Factors influencing calcium influx in endotoxin-challenged fibroblasts

The role of cell density and pH on calcium influx was studied in normal and endotoxin-challenged cultured 3T6 fibroblasts. In normal fibroblasts, at low cell densities, there was no marked difference in calcium influx at pH 6.6, 7.4, and 7.8, whereas at high cell densities, the calcium influx was markedly higher at pH 6.6 as compared to that at pH 7.8. Endotoxin treatment for 4 hr at low cell density and in alkaline pH (7.4-7.8) increased calcium influx in a dose-dependent manner. In contrast, at high cell density and low pH (6.6), endotoxin treatment markedly decreased calcium influx in a dose- and time-dependent manner. These endotoxin-induced changes in calcium influx were not fully compensated by altered calcium efflux because total calcium content of the cells was found to be altered. The efficacy of the endotoxin varied depending on the bacterial source of the endotoxin and the method of purification. There was a relationship between the effect of different endotoxins on the increase in calcium influx and the inhibition of cell proliferation. Endotoxin did not decrease, but slightly increased cell proliferation when added to high cell density cultures even at a concentration of 200 micrograms/ml.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

Lys-bradykinin stimulates Na+ influx and DNA synthesis in cultured human fibroblasts

The effect of Lys-bradykinin on net Na+ influx in serum-deprived cultured human fibroblasts (HSWP cells) was measured. It was found that Lys-bradykinin stimulates net Na+ influx with a K1/2 of 1 nM. When Lys-bradykinin was combined with epidermal growth factor, vasopressin and insulin, the net Na+ influx stimulated was comparable with that measured in response to 10% serum. The above combination of growth factors also was found to stimulate DNA synthesis to 92% of the serum-stimulated levels in HSWP cells and to support cell growth over a period of 6 days. In addition, it was observed that the Na+ influx stimulated by Lys-bradykinin or by the combination of four growth factors was completely inhibited by the amiloride analog benzamil. Thus Lys-bradykinin presumably stimulates the same Na+ transport system as is stimulated by serum. Finally, the present results suggest that an increase in Na+ influx always accompanies DNA synthesis in HSWP cells. On the basis of these observations, it can be hypothesized that Na+ influx is a necessary event to stimulate DNA synthesis.     

Desensitization of the serum effect on Na+ influx in cultured human fibroblasts

Stimulation of an amiloride-sensitive Na+ influx pathway, which mediates Na+/H+ exchange, has been postulated to be an important step in the initiation of DNA synthesis in quiescent human fibroblasts. If the elevation of intracellular Na+ or the alkalinization of intracellular pH resulting from the activation of this system is a trigger for subsequent mitogenic events, then its inactivation may also be important to cellular functions. We investigated the duration of the activation of Na+ influx by serum in human foreskin fibroblasts (HSWP). It was found that activation of Na+ influx by 10% serum was transient, declining with a t 1/2 = 15 min. Similarly, the Na+ content of the cells rose rapidly following serum addition and decreased with a t 1/2 = 15 min. In addition, both the lys-bradykinin- and the vasopressin-stimulated Na+ influx and Na+ content declined with a t 1/2 of approximately 15 min. Similar results were obtained using both Tris-buffered and Hepes-buffered, amino-acid-free EMEM. Finally, the above experiments were repeated under conditions normally used to assess the mitogenic response of cells. It was found that in cells arrested in G0 by serum deprivation in CO2-buffered EMEM, the serum activated Na+ flux was also transient with a t 1/2 of approximately 20 min. The desensitization of cells to serum could be readily (t 1/2 = 20') reversed by a subsequent incubation of cells in serum-free medium. Stimulation of Na+ influx by both the divalent cation ionophore A23187 and the phospholipase activator melittin in also desensitized rapidly, suggesting the process is independent of receptor downregulation. The desensitization during serum preincubation occurred in both low Na+ and low pH medium suggesting that the process is not due to negative feedback on the transport system via a rise in cellular Na+ concentration or a rise in intracellular pH. Although the mechanism of desensitization is at present not known, it is likely to be a physiologically important event.     

Na+ influx and cell growth in cultured human fibroblasts. Effect of indomethacin

The effect of indomethacin on Na+ influx and cell growth in human diploid fibroblasts (HSWP) has been investigated. It was found that both indomethacin and aspirin block serum-stimulated Na+ influx in a dose-dependent manner (Ki = 0.34 +/- 0.04 mM and 11 +/- 1 mM respectively) while having no effect on influx of Na+ in the absence of serum. The Ki for inhibition of [3H]thymidine incorporation into HSWP cells (0.28 +/- 0.02 mM) closely correlated with the Ki for inhibition of Na+ influx. The onset of action of indomethacin is rapid (within 2 min) and inhibition of Na+ flux is readily reversed (within 5 min). Other workers have reported that indomethacin is cytostatic for human fibroblasts presumably via a slowly developing inhibition of "A" system amino acid transport [6]; however, present results indicate that inhibition of Na+ influx in HSWP cells occurs much more rapidly than the inhibition of amino acid transport observed in other human foreskin fibroblasts and therefore may be more closely related to the primary cellular locus of indomethacin action.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons.     

Inhibition by oxytocin of voltage-activated calcium influx in cultured PC12 pheochromocytoma cells.

1. Voltage-activated dihydropyridine-sensitive Ca2+ influx was measured in PC12 pheochromocytoma cells using 45Ca. 2. It has been found that oxytocin inhibits voltage-activated dihydropyridine-sensitive Ca2+ influx with ED50 about 0.30 x 10(-6) M. 3. Tolbutamide (1.3 x 10(-3) M) has no visible effect on both Ca2+ influx itself and on the inhibitory oxytocin effect. 4. External application of Li+ (10 mM) causes a slight shift of ED-curve to lower oxytocin concentrations. 5. It is suggested that the hydrolysis of phosphoinositides may play a role in oxytocin action on Ca2+ influx in PC12 cells.     

Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.     

pH-dependent effect of mitochondria on calcium influx into Jurkat cells; a novel mechanism of cell protection against calcium entry during energy stress

Loss of the mitochondrial membrane potential results in a significant inhibition of calcium influx through calcium release-activated channels (CRAC) in Jurkat cells suspended in the medium of pH lower than 7.4. This effect disappears when the medium pH increases. Alkalinisation of the cytosol achieved by the addition of NH(4)Cl to the cells pretreated with thapsigargin, CCCP and CaCl(2), suspended in the medium of pH 7.2, does not affect CRAC activity, while alkalisation of the extracellular milieu by NaOH results in a strong stimulation of calcium entry. Thus, the mitochondrial effect on CRAC is exclusively related to the extracellular pH.Coupled mitochondria are able to take up Ca(2+) accumulated in the close proximity of CRAC. This protects these channels against feedback inhibition exerted by high [Ca(2+)](c). We conclude that CRAC may exist in two conformations: inhibitable and not inhibitable by cytosolic Ca(2+). Lower extracellular pH promotes the former one. This explains a much higher inhibitory effect of mitochondrial uncouplers on the calcium influx into the cells exposed to pH 7.2 than that observed in the cells suspended in the medium of pH 7.8. This phenomenon may provide an additional mechanism protecting cells against calcium overloading in transient episodes of energy stress.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.     

Cross-linking of IgG receptors inhibits membrane immunoglobulin- stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor- induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg- mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2- loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.     

Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.     

Increased calcium influx in dystrophic muscle

We examined pathways which might result in the elevated resting free calcium [( Ca2+]i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes. We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Ca2+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Ca2+]i levels. Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]i levels were similar, suggesting that calcium-sequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2+]i. The defect appears to be specific for calcium since resting free sodium levels and sodium influx rates in the absence of Na+/K(+)-ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes. These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca2+]i in fibers and myotubes. These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+]i in dystrophic muscle.     

Simulating calcium influx and free calcium concentrations in yeast

Yeast can proliferate in environments containing very high Ca(2+) primarily due to the activity of vacuolar Ca(2+) transporters Pmc1 and Vcx1. Yeast mutants lacking these transporters fail to grow in high Ca(2+) environments, but growth can be restored by small increases in environmental Mg(2+). Low extracellular Mg(2+) appeared to competitively inhibit novel Ca(2+) influx pathways and to diminish the concentration of free Ca(2+) in the cytoplasm, as judged from the luminescence of the photoprotein aequorin. These Mg(2+)-sensitive Ca(2+) influx pathways persisted in yvc1 cch1 double mutants. Based on mathematical models of the aequorin luminescence traces, we propose the existence in yeast of at least two Ca(2+) transporters that undergo rapid feedback inhibition in response to elevated cytosolic free Ca(2+) concentration. Finally, we show that Vcx1 helps return cytosolic Ca(2+) toward resting levels after shock with high extracellular Ca(2+) much more effectively than Pmc1 and that calcineurin, a protein phosphatase regulator of Vcx1 and Pmc1, had no detectable effects on these factors within the first few minutes of its activation. Therefore, computational modeling of Ca(2+) transport and signaling in yeast can provide important insights into the dynamics of this complex system.     

Energized mitochondria increase the dynamic range over which inositol 1,4,5-trisphosphate activates store-operated calcium influx

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.     

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.