Lumenal Ca2 Research Articles

The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Regulation of Ryanodine Receptor Opening by Lumenal Ca2+ Underlies Quantal Ca2+ Release in PC12 Cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.

Calcium signals in the cell nucleus. Strasbourg, France, August 20-23, 1998.

Just as the great explorations revealed a world far more diverse and rich than had been imagined before, a mass of fast accumulating data is disclosing a much more complex and sophisticated picture of the nucleus than hitherto assumed. The nucleus has long been thought of as some sort of enclosure circumscribed by a double membrane and containing the genetic material of the cell. The inner and outer nuclear membranes join periodically at the nuclear pores, forming complexes supposed to create large channels for the free diffusion of ions and small macromolecules. In this view, the flow of calcium between nucleus and cytoplasm would appear unrestricted, with nuclear Ca2+ signals originating from cytosolic Ca2+ waves merely by passive transmission through the nuclear pore complex (NPC). However, recent evidence has changed the mappa mundi of the cell. It has been shown that nuclear Ca2+ can be regulated independently of cytosolic Ca2+ changes. And, just as the Terra Incognita discovered beyond the Ocean Sea proved a fabulous New World, the nuclear envelope (NE) between the cytoplasm and nucleoplasm appears to play an essential role in the regulation of Ca2+ signals inside the nucleus. It contains proteins that regulate and respond to changes in nucleosolic Ca2+ concentration ([Ca2+]nucleosol). Furthermore, several specific Ca2+‐dependent nuclear functions have been described and the list is growing. The regulation of nuclear Ca2+ signals, and the role of nuclear Ca2+ as a specific regulator of nuclear events through Ca2+‐binding proteins, were the main themes of the recent EMBO workshop entitled ‘Calcium signals in the cell nucleus’, organized by A.N.Malviya and coworkers in Strasbourg, France. The existence of Ca2+ signals at the level of the nucleoplasm has never been a real issue, as the …

Ca2+ Regulation of Interactions between Endoplasmic Reticulum Chaperones

Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.

Unitary Ca2+ current through cardiac ryanodine receptor channels under quasi-physiological ionic conditions.

Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1-5 kHz and filtered at 0.2-1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2-30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at approximately 4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 +/- 0.1, 0.65 +/- 0.1, and 0.35 +/- 0.1 pA in 2 mM lumenal Ca2+; and 3.3 +/- 0.4, 2.4 +/- 0. 2, and 1.63 +/- 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 +/- 0.1 and 1.52 +/- 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 +/- 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA.

Active nuclear import and export is independent of lumenal Ca2+ stores in intact mammalian cells.

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (approximately 10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

Regulation of Cardiac Muscle Ca 2+ Release Channel by Sarcoplasmic Reticulum Lumenal Ca 2+

The cardiac muscle sarcoplasmic reticulum Ca 2+ release channel (ryanodine receptor) is a ligand-gated channel that is activated by micromolar cytoplasmic Ca 2+ concentrations and inactivated by millimolar cytoplasmic Ca 2+ concentrations. The effects of sarcoplasmic reticulum lumenal Ca 2+ on the purified release channel were examined in single channel measurements using the planar lipid bilayer method. In the presence of caffeine and nanomolar cytosolic Ca 2+ concentrations, lumenal-to-cytosolic Ca 2+ fluxes ≥0.25 pA activated the channel. At the maximally activating cytosolic Ca 2+ concentration of 4 μM, lumenal Ca 2+ fluxes of 8 pA and greater caused a decline in channel activity. Lumenal Ca 2+ fluxes primarily increased channel activity by increasing the duration of mean open times. Addition of the fast Ca 2+-complexing buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cytosolic side of the bilayer increased lumenal Ca 2+-activated channel activities, suggesting that it lowered Ca 2+ concentrations at cytosolic Ca 2+-inactivating sites. Regulation of channel activities by lumenal Ca 2+ could be also observed in the absence of caffeine and in the presence of 5 mM MgATP. These results suggest that lumenal Ca 2+ can regulate cardiac Ca 2+ release channel activity by passing through the open channel and binding to the channel’s cytosolic Ca 2+ activation and inactivation sites.

Modulation of endoplasmic reticulum calcium pump by Bcl-2.

Members of the bcl-2 gene family encode proteins that function either to promote or to inhibit apoptosis. Despite numerous efforts, the mechanism of action of Bcl-2, an anti-apoptotic protein, is still not clear. In particular, the relation between Bcl-2 and the endoplasmic reticulum (ER) calcium store is not well-understood. In the present work, we examined the effect of Bcl-2 on the ER store. We demonstrate that overexpression of Bcl-2 in breast epithelial cells modulates ER store by upregulating calcium pump (SERCA) expression without affecting the release channel (IP3R). The steady state levels of SERCA2 mRNA and protein were both increased in Bcl-2 expression clones. The increase in SERCA2 protein leads to accelerated calcium uptake and enhanced Ca2+ loading. In addition, we also show the detection of intracellular interaction between Bcl-2 and SERCA molecules by co-immunoprecipitation. Since high lumenal Ca2+ concentration of ER is essential for normal cell functions, the results suggest that Bcl-2 preserves the ER Ca2+ store by upregulating SERCA gene expression as well as by a possible interaction with the pump.

Modelling of some potential effects of lumenal Ca 2+ binding on the kinetics of Ca 2+ release from the endoplasmic reticulum

The time course of Ca 2+ release from intracellular stores during a prolonged exposure to a constant concentration of inositol 1,4,5-trisphosphate does not follow a single exponential but presents a progressive slowing down of the relative rate of efflux. Among the many factors that may contribute to this phenomenon, the possible contribution of lumenal Ca 2+ buffering has been largely neglected. However, since the efflux rate depends on the free Ca 2+ concentration whereas the total Ca 2+ content is mainly determined by bound Ca 2+, simple Ca 2+ efflux kinetics can be expected only if there is a linear relation between free Ca 2+ and bound Ca 2+. Although little is known on lumenal Ca 2+ binding, reasonable assumptions predict that the ratio of (Ca 2+ bound)/(Ca 2+ free) may decrease with increasing free Ca 2+ concentrations, implying a continuously decreasing rate coefficient, as is experimentally observed. Simulated Ca 2+ release curves show significant deviations from a single exponential if high-affinity binding sites are present in addition to low-affinity sites. Although this simple model does not predict several of the experimentally observed properties, it is concluded that a full understanding of Ca 2+ release kinetics requires more detailed information on lumenal Ca 2+ buffering within Ca 2+ stores.

Regulation of Cardiac Muscle Ca 2+ Release Channel by Sarcoplasmic Reticulum Lumenal Ca 2+

Regulation of Cardiac Muscle Ca 2+ Release Channel by Sarcoplasmic Reticulum Lumenal Ca 2+

Fluorescence Probe Study of the Lumenal Ca2+of the Sarcoplasmic Reticulum Vesicles during Ca2+Uptake and Ca2+Release

A limited amount of information is available about the lumenal Ca2+kinetics of the sarcoplasmic reticulum (SR). Incubation of mag-fura-2AM permitted to incorporate a sufficient amount of the probe into the SR vesicles, as determined by Mn2+quenching. Rapid changes in the lumenal [Ca2+] ([Ca2+]lum) during Ca2+uptake and release could be monitored by following the signal derived from the lumenal probe while clamping the extra-vesicular Ca2+([Ca2+]ex) at various desired levels with a BAPTA/Ca buffer. Changes in the [Ca2+]lumduring uptake and release show the characteristics intrinsic to the SR Ca2+pump (the [Ca2+]ex-dependence of the activation and inhibition by thapsigargin) and the Ca2+release channel (blocking by ruthenium red), respectively. A new feature revealed by the [Ca2+]lummeasurement is that during the uptake reaction the free [Ca2+]lumshowed a significant oscillation. Several pieces of evidence suggest that this is due to some interactions between the Ca2+pump and lumenal proteins.

BiP, a Major Chaperone Protein of the Endoplasmic Reticulum Lumen, Plays a Direct and Important Role in the Storage of the Rapidly Exchanging Pool of Ca2+

The activity of BiP, the major chaperone of the endoplasmic reticulum (ER) lumen, is known to be Ca2+-regulated; however, the participation of this protein in the ER storage of the cation has not yet been investigated. Here such a role is demonstrated in human epithelial (HeLa) cells transiently transfected with the hamster BiP cDNA and incubated in Ca2+-free medium, as revealed by two different techniques. In the first, co-transfected aequorin was employed as a probe for assaying either the cytosolic of the mitochondrial free Ca2+ concentration. By this approach higher Ca2+ release responses were revealed in BiP-transfected cells by experiments in which extensive store depletion was induced either by repetitive stimulation with inositol 1,4,5-trisphosphate-generating agonists or by treatment with the Ca2+ ionophore, A23187. In the second technique the cells were loaded at the equilibrium with 45Ca, and the release of the tracer observed upon treatment with thapsigargin, a blocker of the ER Ca2+ ATPases, was larger in BiP-transfected than in control cells. The latter results were obtained also when BiP was overexpressed not via transfection but as a response to ER stress by tunicamycin. These results are sustained by increases of the ER Ca2+ storage capacity rather than by artifacts or indirect readjustments induced in the cells by the overexpression of the chaperone since (a) the exogenous and endogenous BiP were both confined to the ER, (b) the expression levels of other proteins active in the ER Ca2+ storage were not changed, and (c) effects similar to those of wild type BiP were obtained with a deletion mutant devoid of chaperone activity. The specificity of the results was confirmed by parallel 45Ca experiments carried out in HeLa cells transfected with two other Ca2+-binding proteins, calreticulin and CaBP2(ERp72), only the first of which induced increases of Ca2+ capacity. We conclude that BiP has a dual function, in addition to its chaperone role it is a bona fide ER lumenal Ca2+ storage protein contributing, under resting cell conditions, to around 25% of the store, with a stoichiometry of 1-2 moles of calcium/mole of BiP.

Intracellular Association between UDP-glucose:Glycoprotein Glucosyltransferase and an Incompletely Folded Variant of α1-Antitrypsin

Genetic variants of human alpha1-antitrypsin unable to fold into the native structural conformation are poorly secreted from hepatocytes. The molecular chaperone calnexin coimmunoprecipitates with secretion-incompetent variant null(Hong Kong) retained in stably transfected mouse hepatoma cells (Le, A., Steiner, J. L., Ferrell, G. A., Shaker, J. F., and Sifers, R. N. (1994) J. Biol. Chem. 269, 7514-7519). Mobilization of intracellular Ca2+ stores with metabolic poisons diminished interaction with calnexin and coincided with coimmuoprecipitation of a 150-kDa protein (p150). Mobilization of endoplasmic reticulum lumenal Ca2+ with thapsigargin, an inhibitor of the microsomal Ca2+ATPase, gave a similar result. Coimmunoprecipitation of p150 was specifically disrupted in response to incubation of the cell lysate with exogenous CaCl2. Finally, in ECL Western blotting, p150 was recognized by polyclonal antiserum against UDP-glucose:glycoprotein glucosyltransferase that likely functions in glycoprotein folding and quality control (Sousa, M. C., Ferrero-Garcia, M. A., and Parodi, A. J. (1992) Biochemistry 31, 97-105). The data are consistent with a model in which perturbation of endoplasmic reticulum Ca2+ results in a stable physical association between unfolded human alpha1-antitrypsin and UDP-glucose:glycoprotein glucosyltransferase.

Inophore-releasable lumenal Ca 2+ stores are not required for nuclear envelope assembly or nuclear protein import in Xenopus egg extracts

The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca 2+ stores via inositol 1,4,5-trisposphate-gated Ca 2+ channels is required for nuclear assembly in Xenopus egg extracts [1,2]. Other work suggested that lumenal Ca 2+ stores are required for nuclear protein import using vitro [3]. Here, we rigorously tested the role of lumenal Ca 2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca 2+ stores were depleted by pretreating the extracts with Ca 2+ ionophores (ionomycin, A23187) or inhibitors of Ca 2+-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca 2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca 2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca 2+-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca 2+ stores confirs a previous unpublished observation by Greber and Gerace [3], and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca 2+. Finally, we concluded that, contrary to our expectations, lumenal Ca 2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.

Analytical steady-state solution to the rapid buffering approximation near an open Ca2+ channel

We derive an analytical steady-state solution for the Ca2+ profile near an open Ca2+ channel based on a transport equation which describes the buffered diffusion of Ca2+ in the presence of rapid stationary and mobile Ca2+ buffers (Wagner and Keizer, 1994). This steady-state rapid buffering approximation gives an upper bound on local Ca2+ elevations such as Ca2+ puffs or sparks when conditions for the validity of the rapid buffering approximation are met and is an alternative to approximations that assume that mobile buffers are unsaturable. This result also provides an analytical estimate of the cytosolic Ca2+ domain concentration ([Ca2+]d) near a channel pore and shows the dependence of [Ca2+]d on moderate concentrations of endogenous mobile buffer, Ca2+ indicator dye, and bulk cytosolic Ca2+. Assuming a simple relationship between [Ca2+]d and the lumenal depletion domain of an intracellular Ca2+ channel, lumenal and cytosolic Ca2+ profiles are matched to give an implicit analytical expression for the effect of bulk lumenal Ca2+ on [Ca2+]d.

Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells.

We have examined the effects of depleting lumenal Ca2+ on the synthesis, phosphorylation and secretion of caseins in lactating mouse mammary cells by using inhibitors of the endoplasmic reticulum Ca(2+)-ATPase or the ionophore ionomycin in the absence of external Ca2+. Treatment with these drugs resulted in a transient increase in the cytosolic Ca2+ concentration due to Ca2+ mobilization. Protein synthesis over a 1 h period was substantially inhibited by Ca2+ depletion, but in a pulse-chase protocol secretion of pre-synthesized proteins was unaffected by Ca2+ depletion. Analysis of polysome profiles showed that Ca2+ depletion resulted in a loss of polysomes, consistent with an inhibition of initiation of protein synthesis. Neither treatment with Ca(2+)-ATPase inhibitors to deplete endoplasmic reticulum Ca2+ nor treatment with ionomycin/EGTA had any effect on an early phase of phosphorylation of alpha- or beta/gamma-caseins, but Ca2+ depletion resulted in a decrease in a late phase of casein phosphorylation. These results indicate that lumenal Ca2+ is required to maintain protein synthesis in lactating mammary cells but is not required for protein secretion, and that Ca2+ accumulation in the Golgi cisternae is required for a late but not for an early phase of casein phosphorylation.

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.

Apparent nonnuclear regulation of intestinal phosphate transport: effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3.

The steroid hormone 1,25-(OH)2D3 modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium transport (transcaltachia). The present studies determined whether 1,25-(OH)2D3 rapidly enhanced phosphate transport in the perfused duodenal loop of normal chicks. Vascular perfusion with 65 pM 1,25-(OH)2D3 significantly stimulated the appearance of 33P in the venous effluent within 2-8 min, reaching an average of 160% of control levels by 40 min. Lumenal perfusion with hormone failed to augment levels of 33P in the venous effluent. Vascular perfusion with a range of 1,25-(OH)2D3 concentrations yielded an apparent biphasic dose-response curve. Two additional vitamin D metabolites, 24,25(OH)2D3 and 25(OH)D3, which initiate transcaltachia, were tested for their effect on phosphate transport. Neither 6.5 nM 24,25(OH)2D3 nor 100 nM 25(OH)D3 stimulated 33P movement from the lumen to the venous effluent. When duodena were vascularly perfused with 65 pM 1,25-(OH)2D3 and either 6.5 nM 24,25(OH)2D3 or 100 nM 25(OH)D3, enhanced phosphate transport was attentuated or abolished. Phosphate transport was also analyzed at metabolite levels 5-fold lower or higher than those in normal chicks. For 24,25(OH)2D3, 1.3 nM metabolite did not augment phosphate transport, although stimulation did occur at 32.5 nM steroid (180 +/- 0.2% of controls). For 25(OH)D3, no stimulation was observed at 500 nM metabolite, whereas 20 nM steroid resulted in transport that was 160 +/- 0.14% of controls. The presence or absence of lumenal Ca2+ did not influence phosphate transport in duodena vascularly perfused with control medium, 65 pM 1,25-(OH)2D3, or 6.5 nM 24,25(OH)2D3. In contrast, vascular perfusion with 100 nM 25(OH)D3 stimulated phosphate transport when lumenal Ca2+ was present but not when it was absent. The influence of lumenal P(i) on transcaltachia was then studied. Although 65 pM 1,25-(OH)2D3, 6.5 nM 24,25(OH)2D3, and 100 nM 25(OH)D3, initiate transcaltachia in the presence of lumenal P(i), the absence of the anion abolished the stimulatory effects of 1,25-(OH)2D3 and 24,25(OH)2D3 on calcium transport but not those induced by 25(OH)D3. These data suggest a complex regulation of both calcium and phosphate transport based on relative levels of circulating vitamin D metabolites as well as the ionic content of the lumen.

Apparent nonnuclear regulation of intestinal phosphate transport: effects of 1,25-dihydroxyvitamin D3,24,25-dihydroxyvitamin D3, and 25- hydroxyvitamin D3

The steroid hormone 1,25-(OH)2D3 modulates a number of cellular functions through nonnuclear pathways, particularly the stimulation of intestinal calcium transport (transcaltachia). The present studies determined whether 1,25-(OH)2D3 rapidly enhanced phosphate transport in the perfused duodenal loop of normal chicks. Vascular perfusion with 65 pM 1,25-(OH)2D3 significantly stimulated the appearance of 33P in the venous effluent within 2-8 min, reaching an average of 160% of control levels by 40 min. Lumenal perfusion with hormone failed to augment levels of 33P in the venous effluent. Vascular perfusion with a range of 1,25-(OH)2D3 concentrations yielded an apparent biphasic dose-response curve. Two additional vitamin D metabolites, 24,25(OH)2D3 and 25(OH)D3, which initiate transcaltachia, were tested for their effect on phosphate transport. Neither 6.5 nM 24,25(OH)2D3 nor 100 nM 25(OH)D3 stimulated 33P movement from the lumen to the venous effluent. When duodena were vascularly perfused with 65 pM 1,25-(OH)2D3 and either 6.5 nM 24,25(OH)2D3 or 100 nM 25(OH)D3, enhanced phosphate transport was attentuated or abolished. Phosphate transport was also analyzed at metabolite levels 5-fold lower or higher than those in normal chicks. For 24,25(OH)2D3, 1.3 nM metabolite did not augment phosphate transport, although stimulation did occur at 32.5 nM steroid (180 +/- 0.2% of controls). For 25(OH)D3, no stimulation was observed at 500 nM metabolite, whereas 20 nM steroid resulted in transport that was 160 +/- 0.14% of controls. The presence or absence of lumenal Ca2+ did not influence phosphate transport in duodena vascularly perfused with control medium, 65 pM 1,25-(OH)2D3, or 6.5 nM 24,25(OH)2D3. In contrast, vascular perfusion with 100 nM 25(OH)D3 stimulated phosphate transport when lumenal Ca2+ was present but not when it was absent. The influence of lumenal P(i) on transcaltachia was then studied. Although 65 pM 1,25-(OH)2D3, 6.5 nM 24,25(OH)2D3, and 100 nM 25(OH)D3, initiate transcaltachia in the presence of lumenal P(i), the absence of the anion abolished the stimulatory effects of 1,25-(OH)2D3 and 24,25(OH)2D3 on calcium transport but not those induced by 25(OH)D3. These data suggest a complex regulation of both calcium and phosphate transport based on relative levels of circulating vitamin D metabolites as well as the ionic content of the lumen.

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel