Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10–20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo. J. Cell. Physiol. 176:138–149, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Antigen-induced Ca2+ signaling and desensitization in B cells

Cross-linking of B cell surface Ig (sIg) by anti-Ig results in transmembrane signaling. However, the capacity of a thymus-dependent (TD) Ag to mediate B cell signal transduction has been less well documented. Therefore, we examined Ag-induced intracellular free calcium concentration [( Ca2+]) in B cells by using TD Ag that would be expected to either cross-link or not cross-link sIgM and/or induce the coupling of sIgM to FcR. Stimulation of mouse TA3 hybridoma B cell transfectants that express the SP6 anti-TNP specific sIgM with either TNP-OVA or anti-IgM antibodies resulted in a maximal fourfold increase in [Ca2+]i. The net increase in [Ca2+]i in response to TNP-OVA was dependent upon both the Ag dose and the TNP:OVA molar ratio. Because occupancy of several cell-surface receptor types leads to a loss of response to subsequent stimulation by ligand (homologous desensitization), we examined the ability of Ag to induce homologous desensitization of sIgM in these B cells. TNP1-OVA at all concentrations tested (up to 500 micrograms/ml) did not lead to any change in [Ca2+]i or desensitization. Cross-linking of TNP1-OVA (10 micrograms/ml) with F(ab')2 of anti-OVA antibody induced both a rise in [Ca2+]i and homologous desensitization of sIg, suggesting that cross-linking of sIgM by Ag is sufficient to induce both these processes. TNP6-OVA at a concentration of 10 micrograms/ml induced changes in [Ca2+]i and partially desensitized TNP-specific B cells to stimulation by anti-IgM. Interestingly, a high dose (180 micrograms/ml) of TNP6-OVA stimulated minimal changes in [Ca2+]i yet did not lead to desensitization. However, cross-linking of TNP6-OVA at this high dose with F(ab')2 of rabbit anti-OVA elevated [Ca2+]i and elicited partial desensitization. Complete desensitization of sIgM by Ag was achieved when intact (Fc-containing) anti-OVA antibody was used, suggesting that the FcR can play a role in desensitization. Ag- and antibody-mediated desensitization was not caused by steric hindrance of sIg. Thus, we have observed two forms of Ag-induced desensitization of sIgM, both of which involve sIg cross-linking and one of which is mediated by the physiologic coupling of sIg to FcR.     

Ca2+ signaling in the regulation of dendritic cell functions

Dendritic cells (DCs) are highly versatile antigen-presenting cells critically involved in both innate and adaptive immunity as well as maintenance of self-tolerance. DC function is governed by Ca(2+) signaling, which directs the DC responses to diverse antigens, including Toll-like receptor ligands, intact bacteria, and microbial toxins. Ca(2+)-sensitive DC functions include DC activation, maturation, migration, and formation of immunological synapses with T cells. Moreover, alterations of cytosolic Ca(2+) trigger immune suppression or switch off DC activity. Ca(2+) signals are generated by the orchestration of Ca(2+) transport processes across plasma, endoplasmic reticulum, and inner mitochondrial membrane. These processes include active pumping of Ca(2+), Ca(2+)/Na(+) antiport, and electrodiffusion through Ca(2+)-permeable channels or uniporters. Ca(2+) channels in the plasma membrane such as Ca(2+) release-activated Ca(2+) or L-type Ca(2+) channels are tightly regulated by the membrane potential which in turn depends on the activity of voltage-gated K(+) or Ca(2+)-activated nonselective cation channels. The rapidly growing knowledge on the function and regulation of these membrane transport proteins provides novel insight into pathophysiological mechanisms underlying dysfunction of the immune system and opens novel therapeutic opportunity to favorably influence the function of the immune system.     

Ca2+ signaling, intracellular pH and cell volume in cell proliferation

Mitogens control progression through the cell cycle in non-transformed cells by complex cascades of intracellular messengers, such as Ca²⁺ and protons, and by cell volume changes. Intracellular Ca²⁺ and proton concentrations are critical for linking external stimuli to proliferation, motility, apoptosis and differentiation. This review summarizes the role in cell proliferation of calcium release from intracellular stores and the Ca²⁺ entry through plasma membrane Ca²⁺ channels. In addition, the impact of intracellular pH and cell volume on cell proliferation is discussed.     

Automated selective dissociation of cells from different regions of multicellular spheroids

Summary In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions. This work was supported by grants CA-36535, CA-22585, and RR-02845 from the National Institutes of Health, Bethesda, MD, the National Flow Cytometry Resource (NIH grant RR-01315), and by the Department of Energy, Washington, DC.     

Crosstalk between cAMP and Ca2+ signaling in non-excitable cells

An impressive array of cytosolic calcium ([Ca2+](i)) signals exert control over a broad range of physiological processes. The specificity and fidelity of these [Ca2+](i) signals is encoded by the frequency, amplitude, and sub-cellular localization of the response. It is believed that the distinct characteristics of [Ca2+](i) signals underlies the differential activation of effectors and ultimately cellular events. This "shaping" of [Ca2+](i) signals can be achieved by the influence of additional signaling pathways modulating the molecular machinery responsible for generating [Ca2+](i) signals. There is a particularly rich source of potential sites of crosstalk between the cAMP and the [Ca2+](i) signaling pathways. This review will focus on the predominant molecular loci at which these classical signaling systems interact to impact the spatio-temporal pattern of [Ca2+](i) signaling in non-excitable cells.     

G protein-dependent Ca2+ signaling complexes in polarized cells

Polarized cells signal in a polarized manner. This is exemplified in the patterns of [Ca2+]i waves and [Ca2+]i oscillations evoked by stimulation of G protein-coupled receptors in these cells. Organization of Ca(2+)-signaling complexes in cellular microdomains, with the aid of scaffolding proteins, is likely to have a major role in shaping G protein-coupled [Ca2+]i signal pathways. In epithelial cells, these domains coincide with sites of [Ca2+]i-wave initiation and local [Ca2+]i oscillations. Cellular microdomains enriched with Ca(2+)-signaling proteins have been found in several cell types. Microdomains organize communication between Ca(2+)-signaling proteins in the plasma membrane and internal Ca2+ stores in the endoplasmic reticulum through the interaction between the IP3 receptors in the endoplasmic reticulum and Ca(2+)-influx channels in the plasma membrane. Ca2+ signaling appears to be controlled within the receptor complex by the regulators of G protein-signaling (RGS) proteins. Three domains in RGS4 and related RGS proteins contribute important regulatory features. The RGS domain accelerates GTP hydrolysis on the G alpha subunit to uncouple receptor stimulation from IP3 production; the C-terminus may mediate interaction with accessory proteins in the complex; and the N-terminus acts in a receptor-selective manner to confer regulatory specificity. Hence, RGS proteins have both catalytic and scaffolding function in Ca2+ signaling. Organization of Ca(2+)-signaling proteins into complexes within microdomains is likely to play a prominent role in the localized control of [Ca2+]i and in [Ca2+]i oscillations.     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

Ca2+ signaling in prokaryotes

The role of Ca²⁺ ions in the regulation of motility, cell cycle, and division of prokaryotes is discussed, as well as their involvement in the pathogenesis of some infectious diseases. The structural and functional organization of the prokaryotic Ca²⁺ signaling system and the mechanisms of Ca²⁺ membrane transport and homeostasis are described. Special attention is paid to the role of Ca²⁺ cation channels, Ca²⁺ transporters, and Ca²⁺-binding proteins in the regulation of the intercellular Ca²⁺ concentration.     

Homer proteins in Ca2+ signaling by excitable and non-excitable cells

Homers are scaffolding proteins that bind Ca(2+) signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca(2+) signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca(2+) signaling complex in an isoform-specific manner. Homer2 increases the GAP activity of RGS proteins and PLCbeta that accelerate the GTPase activity of Galpha subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP(3)Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Ca(v)1.2 to regulate E-C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca(2+) signaling and create a negative bias that can be reversed by the immediate early gene form of Homer1. Hence, the Homers should be viewed as the buffers of Ca(2+) signaling that ensure a high spatial and temporal fidelity of the Ca(2+) signaling and activation of downstream effects.     

Endoplasmic reticulum Ca2+ store: regulation of Ca2+ release and reuptake by intracellular and extracellular Ca2+ in pancreatic acinar cells

We investigated the effect of cytosolic and extracellular Ca2+ on Ca2+ signals in pancreatic acinar cells by measuring Ca2+ concentration in the cytosol([Ca2+]c) and in the lumen of the ER([Ca2+]Lu). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released Ca2+ mainly from the basolateral ER-rich part of the cell. The rate of Ca2+ release from the ER was highly sensitive to the buffering of [Ca2+]c whereas ER Ca2+ refilling was enhanced by supplying free Ca2+ to the cytosol with [Ca2+]c clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM Ca2+. Elevation of extracellular Ca2+ to 10 mM from 1 mM raised resting [Ca2+]c slightly and often generated [Ca2+]c oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular Ca2+-sensing receptors linked to phospholipase C that mobilize Ca2+ from the ER, exposure of cells to 10 mM Ca2+ did not decrease [Ca2+]Lu but rather raised it. From these findings we conclude that 1) ER Ca2+ release is strictly regulated by feedback inhibition of [Ca2+]c, 2) ER Ca2+ refilling is determined by the rate of Ca2+ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular Ca2+-induced [Ca2+]c oscillations appear to be triggered not by activation of extracellular Ca2+-sensing receptors but by the ER sensitised by elevated [Ca2+]c and [Ca2+]Lu.     

H1 variant synthesis in proliferating and quiescent human cells

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.     

Involvement of Ca2(+)-induced Ca2+ release in the volume regulation of human epithelial cells exposed to a hypotonic medium

Exposure of cultured human epithelial cells (Intestine 407) to a hypotonic solution results in initial osmotic swelling and in a subsequent volume decrease near to the original level. The regulatory volume decrease was inhibited by reduction of the extracellular free Ca2+ concentration to 90 nM. Single epithelial cells responded to a hypotonic challenge with a biphasic increase in the cytosolic free Ca2+ level from about 90 to 200 nM. Both phases of the Ca2+ rise were abolished by reducing the extracellular Ca2+ to 90 nM. In the presence of caffeine (20 mM), the second-phase Ca2+ response to a hypotonic challenge occurred earlier immediately after the first-phase response. The second-phase Ca2+ response was selectively impaired by adenine (10 mM), procaine (1 mM) or ryanodine (5 to 10 microM). These blockers for Ca2(+)-induced Ca2+ release channels inhibited volume regulation after osmotic swelling. It is concluded that Ca2(+)-induced Ca2+ release from a ryanodine-sensitive store is a prerequisite for the volume regulation of human intestinal epithelial cells under hypotonic conditions.