Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.     

Parthenolide inhibits osteoclast differentiation and bone resorbing activity by down-regulation of NFATc1 induction and c-Fos stability,during RANKL-mediated osteoclastogenesis

Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of NF-κB ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and IκB, as well as IκB degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption. [BMB Reports 2014; 47(8): 451-456]     

Age‐dependent alterations in osteoblast and osteoclast activity in human cancellous bone

It is assumed that the activity of osteoblasts and osteoclasts is decreased in bone tissue of aged individuals. However, detailed investigation of the molecular signature of human bone from young compared to aged individuals confirming this assumption is lacking. In this study, quantitative expression analysis of genes related to osteogenesis and osteoclastogenesis of human cancellous bone derived from the distal radius of young and aged individuals was performed. Furthermore, we additionally performed immunohistochemical stainings. The young group included 24 individuals with an average age of 23.2 years, which was compared to cancellous bone derived from 11 body donators with an average age of 81.0 years. In cancellous bone of young individuals, the osteogenesis‐related genes RUNX‐2, OSTERIX, OSTEOPONTIN and OSTEOCALCIN were significantly up‐regulated compared to aged individuals. In addition, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in cancellous bone of young individuals, as well as the WNT gene family member WNT5a and the matrix metalloproteinases MMP‐9. However, quantitative RT‐PCR analysis of BMP‐2, ALP, FGF‐2, CYCLIN‐D1, MMP‐13, RANK, OSTEOPROTEGERIN and TGFb1 revealed no significant difference. Furthermore, Tartrate‐resistant acid phosphatase (TRAP) staining was performed which indicated an increased osteoclast activity in cancellous bone of young individuals. In addition, pentachrome stainings revealed significantly less mineralized bone matrix, more osteoid and an increased bone density in young individuals. In summary, markers related to osteogenesis as well as osteoclastogenesis were significantly decreased in the aged individuals. Thus, the present data extends the knowledge about reduced bone regeneration and healing capacity observed in aged individuals.     

Cytosolic free calcium and ATP in synaptosomes after ischemia

Elevations in cytosolic free calcium ([Ca2+]i) precede electrophysiological alterations due to ischemia in vivo. An in vitro model of these changes would help to elucidate their molecular basis. A model of postdecapitative ischemia was used to study these interactions. Nerve endings (i.e. synaptosomes) were isolated either immediately after decapitation or at various time periods after decapitation. Synaptosomal [Ca2+]i and ATP concentrations were determined during a basal period and following depolarization. K(+)-depolarization produced an initial spike of [Ca2+]i that was followed by a new equilibrium value. Ischemia elevated the basal [Ca2+]i and the new equilibrium [Ca2+]i after KCl but suppressed the [Ca2+]i spike. However, the difference between the basal [Ca2+]i and the new equilibrium [Ca2+]i after K(+)-depolarization did not vary with ischemia. Although ischemia reduced ATP, K(+)-depolarization did not alter ATP concentrations in either the controls or the ischemia group, which suggests that synaptosomal mitochondria can meet an energy demand after ischemia. ATP was inversely related to the basal or the new equilibrium [Ca2+]i following depolarization. These changes in [Ca2+]i may underlie the alterations in neurotransmitter release and cell death following ischemia. This appears to be a useful model in which to study the molecular basis of ischemia induced changes in [Ca2+]i.     

Cytosolic free calcium spiking affected by intracellular pH change

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.     

Cytosolic free calcium concentrations in synaptosomes during histotoxic hypoxia

Altered cytosolic free calcium concentrations ([Ca²⁺]_i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca²⁺]_i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca²⁺]_i. However, with external CaCl₂ concentrations as small as 13 M, KCl depolarization increased [Ca²⁺]_i instantaneously while hypoxia gradually raised [Ca²⁺]_i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K⁺ and KCN on [Ca²⁺]_i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca²⁺]_i due to hypoxia, but enhanced the KCl response. The changes in ATP following K⁺ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca²⁺]_i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca²⁺]_i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca²⁺]_i and the effects were additive with K⁺ depolariration in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca²]_i are due to an inability of the synaptosome to buffer entering calcium.     

Cytosolic free calcium concentrations in avian growth plate chondrocytes

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.     

Aryl hydrocarbon receptor catabolic activity in bone metabolism is osteoclast dependent in vivo

Bone mass is regulated by various molecules including endogenous factors as well as exogenous factors, such as nutrients and pollutants. Aryl hydrocarbon receptor (AhR) is known as a dioxin receptor and is responsible for various pathological and physiological processes. However, the role of AhR in bone homeostasis remains elusive because the cell type specific direct function of AhR has never been explored in vivo. Here, we show the cell type specific function of AhR in vivo in bone homeostasis. Systemic AhR knockout (AhRKO) mice exhibit increased bone mass with decreased resorption and decreased formation. Meanwhile, osteoclast specific AhRKO (AhR^ΔOc/ΔOc) mice have increased bone mass with reduced bone resorption, although the mice lacking AhR in osteoblasts have a normal bone phenotype. Even under pathological conditions, AhR^ΔOc/ΔOc mice are resistant to sex hormone deficiency-induced bone loss resulting from increased bone resorption. Furthermore, 3-methylcholanthrene, an AhR agonist, induces low bone mass with increased bone resorption in control mice, but not in AhR^ΔOc/ΔOc mice. Taken together, cell type specific in vivo evidence for AhR functions indicates that osteoclastic AhR plays a significant role in maintenance of bone homeostasis, suggesting that inhibition of AhR in osteoclasts can be beneficial in the treatment of osteoporosis.     

Cytosolic calcium regulation in rat afferent vagal neurons during anoxia

Sensory neurons are able to detect tissue ischaemia and both transmit information to the brainstem as well as release local vasoactive mediators. Their ability to sense tissue ischaemia is assumed to be primarily mediated through proton sensing ion channels, lack of oxygen however may also affect sensory neuron function. In this study we investigated the effects of anoxia on isolated capsaicin sensitive neurons from rat nodose ganglion. Acute anoxia triggered a reversible increase in [Ca²⁺]_i that was mainly due to Ca²⁺-efflux from FCCP sensitive stores and from caffeine and CPA sensitive ER stores. Prolonged anoxia resulted in complete depletion of ER Ca²⁺-stores. Mitochondria were partially depolarised by acute anoxia but mitochondrial Ca²⁺-uptake/buffering during voltage-gated Ca²⁺-influx was unaffected. The process of Ca²⁺-release from mitochondria and cytosolic Ca²⁺-clearance following Ca²⁺ influx was however significantly slowed. Anoxia was also found to inhibit SERCA activity and, to a lesser extent, PMCA activity. Hence, anoxia has multiple influences on [Ca²⁺]_i homeostasis in vagal afferent neurons, including depression of ATP-driven Ca²⁺-pumps, modulation of the kinetics of mitochondrial Ca²⁺ buffering/release and Ca²⁺-release from, and depletion of, internal Ca²⁺-stores. These effects are likely to influence sensory neuronal function during ischaemia.     

Cytosolic free calcium concentration and intracellular calcium distribution in lymphocytes from cystic fibrosis patients

Lymphocytes prepared from normal individuals and patients with cystic fibrosis (CF) were compared with regard to intercellular Ca²⁺ concentration, distribution, and handling. No difference between control and CF was found in the concentration of cytosolic free Ca²⁺ ($\text{98 ± 5}\text{vs}\text{102 ± 7}\text{nM}$), and no difference was observed in the kinetics with which control and CF cells restored cytoplasmic Ca²⁺ toward normal following a perturbation induced by cold-exposure. However, total intracellular Ca²⁺ is about 25% higher in CF lymphocytes than in control. Of this excess Ca²⁺, about 50% appears to be sequested in mitochondria. This suggests that some difference in Ca²⁺ handling does exist, but the significance of this cystic fibrosis remains to be determined.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption

Background  

Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin α_vβ₃ increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function.     

'Calcium-activated' intracellular calcium elevation: a novel mechanism of osteoclast regulation

The osteoclast is unique in its capacity to resorb bone. An unbalanced increase in this activity causes osteoporosis, a crippling bone disease that poses a major public health problem. Despite this, our understanding of osteoclast regulation is very limited. Calcitonin is the only known physiological inhibitor of osteoclast function. We demonstrate here for the first time that the concentration of calcium ions at the resorptive site directly regulates osteoclast function by modulating the intracellular free calcium concentration. This represents an important feedback mechanism of osteoclast control.     

Cytosolic free calcium increases before and oscillates during frustrated phagocytosis in macrophages

When macrophages and neutrophils are allowed to settle onto an appropriate surface, they attach and spread in a frustrated attempt to phagocytose the substrate. Spreading is associated with extensive rearrangements of the actin cytoskeleton which resemble those occurring during phagocytosis. We have previously shown that spreading in human neutrophils is preceded by an increase in cytosolic-free calcium concentration [( Ca2+]i) (Kruskal, B. A., S. Shak, and F. R. Maxfield. 1986. Proc. Natl. Acad. Sci. USA. 83:2919-2923). To assess the generality of this signal, we measured [Ca2+]i in single thioglycollate- elicited mouse peritoneal macrophages as they spread on an immune complex-coated surface, using fura-2 microspectrofluorometry. A [Ca2+]i increase always precedes spreading. This increase can involve several (up to 8) [Ca2+]i spikes, with an average peak value of 387 +/- 227 nM (mean +/- SD, n = 92 peaks in 24 cells), before spreading is detected. Neither spreading nor the magnitude of these spikes is significantly altered by removal of extracellular calcium. Many of the spreading macrophages exhibit periodic [Ca2+]i increases before and during spreading. The proportion which does so varies among experiments from 0 to 90%, but it is frequently greater than 40%. The largest number of cells (approximately 25%) exhibited only a single peak. In 13 cells that showed more than 10 peaks, the median period was 29 s (range 19-69 s). The average peak [Ca2+]i was 385 +/- 266 nM (mean +/- SD, n = 208 peaks in 14 cells). The calcium producing these increases is derived from intracellular pools. The oscillations occur with spreading on either opsonized or nonopsonized surfaces. The function of these oscillations is not clear, but the large number of cells which exhibit them suggest that they may be important to macrophage function.     

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca²⁺ but not Mg²⁺, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca²⁺ and protein dephosphorylation are factors in Hb induction.