Participation of the adenylate cyclase system in inductive synthesis of acetylcholinesterase in the brain]

The enhancement of the brain acetyl cholinesterase (ACHE) activity in rats by the intraventricular injection of adrenaline or dibutiryl analogue cyclic adenosine-3',5'-monophosphate (cAMP) was shown to be due to the enzyme inductive synthesis. ACHE induction manifests itself more in the subcortical white matter than in the cortex. The stimulating effect of adrenaline on the ACHE activity is suppressed under the beta-adrenoreceptor block, while the cAMP effect remains unchanged. On the contrary, the block of the alpha-adrenoreceptors stimulates the enzyme synthesis induction. The effects of adrenaline and cAMP are of the same direction and are realized through the beta-adrenoreceptors. The enhancement of ACHE activity during the block of the alpha-adrenoreceptors is accounted for by the elimination of their inhibitory influence on the beta-adrenoreceptors.     

cAMP Level Modulates Scleral Collagen Remodeling,a Critical Step in the Development of Myopia

The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP). We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs). Form-deprived myopia (FDM) was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC) activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.     

Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes

Intracellular collagen degradation in normal rat hepatocytes was exponetially stimulated by db-cAMP (10–100 µM). The effect was manifested as a decrease (p < 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only upto a threshold concentration (16.2 ± 1.3 p moles/10⁶ cells) elicited by 100 µM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10–50 µM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p < 0.05) in hepatocytes from rats administered CCL₄. However, the extent of stimulation was significantly (p < 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.Abbreviations cAMP 3,5 cyclic Adenosine Monophosphate - db-cAMP dibutyryl cyclic Adenosine Monophosphate - TCA Trichloroacetic Acid - Coll. Collagen - DMEM Dulbecoo's Minimal Essential Medium     

Postradiation change in the body's reaction to adrenaline

In experiments with albino mongrel rats weighing 220-260 g a study was made of the influence of adrenaline on the cyclic nucleotide content of blood plasma at early times following 60Co-gamma-irradiation (75 Gy). The absence of the organism response to adrenaline is perhaps due to the development of postirradiation desensitization of beta-adrenergic system and inability to induce cAMP formation in a cell. The data are submitted confirming the influence of levamisole on the cyclic nucleotide level. The level of cGMP decreased in thymocytes and increased in blood plasma after the administration of the drug to irradiated animals.     

Effect of exercise on plasma cyclic AMP

The purpose of this study was to confirm the relationship between cyclic AMP(cAMP) level in plasma and changes of hormones concentrations in blood, during and after physical exercise. The results were as follows: At rest, plasma cAMP were 23.1 p mole/ml on the average and decreased after glucose loading. The level in plasma increased in proportion to the intensity of exercises. Under the 50% condition of the maximal intensity, cAMP level in plasma was about 40 p mole/ml and the contents of both thyroxine and growth hormone in serum clearly increased. And, under the 70% of the maximal, the contents of both adrenaline and noradrenaline in serum as well as that of cAMP in plasma increased. Plasma cAMP level also increased by prolongation of exercise (ca 45 p mole/ml). And when exercise lasted over 1.5 hrs, plasma glucagon level began to rise. The effect of carbohydrate load to lower the levels of plasma cAMP were also found during physical exercise. These results suggested that the cAMP level in plasma was affected, not only by the some regulating factors of glycolytic activities such as adrenaline and glucagon, but also by the production of thyroxine and growth hormone at the onset of exercise.     

Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model

ABSTRACT

During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5?Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5?Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13?Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.     

Prostaglandin deficiency promotes sensitization of adenylyl cyclase

Inhibition of prostaglandin synthesis by the drug indomethacin suppresses the synthesis of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), and leads to a metabolic state comparable to type II diabetes. It was of interest whether prostaglandin-deficiency likewise causes sensitization of adenylyl cyclase, as this has been reported for the diabetic state. In liver plasma membranes of indomethacin-treated male rats, basal and forskolin-stimulated cyclic AMP synthesis remained unchanged when compared to untreated control rats. In control rats, stimulation of cyclic AMP synthesis by fluoride (2.2-fold) or glucagon (3.5-fold) was much lower than stimulation by forskolin (6.6-fold). In contrast, in indomethacin-treated rats, stimulation of cAMP synthesis by fluoride (4.6-fold) or glucagon (5.2-fold) nearly matched the stimulation by forskolin (6.4-fold). The level of alpha1-adrenergic receptors was slightly reduced, from 450 to 320 fmol/mg protein, by the indomethacin treatment. Independent of the treatment by indomethacin, stimulation of cyclic AMP synthesis by adrenaline failed, in agreement with the low density of adrenergic beta-receptors. In conclusion, PGE deficiency sensitizes adenylyl cyclase in rat liver for G protein-coupled receptors (glucagon) and also for fluoride.     

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.     

Impairment of chondrocyte biosynthetic activity by exposure to 3-tesla high-field magnetic resonance imaging is temporary

The influence of magnetic resonance imaging (MRI) devices at high field strengths on living tissues is unknown. We investigated the effects of a 3-tesla electromagnetic field (EMF) on the biosynthetic activity of bovine articular cartilage. Bovine articular cartilage was obtained from juvenile and adult animals. Whole joints or cartilage explants were subjected to a pulsed 3-tesla EMF; controls were left unexposed. Synthesis of sulfated glycosaminoglycans (sGAGs) was measured by using [³⁵S]sulfate incorporation; mRNA encoding the cartilage markers aggrecan and type II collagen, as well as IL-1β, were analyzed by RT–PCR. Furthermore, effects of the 3-tesla EMF were determined over the course of time directly after exposure (day 0) and at days 3 and 6. In addition, the influence of a 1.5-tesla EMF on cartilage sGAG synthesis was evaluated. Chondrocyte cell death was assessed by staining with Annexin V and TdT-mediated dUTP nick end labelling (TUNEL). Exposure to the EMF resulted in a significant decrease in cartilage macromolecule synthesis. Gene expression of both aggrecan and IL-1β, but not of collagen type II, was reduced in comparison with controls. Staining with Annexin V and TUNEL revealed no evidence of cell death. Interestingly, chondrocytes regained their biosynthetic activity within 3 days after exposure, as shown by proteoglycan synthesis rate and mRNA expression levels. Cartilage samples exposed to a 1.5-tesla EMF remained unaffected. Although MRI devices with a field strength of more than 1.5 T provide a better signal-to-noise ratio and thereby higher spatial resolution, their high field strength impairs the biosynthetic activity of articular chondrocytes in vitro. Although this decrease in biosynthetic activity seems to be transient, articular cartilage exposed to high-energy EMF may become vulnerable to damage.     

Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.     

Regulation of the cell cycle of B-lymphocytes in mice by substances elevating the levels of intracellular cAMP and cGMP

Spontaneous velocity sedimentation of B lymphocytes activated by intraperitoneal injection of ovalbumin into mice was used to obtain cell cycle synchronized cells, evidenced by differences in the incorporation of labeled precursors of protein and nucleic synthesis (14C-methionine and 3H-thymidine). The effects of acetylcholine and adrenaline, cAMP and cGMP on the intensity of 3H-thymidine incorporation into mouse B lymphocytes and on the amount of the cells entering mitosis were examined. It was shown that acetylcholine is capable of stimulating whereas adrenaline of inhibitin B lymphocyte entry into the stage of DNA synthesis and egress of these cells from the stage of DNA synthesis to the stage of mitosis. Adrenaline was found to have a reciprocal action. The acetylcholine effect could be mimetized by exogenous cGMP, that of adrenaline by cAMP. Stimulation of the G1/S transition was mediated by intracellular calcium ions but did not depend on exocellular calcium.     

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.     

Inhibitory effects of glucocorticoids on collagen synthesis by mouse sponge granulomas and granuloma fibroblasts in culture.

The basis for the glucocorticoid-mediated decrease in tissue collagen was studied in mouse granulomas and in primary granuloma fibroblast cultures. Injection of mice for 12 days with dexamethasone (0.35 mg/kg body weight) resulted in a 50--70% inhibition of collagen synthesis and accumulation in polyvinyl sponge-induced granulomas whereas total protein synthesis was inhibited by only about 25%. The decreased collagen content of the granuloma was accounted for by both a reduced fibroblast number and diminished synthesis per cell. Growth rates, total protein synthesis and collagen synthesis were the same in granuloma fibroblast cultures derived from control or steroid-treated mice. However, addition of 3.10(-7) M hydrocortisone to the culture medium caused a 30--50% inhibition of both collagen and non-collagen protein synthesis in firbroblasts from either source. These inhibitory effects were dose- and time-dependent with a lag time of 12--24 h. Prolyl hydroxylase activity was reduced both in sponge granulomas from glucocorticoid-treated mice and in hydrocortisone-treated fibroblast cultures. However, protein synthesis was inhibited to the same extent as the inhibition of prolyl hydroxylase activity and there was no effect on peptidyl prolyl hydroxylation. These results indicate that the glucocorticoid-induced reduction of collagen synthesis and accumulation observed in mouse granulomas and primary granuloma fibroblast cultures is not specific for this protein. Furthermore, glucocorticoid-induced inhibition of collagen synthesis cannot be attributed to underhydroxylation of collagen prolyl residues.     

Modulation of in vitro erythropoiesis: enhancement of erythroid colony growth by cyclic nucleotides.

Mammalian erythropoiesis, as assayed by erythroid colony formation in vitro, is enhanced by cyclic adenosine nucleotides and agents which are capable of raising intracellular cyclic AMP (cAMP) levels. With canine marrow cells as target, this enhancement was shown to be specific for cAMP and its mono- and dibutyryl derivatives. Adenosine and its derivatives, such as AMP, ADP and ATP, and other cyclic nucleotides, such as cGMP, dibutyryl-cGMP, cCMP and cIMP and sodium butyrate were inactive. The phosphodiesterase inhibitor, RO-20-1724, and the adenyl cyclase stimulator, cholera enterotoxin, both markedly increased colony numbers. Studies with tritiated thymidine showed that about 50% of the cells responding to either erythropoietin (ESF) or dibutyryl cAMP (db-cAMP) were in DNA synthesis. However, by unit gravity sedimentation velocity analysis, the peak of ESF-responsive colony forming cells sedimented more rapidly (8-7 +/- 0-2 mm/hr) than the peak of db-cAMP-responsive cells (7-5 +/- 0 mm/hr). These results demonstrate that adenyl cyclase-linked mechanisms influence in vitro erythropoietic proliferation and suggest that other hormones and simple molecules might interact with surface receptors and thus modulate the action of ESF at the cellular level.     

Effect of thyroid state on cyclic AMP-mediated induction of hepatic phosphoenolpyruvate carboxykinase.

Hepatic phosphoenolpyruvate carboxykinase (PEPCK) is significantly increased in the hyperthyroid starved rat, and moderately decreased in the hypothyroid starved rat. As tri-iodothyronine by itself has only a small and sustained effect on the induction of this enzyme, as was previously shown in the isolated perfused organ, the effect of hypo- and hyper-thyroidism on the increase in cytosolic PEPCK provoked by dibutyryl cyclic AMP (Bt2cAMP) was investigated in vivo and in the isolated perfused liver. Compared with euthyroid fed controls, in hypothyroid fed rats Bt2cAMP provoked in 2 h only a small increase in translatable mRNA coding for PEPCK. In contrast, in hyperthyroid animals PEPCK mRNA as measured by translation in vitro was already increased in the fed state, and further enhanced by Bt2cAMP injection to values as in euthyroid controls. Under all thyroid states a close correlation between PEPCK mRNA activity and PEPCK synthesis was observed. In the isolated perfused liver from the hyperthyroid fed rat, the increase in PEPCK provoked by Bt2cAMP or Bt2cAMP + isobutylmethylxanthine was considerably enhanced compared with those obtained in livers of hypothyroid rats. Also, adrenaline provoked a stimulated induction of PEPCK in hyperthyroid rats compared with hypothyroid rats. To summarize, our data indicate that the primary action of thyroid hormones on the synthesis of hepatic cytosolic PEPCK is to accelerate the cyclic AMP- or adrenaline-induction of the enzyme, acting primarily at a pretranslational level.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50–800 μg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H₂O₂, 50–200 μM), or tumor necrosis factor α (TNFα, 40 ng/ml) for 24–48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [³H]-thymidine or [³H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 μg/ml) and double therapeutic (100 μg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200–800 μg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 μg/ml) significantly inhibited TNFα-induced apoptosis in myocytes, with 12.3±1.46% cells being apoptosis in CP treatment group and 37.0±7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by ～50% and ～90% reduction of AII-induced collagen synthesis at 50 and 100 μg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.     

Cerium depresses endocardial endothelial cell-mediated proliferation of cardiac fibroblasts

Cerium has been implicated in the pathogenesis of cardiac disorders such as acute myocardial infarction and endomyocardial fibrosis (EMF). A geochemical hypothesis for the causation of EMF linked the cardiac lesions to magnesium deficiency consequent to malnutrition and increased cardiac levels of cerium derived from monazite soils in the coastal regions of the tropics. We tested the hypothesis that the stimulus for fibroblast proliferation and enhanced collagen synthesis in EMF is derived from cardiac endothelial cells activated or injured by cerium. We explored whether endocardial endothelial cells exposed to cerium secrete factors responsible for the increased proliferation and collagen synthesis in cardiac fibroblasts. Our results suggest that the growth response of cardiac fibroblasts to cerium is not mediated through growth factors secreted by endocardial endothelium and that the cardiac lesions in EMF result from direct stimulation of subendocardial fibroblasts by cerium.     

Effects of extremely low frequency electromagnetic field (EMF) on collagen type I mRNA expression and extracellular matrix synthesis of human osteoblastic cells

Human osteoblastic cells were grown in a three-dimensional (3-D) cell culture model and used to test the effects of a 20 Hz sinusoidal electromagnetic field (EMF; 6 mT and 113 mV/cm max) on collagen type I mRNA expression and extracellular matrix formation in comparison with the effects of growth factors. The cells were isolated from trabecular bone of a healthy individual (HO-197) and from a patient presenting with myositis ossificans (MO-192) and grown in a collagenous sponge-like substrate. Maximal enhancement of collagen type I expression after EMF treatment was 3.7-fold in HO-197 cells and 5.4-fold in MO-192 cells. Similar enhancement was found after transforming growth factor-β (TGF-β) and insulin-like growth factor-I (IGF-I) treatment. Combined treatment of the cells with EMF and the two growth factors TGF-β and IGF-I did not act synergistically. MO-192 cells produced an osteoblast-characteristic extracellular matrix containing collagen type I, alkaline phosphatase, and osteocalcin, together with collagen type III, TP-1, and TP-3, two epitopes of an osteoblastic differentiation marker. The data suggest that the effects of EMFs on osteoblastic differentiation are comparable to those of TGF-β and IGF-I. We conclude that EMF effects in the treatment of skeletal disorders and in orthopedic adjuvant therapy are mediated via enhancement of collagen type I mRNA expression, which may lead to extensive extracellular matrix synthesis. Bioelectromagnetics 19:222–231, 1998. © 1998 Wiley-Liss, Inc.     

Effect of cyclic adenosine-3,5-monophosphate and its dibutyryl analog on macromolecule biosynthesis in actively proliferating cells

The effect of cAMP and its dibutyryl analogue on the biosynthesis of nucleic acids and protein in active proliferating cells was studied. It was shown that cAMP (10(-3)--10(-4)M) caused stimulation of the biosynthesis of DNA and RNA in Ehrlich's ascites carcinoma (EAC) cells and intensification of collagen biosynthesis in the chick embryo cartilage tissue in vitro. Dibutyryl -- cAMP (10(-3)--10(-4)M) has an inhibitory action on the biosynthesis of macromolecules both in EAC cells and embryonic cartilage tissue. Addition of cAMP phosphodiesterase inhibitors together with cAMP to the incubation media prevents the stimulation of macromolecular biosynthesis observed under the influence of cAMP. Studies on cAMP metabolism revealed that this compound is rapidly catabolized to AMP and adenosine. The latter enters the cells and incorporates into the adenyl nucleotide intracellular pool. The stimulant action of exogenous cAMP is related to its extracellular metabolism rather than to the intracellular effects of the nucleotide.