measured using qPCR.ResultsLinear regression analysis modeling Ca flux (nmol/cm2/hr) by treatment using vehicle control as the reference group elicited significant increases with 80mM raffinose (β= 66.7, t= 3.2, p< 0.05) and 5mM leucine (β= 67.6, t= 1.9, p≤0.05), whereas 10mM phenylalanine resulted in a significant decrease in Ca flux (β= −89.3, t=−1.9, p< 0.05). No significant changes were observed with mixed amino acidor lysine treatment. The effects of amino acids on Ca flux were not associated with changes in mRNA expression of the Ca permissive tight junction proteins claudins‐2 and 12.ConclusionsThese data demonstrate that amino acids have differential effects on paracellular Ca flux in a model of intestinalabsorptive epithelia. This suggests that specific amino acid constituents of dietary protein may be partially responsible for protein‐mediated increases inintestinal Ca absorption. Gene expression of claudins‐2 and 12 did not explain aminoacid induced changes in Ca flux. Future research will investigate posttranscriptional effects of amino acids on tight junction proteins in anintestinal epithelial model.Support or Funding InformationResearch supported by: USAMRMC. The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.

Diadenoine polyphosphate-induced increase in cytosolic free calcium in vascular smooth muscle cells

To evaluate the effects of two new endogenous vasoconstricting substances, diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) on the cytosolic free calcium concentration in vascular smooth muscle cells. Spectrofluorophotometric measurements of cytosolic Ca2+ were conducted in monolayers of cultured rat vascular smooth muscle cells using the calcium-sensitive fluorescent dye fura-2. The resting Ca2+ concentration in vascular smooth muscle cells was 80 +/- 5 nmol/l (mean +/- SEM; n = 39). The addition of 10 mu mol/l AP5A or AP6A significantly increased Ca2+ in vascular smooth muscle cells to 248 +/- 55 and 358 +/- 124 nmol/l, respectively. The sustained increase in Ca2+ after administration of AP5A or AP6A was 143 +/- 40 and 148 +/- 57 nmol/l, respectively. Diadenosine polyphosphates induce a transplasmamembrane calcium influx as detected by experiments in the absence of external calcium or by using the manganese quenching technique to report unidirectional calcium fluxes. The effects of diadenosine polyphosphates were compared to those of the well known vasoconstrictor angiotensin II, which increased Ca2+ in vascular smooth muscle cells by 1053 +/- 174 nmol/l. Diadenosine polyphosphates increase Ca2+ in vascular smooth muscle cells, thereby regulating the contractility of vascular smooth muscle and subsequently blood pressure.

Calcium absorption by fish intestine: The involvement of ATP-and sodium-dependent calcium extrusion mechanisms

Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia, Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca2+ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca2+ pump (Vm: 0.63 nmol.min-1.mg-1, Km:27 nM Ca2+) is calculated to be 2.17 nmol.min-1.mg-1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na+/Ca2+ exchanger with high affinity for Ca2+ (Vm:7.2 nmol.min-1.mg-1, Km:181 nM Ca2+) is calculated to be 13.6 nmol.min-1.mg-1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na+/Ca2+ exchanger. The high affinity for Ca2+ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na+/Ca2+ exchanger will contribute substantially to Ca2+ extrusion in the fish enterocyte. Further evidence for an important contribution of Na+/Ca2+ exchange to Ca2+ extrusion was obtained from studies in which the simultaneous operation of ATP- and Na(+)-gradient-driven Ca2+ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca2+ transporting cell, in which Na+/Ca2+ exchange activity with high affinity for Ca2+ extrudes Ca2+ from the cell.

Comparison of the enhanced steady-state diffusion of calcium by calbindin-D9K and calmodulin: Possible importance in intestinal calcium absorption

The diffusion of calcium was measured using the unidirectional flux of 45Ca across an aqueous layer. The aqueous layer was bounded by two dialysis membranes and convection was eliminated by gelling the aqueous layer with agarose. The apparent self-diffusion coefficient was determined by the dependence of the tracer flux on the diffusion distance. The apparent self-diffusion coefficient increased linearly with the concentration of calbindin-D9K and calmodulin, but the effect of calmodulin was markedly less than that of calbindin-D9K. This difference is attributed to the lower association constant for calmodulin. The ion-exchange resin Chelex-100 also increased the steady-state flux of 45Ca, but the effect of Chelex-100 was much less efficient than the effect of calbindin-D9K. The mechanism of enhanced diffusion was attributed to an enhanced gradient of total 45Ca. These results indicate that the steady-state unidirectional calcium flux is a superposition of free calcium diffusion and bound calcium diffusion, with only a small contribution due to a ‘bucket brigade’ mechanism. We suggest that this phenomenon may be important in calcium absorption across the intestine.

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout.

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.

Calcium transport across the colon ascendens and the influence of 1,25-dihydroxyvitamin D3 and dexamethasone.

Concentration dependence of unidirectional calcium fluxes across the rat colon ascendens were measured in a modified Ussing chamber. Both the mucosa (m) to serosa (s) and the s to m calcium flux exhibited saturation kinetics. The maximum transport rates and the affinity to the transporter of calcium was higher in the m to s direction than that from s to m, resulting in a remarkable net calcium absorption. The results obtained from measurements of unidirectional calcium fluxes in dependence on clamped transepithelial potentials showed that: (i) calcium transport in both direction had a voltage-independent component; (ii) the voltage-independent, i.e. non diffusive fraction of the m to s calcium flux was 3.2 times greater than that in the opposite direction; (iii) the voltage dependent, i.e. diffusional fraction of the m to s calcium flux, was about two times greater than the voltage-dependent fraction of the calcium flux in the s to m direction; and (iv) in the m to s direction 62%, and the s to m direction 73%, of the total unidirectional flux was voltage-dependent. Dexamethasone, known to enhance sodium and water absorption in the colon, had no significant influence on net calcium absorption but increased the unidirectional calcium fluxes in both directions. The increase in unidirectional calcium fluxes parallel to that of the extracellular marker mannitol suggests that dexamethasone has no influence on the transcellular calcium transport but increases the calcium flux along the paracellular way. Amiloride had no influence on the dexamethasone-induced changes of the epithelial electrical parameters as distinguished from the colon descendens.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and paracellular calcium transport in the rat ileum and the influence of 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone.

Concentration dependence of unidirectional calcium fluxes across the rat ileum freed from the serosa and the muscularis externa were measured in a modified Ussing-chamber. Mucosa (m) to serosa (s) calcium flux showed a saturable component, whereas s to m calcium flux was linearly related to the calcium concentration between 0.125 mmol/l and 5 mmol/l. At all calcium concentrations used net secretion of calcium was observed. The s to m flux of the simultaneously measured paracellular marker mannitol at all calcium concentrations was remarkably higher than the m to s flux, resulting in net mannitol secretion. The results obtained from the calcium fluxes when clamping the transepithelial electrical potential agree well with those of the concentration dependence of the calcium fluxes: 1. Only m to s flux has a voltage independent, transcellular component. 2. Calcium s to m flux is totally voltage dependent, i.e. diffusive. 3. Diffusional s to m calcium flux is about 80% greater than the diffusional fraction of the m to s flux. Omitting glucose from the bathing solution effected a decrease of the transepithelial electrical potential and of the short circuit by 91% and 85% respectively; net calcium secretion was almost abolished and net mannitol secretion remarkably reduced. Addition of glucose, which stimulates water absorption in the ileum as a metabolic substrate, activated m to s but significantly more pronounced s to m calcium flux parallel to that of mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the paracellular pathway in the net transport of calcium across the colonic mucosa.

Concentration dependent calcium fluxes across the colon descendens of the rat were measured in a modified Ussing chamber. Mucosa (m) to serosa (s) calcium flux showed a saturable component, whereas s to m calcium flux was linearly related to the calcium concentration. At low calcium concentrations net absorption and at concentration above 2.5 mmol/l net secretion of calcium was observed. The results obtained from the unidirectional calcium fluxes when clamping the transepithelial electrical potential agree well with those of the concentration dependence of the calcium fluxes: Only m to s flux has a voltage independent component. Calcium s to m movement is totally voltage dependent. Diffusional s to m calcium flux is greater than the diffusional fraction of the m to s calcium flow. Dexamethasone, known to stimulate water absorption in the colon descendens by an activation of sodium transport, had no effect on the cellular mediated m to s calcium transport but significantly increased paracellular s to m flux parallel to that of the extracellular marker mannitol. This increase in paracellular s to m calcium and mannitol flux was completely abolished by amiloride, which is known to suppress the dexamethasone-induced stimulation in sodium and water absorption. The results demonstrate that the increased paracellular s to m calcium and mannitol flow is oppositely directed to the dexamethasone-induced net fluid movement as it could be expected on the basis of Ussing's "anomalous solvent drag" effect.

Effects of Mg 2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg 2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg 2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg 2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg 2+). Calcium uptake velocity in light vesicles correlated with Ca 2+-dependent ATPase activity, suggesting that Mg 2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca 2+-dependent ATPase activity, although a Mg 2+-dependent increase in calcium influx was observed. Thus, Mg 2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg 2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca 2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg 2+ suggested that ruthenium red (0.5 μ M) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.

The defect of intestinal calcium transport in hyperthyroidism and its response to therapy.

Skeletal demineralization occurs in thyrotoxicosis. Fecal calcium excretion may be enhanced, and calcium balance tends to be negative. We investigated intestinal calcium transport in 12 hyperthyroid patients. Absorption was measured by segmental perfusion in the proximal jejunum at calcium concentrations commensurate with those in the fasting and postprandial states. At low luminal concentrations, under conditions where calcium is transported predominantly by active processes, the calcium absorption rate was reduced though not abolished in hyperthyroid patients (16 +/- 4 (SE) mumol/h . 30 cm segment) as compared to normal subjects (71 +/- 8 mumol/h; P less than 0.001). When perfusate calcium was raised to 5 mmol/liter there was little increment of the net absorption rate in the hyperthyroid group (45 +/- 11 mumol/h), whereas that in the normal subjects rose to 183 +/- 17 mumol/h. Likewise, the unidirectional calcium flux out of the lumen was low in hyperthyroidism (43 +/- 7 mumol/h), suggesting that low net absorption rates were not due to transmucosal calcium loss. D-Xylose permeation was similar in all study groups. Treatment of the thyroid disease led to a marked increase in calcium absorption rates from 33 +/- 10 to 124 +/- 20 mumol/h (at 2 mmol/liter P less than 0.001; n = 5) into the range of values in normal subjects (124 +/- 9 mumol/h). Circulating levels of 1,25-dihydroxyvitamin D were low in hyperthyroid patients (38 +/- 11 pg/ml) and increased during treatment to 63 +/- 11 pg/ml (P less than 0.025; n = 9), whereas 25-hydroxyvitamin D was normal and remained unchanged. We conclude that intestinal calcium transport, particularly its active component, is reversibly decreased in hyperthyroidism. The association with low plasma levels of the active vitamin D metabolite suggests that the decrease in calcium absorption may be related to calcium-regulating mechanisms as a consequence of the net calcium efflux from bone in this disease.

Unidirectional calcium and nucleotide fluxes in sarcoplasmic reticulum. I. Interpretation of flux ratios for different reaction schemes

The relation between unidirectional calcium and nucleotide fluxes was examined for different ATPase reaction schemes of the sarcoplasmic reticulum. The schemes considered differed in the order of sorption and desorption of calcium, ATP, and ADP. The results suggest that the theoretical relation between calcium and nucleotide fluxes depends on the reaction scheme and that experimental measurements can distinguish among them. The results obtained are generally valid and do not depend on assumptions of equilibrium or pseudoequilibrium between intermediate states of the pump.

Measurement of facilitated calcium diffusion by a soluble calcium-binding protein

The flux of calcium through an aqueous compartment was determined in a flow-dialysis cell in which two dialysis membranes separated the middle aqueous compartment from two outer compartments. The contribution of convection to the total calcium flux was large but could be removed by addition of 1% agar. The flux of calcium through the gelled aqueous compartment agreed with theoretical expectations. The self-diffusion coefficient for calcium from these results was calculated to be 0.81·10 −5 cm 2·s −1. Carp parvalbumin significantly enhanced the calcium flux at 2.3·10 −6M free calcium. The calcium flux increased linearly with parvalbumin concentration. These observations are consistent with the hypothesis that the overall unidirectional calcium flux J is the sum of free calcium diffusion and protein-calcium diffusion: J = D[Ca] + D′[CaPr]. The value of D′, the self-diffusion coefficient for parvalbumin, was calculated from the flux data to be 13.7·10 −7 cm 2·s −1.

Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results

Unidirectional calcium influx and efflux were evaluated in cardiac sarcoplasmic reticulum (SR) by 45Ca-40Ca exchange at steady state calcium uptake in the absence of calcium precipitating anions. Calcium efflux was partitioned into a pump-mediated efflux and a parallel passive efflux by separately measuring passive efflux referable to the steady state. Unidirectional and net ATP-ADP fluxes were measured using [3H]-ATP----ADP and [3H]-ADP----ATP exchanges. Methods are presented that take into account changing specific activities and sizes of the nucleotide pools during the measurement of nucleotide fluxes. The contribution of competent and incompetent vesicles to the unidirectional and net nucleotide fluxes was evaluated from the specific activity of these fluxes in incompetent vesicles and from the fraction of vesicles that were incompetent. The results indicate that, in cardiac SR, unidirectional calcium fluxes are larger than the unidirectional nucleotide fluxes contributed by competent vesicles. Because the net ATPase rate of competent vesicles is similar to the parallel passive efflux, it appears that cardiac SR Ca-ATPase tightly couples ATP hydrolysis to calcium transport even at static head, with a coupling ratio near 1.0.

Effects of lactose on calcium absorption and secretion by rat ileum.

The direct effects of lactose on net intestinal calcium absorption were determined by measuring unidirectional steady-state calcium fluxes in vitro under short-circuited conditions in segments of rat ileum. The isosmotic mucosal additions in segments of rat ileum. The isosmotic mucosal addition of lactose (160 mM) increased net calcium absorption (J net) by increasing the absorptive flux from mucosa to serosa (Jm----s) and reducing the secretory flux from serosa to mucosa (Js----m). Lactose also reduced tissue conductance and short-circuit current and reversed tissue polarity. 1,25-Dihydroxyvitamin D3 administration (50 ng/day for 4 days) increased J net from secretion to no net flux (Jm----s = Js----m), and lactose increased J net further to net absorption. Removal of sodium from the medium, like lactose addition, increased J net by increasing Jm----s and reducing Js----m. The replacement of medium sodium with choline abolished a further increase of J net by lactose. These results show that lactose increases net calcium absorption in the absence of transepithelial electrochemical or osmotic gradients. Transcellular calcium transport may be stimulated by lactose by hyperpolarization of the brush border as a result of reduced mucosal sodium.

High-affinity Ca-Mg-ATPase along the rabbit nephron.

Maintenance of cell calcium homeostasis and transepithelial transport of this cation require its extrusion from the cell against a steep electrochemical gradient. Because it has been proposed that a membrane Ca-ATPase activated by micromolar concentrations of Ca2+ prevailing in the cell participates in these processes, we attempted in this study to determine whether such an enzyme is present in the rabbit nephron. A magnesium-dependent ATPase, maximally activated by Ca2+ (Ca-Mg-ATPase) concentrations between 1.1 and 2.3 microM (apparent Km = 0.3-0.4 microM), was found in all segments of the nephron. Ca-Mg-ATPase (pmol.mm-1.h-1) was highest in the distal convoluted tubule (243) and cortical collecting tubule (208), intermediate in the proximal convoluted tubule (140) and medullary thick ascending limb of Henle's loop (135), and lower in the pars recta (97), cortical thick ascending limb (50), and medullary collecting tubule (51). The enzyme was insensitive to ouabain and vanadate, but was inhibited by ruthenium red in a dose-dependent manner (Ki congruent to 2.10(-6) M). Sodium azide, an inhibitor of mitochondrial ATPase, did not affect Ca-Mg-ATPase, suggesting that the enzyme was located in the plasma membrane. The Ca-Mg-ATPase activity measured in most segments of the rabbit nephron in this study appears sufficient to account in theory for the active component of the unidirectional (lumen-to-bath) calcium flux found in the corresponding region of the nephron with in vitro single tubule microperfusion techniques.

Voltage-dependent calcium movement across the cortical collecting duct.

Cortical collecting ducts were dissected from rabbit kidneys and perfused in vitro. Unidirectional transepithelial calcium fluxes from bath-to-lumen and lumen-to-bath were measured with 45Ca. Transepithelial voltage was varied over a wide range by pharmacologic manipulations. With lumen-negative voltages net calcium secretion from bath to lumen, which varied directly with the voltage, was observed. At voltages near 0 there was no measurable net flux. When the voltage was made positive, the direction of net calcium transport reversed (i.e., absorption from lumen to bath). Calcium permeability, calculated from the dependence of net flux on voltage, was 1.4 X 10(-7) cm/s, which is less than 2% of the calcium permeability previously determined in the cortical thick ascending limb of Henle's loop. Analysis of the calcium flux ratios revealed apparent interdependence of the bidirectional fluxes consistent with exchange diffusion but no evidence for active calcium transport. We conclude that there is a small, but measurable, component of passive net calcium flux driven by the transepithelial voltage across the cortical collecting duct.

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca 2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K + gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K + gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca 2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca 2+-activated ATPase protein.

Voltage dependence of calcium transport in the thick ascending limb of Henle's loop

Thick ascending limbs of Henle's loop were dissected from rabbit kidneys and perfused in vitro. Unidirectional transepithelial calcium fluxes from lumen-to-bath and bath-to-lumen were measured with 45Ca using different solutions that caused the transepithelial voltage to vary over a wide range. With lumen-positive voltages there was net calcium absorption from lumen to bath which varied directly with the voltage. With voltage near zero there was no measurable net flux. When the voltage was made negative, the direction of net calcium transport reversed (i.e., secretion from bath to lumen). The presence or absence of bicarbonate in the lumen did not affect the calcium fluxes. Calcium permeability, calculated from the dependence of net flux on voltage, was 7.7 x 10(-6) cm/s, which is approximately 25% of the sodium permeability previously determined in this segment. Analysis of the calcium flux ratios revealed interdependence of the bidirectional fluxes consistent with single-file diffusion but no evidence for active calcium transport. We conclude that there is an important component of passive net calcium transport driven by the voltage in this segment.

Jejunal and ileal absorption in patients with chronic renal disease. Effect of 1alpha-hydroxycholecalciferol.

Calcium absorption in 30-cm segments of small intestine was measured by constant perfusion of test solutions containing different concentrations of calcium gluconate. In both the jejunum and ileum, calcium absorption rates increased progressively as luminal calcium concentration was increased stepwise between 1 and 20 mM. Although calcium transport was not saturable within these limits, unidirectional flux ratios of calcium (47Ca) suggest that calcium absorption is active in both the jejunum and ileum. Calcium absorption in patients with chronic renal disease was markedly depressed in both regions of the small intestine. This was due to decreased flux out of the lumen; flux in the reverse direction was normal. Flux ratios in the renal disease patients showed no evidence for active calcium transport. Treatment of these patients for 1 wk within 2 mug/day of 1alpha-hydroxycholecalciferol [1alpha-(OH)-D3] restored net calcium absorption and unidirectional calcium flux out of the lumen to normal values in the jejunum; in the ileum, 1alpha-(OH)-D3 increased calcium absorption 60-83% of normal at the various luminal calcium concentrations. 1alpha(OH)-D3 had no effect on unidirectional calcium flux into the lumen or on xylose and electrolyte absorption in either area of the small intestine.

Jejunal absorption and secretion of calcium in patients with chronic renal disease on hemodialysis.

10 patients with chronic renal disease on hemodialysis and 8 normals were studied by constant jejunal perfusion of calcium gluconate solutions, using polyethylene glycol as a nonabsorbable marker. Results in normals indicated that calcium absorption from 1 and 5 mM calcium solutions is mainly active. Absorption from 5, 15, and 20 mM solutions was a linear function of luminal calcium concentration, suggesting that the active transport carrier is saturated when luminal calcium concentration is greater than 5 mM and indicating that the increment in absorption at higher luminal concentrations is mainly the result of passive absorption. With 1 mM calcium, normals absorbed calcium against a concentration gradient, whereas the patients secreted calcium. Absorption in the patients was much less than normal with 5, 15, and 20 mM luminal calcium concentrations; however, the slope of this linear (passive) portion of the curve was normal. Unidirectional calcium fluxes were measured with calcium-47. Flux out of the lumen was depressed 2.5-fold in the patients, but flux into the lumen was normal. Xylose, urea, and tritiated water were absorbed normally, indicating no generalized abnormality of jejunal transport in these patients. Endogenous calcium secretion, estimated by the amount of calcium added to a calcium-free solution, was normal in the dialysis patients. These results indicate that active calcium absorption is markedly depressed in patients with chronic renal disease who are receiving hemodialysis therapy. On the other hand, passive calcium movement and endogenous calcium secretions are normal.