Inverse correlation of intracellular calcium and cyclic AMP levels in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common renal tumour in adults. Altered levels of secondary messengers, that is, intracellular calcium and cyclic AMP (cAMP), have been implicated in the pathogenesis of various malignancies. In the present study, we measured levels of intracellular calcium and cAMP in RCC. The intracellular calcium level was significantly reduced, whereas the cAMP level was significantly augmented in RCC as compared with adjacent grossly normal renal parenchyma. Copyright © 2012 John Wiley & Sons, Ltd.     

Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes

Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx.     

Effect of intracellular cyclic AMP injection on calcium current in identifiedHelix pomatia neurons

The effect of intracellular injection of cyclic AMP (cAMP) and extracellular application of theophylline on the inward calcium current was investigated in neurons RPa3 and LPa3 ofHelix pomatia. Iontophoretic injection of cyclic AMP (current 10–35 nA, duration about 1 min) led to a decrease in amplitude of the calcium current to a new stationary level, which depended on the injection current. After the end of injection the calcium current was restored to its initial level. Current-voltage characteristic curves of the calcium current were not shifted along the voltage axis by cAMP injection, indicating that the reduction in this current was connected with a change in maximal calcium conductance. An increase in the frequency of depolarizing shifts from 0.1 to 0.5 Hz caused a decrease in the calcium current but did not affect the time course of the decrease in calcium current in response to injection of cAMP or the time course of its recovery after the end of injection. Theophylline an inhibitor of cyclic nucleotide phosphodiesterase, in a concentration of 1 mM in the external solution, lowered the amplitude of the calcium current by 50–75% of its initial value. In 40% of neurons, abolition of the action of theophylline by rinsing was incomplete, but in the rest the effect of theophylline was irreversible. It is postulated on the basis of the results that cytoplasmic compounds take part in regulation of the calcium current of molluscan neurons. The possible physiological role of this process is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 290–297, May–June, 1982.     

A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants

An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.     

Modulation of nicotinic acetylcholine receptors by intracellular calcium and cyclic AMP in Lymnaea stagnalis neurones.

Acetylcholine (ACh)-receptor ion channels were investigated under the modulatory action of calcium and cyclic AMP in completely isolated Lymnaea stagnalis neurones using the noise analysis technique. Elevation of the intracellular Ca2+ concentration in dialyzed neurones produced a reduction in the amplitude of ACh induced current accompanied by slight decrease in the mean channel open time and a simultaneous 1.5-fold increase in mean channel conductance. Direct introduction of cyclic AMP into neurones or elevation of intracellular cyclic AMP level by application of serotonin or forskolin produced 20-40% reduction in ACh-induced conductance without significant effect on the measured parameters of the ion channels. The inhibitory effects of calcium and cyclic AMP appear to be independent. Our findings indicate that reduction in ACh induced conductance under calcium and cyclic AMP modulation results from an alteration in the channel gating mechanism. Since the efficiency of ion transfer is independent of cyclic AMP, and it even rises with the elevation of calcium concentration, the inhibition of ACh responses may be accounted for by a decrease in the rate constant for channel opening, so that channels activated by acetylcholine remain in a closed state over longer intervals.     

Mechanical and biochemical characterization of cartilage explants in serum-free culture

Allografts of articular cartilage are both used clinically for tissue-transplantation procedures and experimentally as model systems to study the physiological behavior of chondrocytes in their native extracellular matrix. Long-term maintenance of allograft tissue is challenging. Chemical mediators in poorly defined culture media can stimulate cells to quickly degrade their surrounding extracellular matrix. This is particularly true of juvenile cartilage which is generally more responsive to chemical stimuli than mature tissue. By carefully modulating the culture media, however, it may be possible to preserve allograft tissue over the long-term while maintaining its original mechanical and biochemical properties. In this study juvenile bovine cartilage explants (both chondral and osteochondral) were cultured in both chemically defined medium and serum-supplemented medium for up to 6 weeks. The mechanical properties and biochemical content of explants cultured in chemically defined medium were enhanced after 2 weeks in culture and thereafter remained stable with no loss of cell viability. In contrast, the mechanical properties of explants in serum-supplemented medium were degraded by ( approximately 70%) along with a concurrent loss of biochemical content (30-40% GAG). These results suggest that long-term maintenance of allografts can be extended significantly by the use of a chemically defined medium.     

Coupling plowing of cartilage explants with gene expression in models for synovial joints

Articular cartilage undergoes complex loading modalities generally including sliding, rolling and plowing (i.e. the compression by a condyle normally to the tissue surface under simultaneously tangential displacement, thus generating a tractional force due to tissue deformation). Although in in vivo studies it was shown that excessive plowing can lead to osteoarthritis, little quantitative experimental work on this loading modality and its mechanobiological effects is available in the literature. Therefore, a rolling/plowing explant test system has been developed to study the effect on pristine cartilage of plowing at different perpendicular forces. Cartilage strips harvested from bovine nasal septa of 12-months-old calves were subjected for 2h to a plowing-regime with indenter normal force of 50 or 100 N and a sliding speed of 10 mm s(-1). 50 N produced a tractional force of 1.2±0.3N, whereas 100 N generated a tractional force of 8.0±1.4N. Furthermore, quantitative-real-time polymerase chain reaction experiments showed that TIMP-1 was 2.5x up-regulated after 50 N plowing and 2x after 100 N plowing, indicating an ongoing remodeling process. The expression of collagen type-I was not affected after 50 N plowing but it was up-regulated (6.6x) after 100 N plowing, suggesting a possible progression to an injury stage of the cartilage, as previously reported in cartilage of osteoarthritic patients. We conclude that plowing as performed by our mimetic system at the chosen experimental parameters induces changes in gene expression depending on the tractional force, which, in turn, relates to the applied normal force.     

Vasopressin gene expression is stimulated by cyclic AMP in homologous and heterologous expression systems

The possible role of cyclic AMP (cAMP) in the regulation of the vasopressin (VP) gene was tested in two cellular expression systems: one cell line with endogenous VP expression and the other which was transiently with a VP promoter-luciferase fusion gene. 8,Bromo-cAMP stimulated the VP mRNA content about 4-fold in the human VP-expressing small cell lung carcinoma cell line GLC-8. The luciferase activity in P19 embryonal carcinoma cells which were transiently transfected with -174 to +44 of the 5'-flanking region of the human VP gene linked to the firefly luciferase gene, was stimulated about 2-fold by the cAMP analogue. The results indicate that cAMP plays a role in the upregulation of the VP gene and hence point to several putative nucleotide motives in the promoter functionally conferring this response.     

Arachidonic acid induces mobilization of calcium stores and c-jun gene expression: evidence that intracellular calcium release is associated with c-jun activation.

Arachidonic acid (AA) plays a signaling role in the induction of several genes. We previously demonstrated that AA induces c-jun gene expression in the stromal cell line +/+.1 LDA 11 by a signaling pathway involving activation of the c-jun amino-terminal kinase (JNK). This study investigated the role of calcium in AA signaling of c-jun activation in +/+.1 LDA 11 cells. AA (10-50 microM) caused a rapid dose-dependent rise in cytosolic calcium. AA-induced calcium mobilization involved both influx of extracellular calcium and the release of intracellular calcium. The importance of calcium was investigated by variation of the extracellular calcium concentration, chelation of intracellular calcium and by calcium ionophore-induced influx of extracellular calcium. AA-induced c-jun gene expression and increased luciferase activity of a construct containing the high affinity AP-1 binding site was decreased in cells preincubated with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)-eThane-N,N,N',N',-tetraacetic acid tetra(aceToxymethyl-esTer) (BAPTA-AM, 10 microM) prior to stimulation with AA. Similarly, chelation of intracellular calcium decreased AA-induced JNK activation. On the contrary, changes in the extracellular calcium concentration had no effect. Also, ionophore A23187 failed to induce c-jun and JNK activation either alone than in combination with AA. These results suggested that calcium was required for AA-dependent activation of c-jun, but that calcium alone was insufficient to induce activation of c-jun. Thus, release of calcium from intracellular stores is implicated in the signaling pathway of AA-induced c-jun activation in stromal cells.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

Spatial expression patterns of genes involved in cyclic AMP responses in Dictyostelium discoideum development

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G-protein alpha2 and beta subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA-C) and cAMP phosphodiesterases (PDE and REG-A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal-regulated kinase-2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA-C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis.     

Interaction of calcium antagonists with cyclic AMP phosphodiesterases and calmodulin

The calcium antagonists, nimodipine and nicardipine, competitively inhibited calmodulin-sensitive and calmodulin-insensitive forms of cyclic AMP phosphodiesterase, with IC_50's in the micromolar range. Verapamil showed similar inhibitory potency against calmodulin-insensitive phosphodiesterases, but in marked contrast, it was a very weak inhibitor (30–100 times less potent) against calmodulin-sensitive forms of the enzyme. Verapamil and nimodipine both antagonized the calmodulin stimulation of phosphodiesterase. Through use of hydrophobic fluorescent probes, verapamil, and another calmodulin antagonist, proadifen, were shown to interact directly with calmodulin in a manner that differed from the interaction of calmodulin with trifluoperazine.     

The regulation of cell proliferation by calcium and cyclic AMP

Calcium, in partnership with cyclic AMP, controls the proliferation of non-tumorigenic cells in vitro and in vivo. While it does not seem to be involved in the proliferative activation of cells such as hepatocytes (in vivo) or small lymphocytes (in vitro), it does control two later stages of prereplicative (G1) development. It must be one of the very many regulatory and permissive factors affecting early prereplicative development, because severe calcium deprivation reversibly arrests some types of cell early in the G1 phase of their growth-division cycle in vitro. However, calcium more specifically and much more often regulates a later (mid or late G1) stage of prereplicative development. Thus, regardless of its severity or the type of cell, calcium deprivation in vitro or in vivo reversibly stops proliferative development at that part of the G1 phase in which the cellular cyclic AMP content transiently rises and the synthesis of the four deoxyribonucleotides begins. The evidence points to calcium and the cyclic AMP surge being co-generators of the signal committing the cell to DNA synthesis. The evidence is best explained so far by the cyclic AMP surge causing a surge of calcium ions which combine with molecules of the multi-purpose, calcium-dependent, regulator protein calmodulin (CDR) somewhere between the cell surface and the cytosol. The resulting Ca-calmodulin complexes then stimulate many different (and possibly membrane-associated) enzymes such as protein kinases, one of which produces the DNA-synthetic initiator. Calcium has little or no influence on the proliferation of tumor cells. Some possible explanations of this very important loss of control are considered.