Nucleoplasmic Ca2 Research Articles

Presence of a Putative Vesicular Inositol 1,4,5-Trisphosphate-Sensitive Nucleoplasmic Ca2+ Store

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are widely localized in both the heterochromatin and euchromatin regions. We found recently the presence of nucleoplasmic complexes that are composed of phospholipids, IP(3)R/Ca(2+) channels, and Ca(2+) storage protein chromogranin B (CGB). Close examination and 3D image reconstruction of these complexes revealed numerous vesicular structures with an average diameter of approximately 50 nm that are primarily interspersed between the heterochromatins. IP(3) rapidly released Ca(2+) from these structures, but other inositol phosphates, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, failed to release Ca(2+). Addition of heparin or IP(3)R antibody blocked the IP(3)-induced Ca(2+) releases, indicating the release of Ca(2+) through the IP(3)R/Ca(2+) channels. Given the presence of the IP(3)R/Ca(2+) channels and Ca(2+) storage protein CGB in these vesicular structures, we postulate that these vesicles are the IP(3)-sensitive nucleoplasmic Ca(2+) stores. Abundance of the vesicular Ca(2+) stores between the heterochromatins appeared to imply critical roles these vesicular Ca(2+) stores play in controlling the Ca(2+) concentrations of the chromosomes.

Presence of a Nucleoplasmic Complex Composed of the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel, Chromogranin B, and Phospholipids

Although the inositol 1,4,5-trisphosphate (IP(3)) induced nuclear Ca(2+) releases have been shown to play key roles in nuclear functions, the presence and operation of the IP(3)-dependent Ca(2+) control mechanism in the nucleoplasm have not been shown. Recently, we found the presence of a high-capacity, low-affinity Ca(2+)-storage protein chromogranin B (CGB) and all three IP(3) receptor (IP(3)R) isoforms in the nucleoplasm, localizing widely in both the heterochromatin and euchromatin regions. In view of the essential role of CGB-IP(3)R coupling in IP(3)-dependent Ca(2+) release in the endoplasmic reticulum, the potential coupling between CGB and the IP(3)Rs in the nucleoplasm was investigated. Hence, we found in the present study the presence of a nucleoplasmic complex, which is composed of the IP(3)R, CGB, and phospholipids, with an estimated molecular mass of approximately 2-3 x 10(7) Da, suggesting the possibility of the presence of an IP(3)-sensitive Ca(2+) store in the nucleoplasm. Moreover, double-labeling immunogold electron microscope studies showed the colocalization of all three IP(3)R isoforms with CGB to the extent that the majority of each IP(3)R isoform-labeling gold particles found in the nucleoplasm was literally next to the CGB-labeling gold particles. In line with the potential existence of an IP(3)-dependent vesicular nucleoplasmic Ca(2+) store, our preliminary results indeed showed a sudden release of Ca(2+) from a putative nucleoplasmic Ca(2+) store in response specifically to IP(3) but not to inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate.

Characteristics of Prolonged Ca 2+ Release Events Associated With the Nuclei in Adult Cardiac Myocytes

Confocal microscopy was used to study the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78+/-0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2+/-0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0+/-0.2 microm) was also significantly greater than that of cytosolic Ca2+ sparks (2.6+/-0.05 microm) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.

Activation of sarcolemma and nuclear membranes ET-1 receptors regulates transcellular calcium levels in heart and vascular smooth muscle cells.

The use of an ET-1 fluorescent probe in human heart and vascular smooth muscle cells showed that ET-1 receptors are present at both the sarcolemma and nuclear envelope membranes. The use of immunofluorescence studies showed that the ETA receptor was mainly present at the sarcolemma and cytosolic levels. However, the ETB receptor was present at the sarcolemma and the cytosol, as well as the nuclear envelope membranes and the nucleoplasm. In addition, ET-1 immunoreactivity was seen in the cytosol and the nucleus. Using Ca2+ fluorescent probes such as Fluo-3, Indo 1, and yellow cameleon, as well as confocal microscopy three-dimensional image measurement technique, stimulation of ET-1 receptors at the sarcolemma membranes induced an increase of cytosolic and nuclear free Ca2+ levels. This effect of extracellular ET-1 was blocked by removal of extracellular calcium. Direct stimulation of ET-1 receptors at the nuclear envelope membranes also induced an increase of intranuclear free Ca2+ level. Our results suggest that the stimulation of sarcolemmal Ca2+ influx by ET-1 seems to be due to the activation of ETA and ETB receptors. However, the increase of nucleoplasmic Ca2+ levels by cytosolic ET-1 seems to be mediated via the activation of ETB receptors. Activation of nuclear membranes ETB receptors seems to prevent nuclear Ca2+ overload and may protect the cell from apoptosis.

Nuclear Lipid Signaling

Abundant evidence now supports the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope as part of a bilayer membrane, but are actually within the nucleus in the form of proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet entirely clear. Generation of DAG within the nucleus is believed to recruit protein kinase C (PKC) to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P3 may mobilize Ca2+ from the space between the nuclear membranes and thus increase nucleoplasmic Ca2+. Less well understood are the increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus, (i) two routes of synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; (ii) two sources of DAG, one from the PI-PLC pathway and the other probably from phosphatidylcholine; (iii) several isoforms of PKC translocating to nuclei; (iv) increases in activity of the PI-PLC pathway at two points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P3, which may have several functions, including a role in the transfer of mRNA out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and, in particular, 3-phosphorylated inositol lipids, which are now emerging as possible major players in nuclear signaling.

IP(3) receptor function and localization in myotubes: an unexplored Ca(2+) signaling pathway in skeletal muscle.

We present evidence for an unexplored inositol 1,4,5-trisphosphate-mediated Ca(2+) signaling pathway in skeletal muscle. RT-PCR methods confirm expression of all three known isotypes of the inositol trisphosphate receptor in cultured rodent muscle. Confocal microscopy of cultured mouse muscle, doubly labeled for inositol receptor type 1 and proteins of known distribution, reveals that the receptors are localized to the I band of the sarcoplasmic reticulum, and this staining is continuous with staining of the nuclear envelope region. These results suggest that the receptors are positioned to mediate a slowly propagating Ca(2+) wave that follows the fast Ca(2+) transient upon K(+) depolarization. This slow wave, imaged using fluo-3, resulted in an increase in nucleoplasmic Ca(2+) lasting tens of seconds, but not contraction; the slow wave was blocked by both the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and the phospholipase C inhibitor U-73122. To test the hypothesis that these slow Ca(2+) signals are involved in signal cascades leading to regulation of gene expression, we assayed for early effects of K(+) depolarization on mitogen-activated protein kinases, specifically extracellular-signal related kinases 1 and 2 and the transcription factor cAMP response element-binding protein (CREB). Within 30-60 seconds following depolarization, phosphorylation of both the kinases and CREB was evident and could be inhibited by 2-aminoethoxydiphenyl borate. These results suggest a signaling system mediated by Ca(2+) and inositol trisphosphate that could regulate gene expression in muscle cells.

Nuclear lipid signaling.

Abundant evidence now supports the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope as part of a bilayer membrane, but are actually within the nucleus in the form of proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet entirely clear. Generation of DAG within the nucleus is believed to recruit protein kinase C (PKC) to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P3 may mobilize Ca2+ from the space between the nuclear membranes and thus increase nucleoplasmic Ca2+. Less well understood are the increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus, (i) two routes of synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]; (ii) two sources of DAG, one from the PI-PLC pathway and the other probably from phosphatidylcholine; (iii) several isoforms of PKC translocating to nuclei; (iv) increases in activity of the PI-PLC pathway at two points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P3, which may have several functions, including a role in the transfer of mRNA out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and, in particular, 3-phosphorylated inositol lipids, which are now emerging as possible major players in nuclear signaling.

Nuclear lipid signaling.

There is now abundant evidence for the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope, but are actually within the nucleus, presumably not in a bilayer membrane but instead forming proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol and inositol-1,4,5-trisphosphate [Ins(1,4,5)P(3)]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet clear. Generation of diacylglycerol within the nucleus is believed to recruit protein kinase C to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P(3) may mobilize Ca(2+) from the space between the nuclear membranes and thus increase nucleoplasmic Ca(2+). Less well understood are an increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus: (i) two separate routes of synthesis of phosphatidylinositol-4,5-bisphosphate; (ii) two different sources of diacylglycerol, one being from the PI-PLC pathway, and the other probably from phosphatidylcholine; (iii) several different isoforms of PKC translocating to the nuclei; (iv) increases in activity of the PI-PLC pathway at two different points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P(3), which may have several functions, including a role in the transfer of messenger RNA (mRNA) out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and 3-phosphorylated inositol lipids.

Nuclear Lipid Signaling

There is now abundant evidence for the existence of phospholipids in the nucleus that resist washing of nuclei with detergents. These lipids are apparently not in the nuclear envelope, but are actually within the nucleus, presumably not in a bilayer membrane but instead forming proteolipid complexes with unidentified proteins. This review discusses the experimental evidence that attempts to explain their existence. Among these nuclear lipids are the polyphosphoinositol lipids which, together with the enzymes that synthesize them, form an intranuclear phospholipase C (PI-PLC) signaling system that generates diacylglycerol and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The isoforms of PI-PLC that are involved in this signaling system, and how they are regulated, are not yet clear. Generation of diacylglycerol within the nucleus is believed to recruit protein kinase C to the nucleus to phosphorylate intranuclear proteins. Generation of Ins(1,4,5)P3 may mobilize Ca2+ from the space between the nuclear membranes and thus increase nucleoplasmic Ca2+. Less well understood are an increasing number of variations and complications on the "simple" idea of a PI-PLC system. These include, all apparently within the nucleus: (i) two separate routes of synthesis of phosphatidylinositol-4,5-bisphosphate; (ii) two different sources of diacylglycerol, one being from the PI-PLC pathway, and the other probably from phosphatidylcholine; (iii) several different isoforms of PKC translocating to the nuclei; (iv) increases in activity of the PI-PLC pathway at two different points in the cell cycle; (v) a pathway of phosphorylation of Ins(1,4,5)P3, which may have several functions, including a role in the transfer of messenger RNA (mRNA) out of the nucleus; and (vi) the possible existence of other lipid signaling pathways that may include sphingolipids, phospholipase A2, and 3-phosphorylated inositol lipids.

Nucleoplasmic Ca2+loading is regulated by mobilization of perinuclear Ca2+

Regulation of nucleoplasmic calcium (Ca2+) concentration may occur by the mobilization of perinuclear luminal Ca2+pools involving specific Ca2+pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca2+, we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca2+fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca2+channels (RyR2), sarco/endoplasmic reticulum Ca2+ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca2+channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca2+from 100 to 1600 nM Ca2+was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca2+loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca2+loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca2+loading at low concentrations of extranuclear added Ca2+. Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca2+fluxes.

Novel biochemical and functional insights into nuclear Ca(2+) transport through IP(3)Rs and RyRs in osteoblasts.

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.

Presence of Functional Endothelin-1 Receptors in Nuclear Membranes of Human Aortic Vascular Smooth Muscle Cells

Summary: Our previous work showed that the nucleus plays a role in excitation-contraction coupling and that the channels and receptors could be present at the nuclear membrane. In the study reported here, the objective was to test the hypothesis that endothelin-1 (ET-1) receptors are functional at the level of the nuclear membranes and that their stimulation importantly regulates free nucleoplasmic Ca2+ level. Using a Fluo-3 Ca2+ measurement technique in human vascular smooth muscle cells (HVSMC), superfusion with increasing concentrations of extracellular ET-1 induced a dose-dependent sustained increase of free cytosolic ([Ca]c), nuclear ([Ca]n) Ca2+ and contraction with an EC50 near 3 × 10−10 M. Like the extracellular ET-1, the cytosolic application of ET-1 using the perforated sarcolemma membrane technique, induced a dose-dependent increase of nuclear free calcium of HVSMC with an EC50 of 2 × 10−11 M. These results strongly suggest that ET-1 receptors are functional at the level of the nuclear membranes. Furthermore, the sensitivity of ET-1 receptors at the nuclear membrane level seems to be higher than that of the receptors at the sarcolemma membrane. Finally, our results suggest that cytosolic ET-1 may play a role in preventing HVSMC nuclear calcium overload, thus protecting the cells from apoptosis.

Presence of functional endothelin-1 receptors in nuclear membranes of human aortic vascular smooth muscle cells.

Our previous work showed that the nucleus plays a role in excitation-contraction coupling and that the channels and receptors could be present at the nuclear membrane. In the study reported here, the objective was to test the hypothesis that endothelin-1 (ET-1) receptors are functional at the level of the nuclear membranes and that their stimulation importantly regulates free nucleoplasmic Ca2+ level. Using a Fluo-3 Ca2+ measurement technique in human vascular smooth muscle cells (HVSMC), superfusion with increasing concentrations of extracellular ET-1 induced a dose-dependent sustained increase of free cytosolic ([Ca]c), nuclear ([Ca]n) Ca2+ and contraction with an EC50 near 3 x 10(-10) M. Like the extracellular ET-1, the cytosolic application of ET-1 using the perforated sarcolemma membrane technique, induced a dose-dependent increase of nuclear free calcium of HVSMC with an EC50 of 2 x 10(-11) M. These results strongly suggest that ET-1 receptors are functional at the level of the nuclear membranes. Furthermore, the sensitivity of ET-1 receptors at the nuclear membrane level seems to be higher than that of the receptors at the sarcolemma membrane. Finally, our results suggest that cytosolic ET-1 may play a role in preventing HVSMC nuclear calcium overload, thus protecting the cells from apoptosis.

A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis.

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.

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 …

Novel mechanisms of calcium handling by the osteoclast: A review-hypothesis.

The osteoclast is a cell that is unique in its ability to resorb bone and, in doing so, becomes exposed to unusually high millimolar Ca2+ concentrations. It is generally accepted that, during resorption, osteoclasts can "sense" changes in their ambient Ca2+ concentration. This triggers a sharp cytosolic Ca2+ increase through both Ca2+ release and Ca2+ influx. The change in cytosolic Ca2+ is transduced finally into inhibition of bone resorption. It has been shown that a type 2 ryanodine receptor isoform, expressed uniquely in the plasma membrane, functions as a Ca2+ influx channel and possibly as a Ca2+ sensor. Ryanodine receptors are ordinarily Ca2+ release channels that have a microsomal membrane location in a wide variety of eukaryotic cells, including the osteoclasts. However, only recently has it become obvious that ryanodine receptors are also expressed in osteoclast nuclear membranes, at which site they probably gate nucleoplasmic Ca2+ influx. Nucleoplasmic Ca2+ in turn regulates key nuclear processes, including gene expression and apoptosis. Here, we review the potential mechanisms underlying the recognition, movement, and effects of Ca2+ in the osteoclast. We will also speculate on the general biological significance of the unique processes used by the osteoclast to handle high Ca2+ loads during bone resorption.

Emerging Insights into the Role of Calcium Ions in Osteoclast Regulation

Osteoclasts are exposed to unusually high, millimolar, Ca2+ concentrations and can "sense" changes in their ambient Ca2+ concentration during resorption. This results in a sharp cystolic Ca2+ increase through both Ca2+ release and Ca2+ influx. The rise in cystolic Ca2+ is transduced finally into an inhibition of bone resorption. We have shown that a type 2 ryanodine receptor isoform, expressed uniquely in the osteoblast plasma membrane, functions as a Ca2+ influx channel, and possibly as a Ca2+ sensor. Ryanodine receptors are ordinarily microsomal membrane Ca2+ release channels. They have only recently been shown to be expressed a other sites, including nuclear membranes. At the latter site, ryanodine receptors gate nucleoplasmic Ca2+ influx. Nucleoplasmic Ca2+, in turn, regulates key nuclear processes, including gene expression and apoptosis. Here, we review potential mechanisms underlying the recognition, movement, and actions of Ca2+ in the osteoclast.

A Ca 2+- and voltage-dependent cation channel in the nuclear envelope of red beet

The patch-clamp technique was applied to study ion conductances in various configurations of the nuclear envelope of non-enzyme-treated red beet ( Beta vulgaris L.) nuclei. With excised patches a non-selective cation channel was observed, that was activated by micromolar concentrations of Ca 2+ on the nucleoplasmic side of the envelope. The channel activity was also voltage-dependent and the voltage threshold of channel activation changed with the nucleoplasmic Ca 2+ concentration. The most prominent conductance level was 110±22 pS with 150 mM KCl in the bath and pipette. The channel was permeable to small cations: permeabilities relative to K + were P K≅ P Na=1, P Cs=0.3, but P Cl=0.09. Calcium ions also permeated the channel with P Ca=0.43, estimated from reversal potential, or 0.14, estimated from conductance ratio. Zn 2+ (1 mM) when applied to the cytoplasmic side of the envelope blocked the channel activity completely, while amiloride (2 mM) reduced the channel current by 86% from the nucleoplasmic side. The properties of the whole-nucleus current (voltage-, time- and Ca 2+-dependence) paralleled those observed with excised patches. The channel may provide a Ca 2+-regulated pathway for passive diffusion of cations across the nuclear envelope and thus may play an important role in Ca 2+-dependent nuclear processes ranging from gene transcription to apoptosis.

Basic FGF Enhances Calcium Permeable Channel Openings in Adult Rat Cardiac Myocytes: Implication in the bFGF-induced Increase of Free Ca2+Content

Basic fibroblast growth factor (bFGF) has been implicated in the changes in gene expression that, under pathological conditions such as ischemia or volume overload, lead to adult cardiomyocyte hypertrophy. In many tissues, one of the first events following cell activation by growth factors is an enhancement of the intracellular free calcium concentration, generated by fluxes from internal storage compartments and through channels of the plasma membrane. The present study was undertaken to determine whether cardiac myocytes isolated from adult rat ventricles express Ca2+-permeable channels activated by bFGF. Using the cell-attached mode of the patch-clamp technique, we observed that bFGF (from 0.1–10 nm) induced an increase of fast burst openings, mediated by Ca2+-permeable channels with low conductance (15 pS) and voltage-independence. Inside-out patch-clamp experiments revealed that inositol 1,4,5-trisphosphate (5μm) enhanced the opening of Ca2+-permeable channels with similar properties as the bFGF-induced channels, indicating that IP3may be a second messenger of this process. Confocal fluorescence imaging of intracellular free calcium provided direct evidence that bFGF induced an increase of cytoplasmic and nucleoplasmic free Ca2+concentrations which were generated, in part, by Ca2+influx through the plasma membrane. In conclusion, this study supports the presence, in the plasma membrane of adult cardiac myocytes, of messenger-activated calcium channels which could play key roles in the calcium-dependent pathways that are activated in response to growth factors.

Does the nuclear envelope contain two types of ligand-gated Ca 2+ release channels?

The nuclear envelope is composed of two membranes deliminating a perinuclear space which displays functional properties similar to those of a Ca 2+-storing compartment. ATP-driven Ca 2+ uptake and InsP 3-induced Ca 2+ release processes have been described in isolated nuclei. Recently, it was reported that cADP-ribose and InsP 3 can trigger a nucleoplasmic Ca 2+ increase. It was hypothesized that the inner nuclear membrane possesses Ca 2+ channels that are regulated by ryanodine or InsP 3. Radio-ligand binding assays and Western blot experiments were performed in order to investigate their presence in sheep cardiac and rat liver nuclear envelopes. Ryanodine receptors (RyR) were not detected in liver nuclear envelopes by either binding assay or Western blot analysis. However, cardiac nuclear envelopes were found to retain a very low level of specific ryanodine binding, which was not detected on immuno-blots obtained with three types of isoform-specific RyR antibodies. In contrast, nuclear InsP 3-binding sites were consistently detected in both cardiac and liver nuclear envelopes. Altogether, these results provide evidence for the major contributor InsP 3-gated Ca 2+ channels in control of Ca 2+ release from the perinuclear space in liver and cardiac cells.