Determination of the site of action of calcitonin gene-related peptide in the alteration of intracellular calcium levels in adult and neonatal rodent myocytes

The purpose of this study is to elucidate the mechanism of action and site of action of calcitonin gene-related peptide (CGRP) and its effects on calcium concentrations in two types of cardiomyocytes, neonatal and adult, by employing real-time fluorescence imaging. CGRP caused an increase in intramyocytic calcium with adult cells, but a decrease with neonates. Treatment of adult myocytes with ouabain and ryanodine yielded results suggesting that CGRP action is not at the ryanodine receptor (RyR) and does not involve Na+ +K+ ATPase. Furthermore, in neonatal cardiomyocytes CGRP caused a reduction in intramyocytic calcium levels, and challenges with ryanodine and ouabain gave results supporting the hypothesis that CGRP acts at the sarcolemmal L-type calcium channel. Employing real-time fluorescence measurements in cultured, dedifferentiated adult cardiomyocytes, which are known to express a fetal phenotype and exhibit neonatal-like calcium transients, our acquisitions demonstrated a major reduction in intracellular calcium levels. Finally, our collaborative studies in human myocardium using fluorescence deconvolution microscopy revealed that CGRP localization was found in a pattern similar to that of the sarcolemmal L-type calcium channel.     

Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes

Ginsengs are widely used to improve cardiac health and circulation. Loosely termed as ginsengs, Asian (Panax), Siberian and Ashwagandha (Indian Ginseng) Indian ginsengs are prepared from different plants. We tested the popular belief of cardiotonic effects of ginsengs using both neonatal and adult rat cardiomyocytes, comparing extracts from the three ginsengs. Addition of 10% v/v of extract (100 microl of extract/ml of culture medium) of each of the ginsengs resulted in a rapid (<10 s) cessation of beating in neonatal cardiomyocytes due to calcium overload, while sequential dilutions revealed that treatment with a low dose (0.01% v/v, 0.1 microl/ml of the medium) resulted in constant, regular beats (transients), and a slight elevation of diastolic calcium without overload. Addition of extracts to sparking, calcium-tolerant adult cardiomyocytes resulted in initiation of calcium transients, and adult cells were able to tolerate exposure to high concentrations of extract. Cardiotonic effects in adult cells (cardiotoxicity in neonatal cells) were most profound with Asian ginseng (2.6 times that of Siberian ginseng, 1.6 times that of Indian ginseng) probably due to the active ingredients (ginsenosides in Asian, eleutherosides in Siberian and withanolides in Indian) being structurally different. We conclude that fully developed cardiomyocytes are able to accommodate higher doses of ginseng than neonatal cells, and that the effects of ginseng on newly formed, developing myocytes, could be extremely deleterious to the fetus. However, for adults, ginseng might well be a 'tonic' in its ability to increase beating and intramyocytic calcium levels.     

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes

Abstract

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15?Hz, 50?Hz, 75?Hz and 100?Hz) and at a flux density of 2?mT. Intracellular calcium concentration ([Ca²⁺]_i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10?mM) was carried out to verify the function of the cardiac Na⁺/Ca²⁺ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca²⁺-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca²⁺]_i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.     

Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.     

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.     

牛磺酸镁对哇巴因致豚鼠心肌细胞心律失常模型钙离子通道的作用机制分析

摘要目的：研究牛磺酸镁（TMCC）对哇巴因致豚鼠心肌细胞心律失常模型钙离子通道的作用机制。方法：运用全细胞膜片钳技术分别记录TMCC和胺碘酮对正常心肌细胞和哇巴因导致的心律失常心肌细胞模型钙离子通道的作用。结果：5bμmol／L哇巴因使心肌细胞钙离子通道电流（ICa-L）减小。200和400μmol／L可以明显使Ic}L恢复。24．26μmol／L胺碘酮使IM进一步减少（P〉0．05）。结论：400μmol／LTMCC可以明显加大正常细胞的b。,起到促进钙内流的作用,并且增强哇巴因致豚鼠心律失常心肌细胞异常减少的电流。     

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.     

MMP-9 Gene Ablation and TIMP-4 Mitigate PAR-1-Mediated Cardiomyocyte Dysfunction: A Plausible Role of Dicer and miRNA

Although matrix metalloproteinase-9 (MMP‐9) is involved in cardiomyocytes contractility dysfunction, tissue inhibitor of metalloproteinase-4 (TIMP‐4) mitigates the effect of MMP‐9, and proteinase-activated receptor-1 (PAR‐1, a G-protein couple receptor, GPCR) is involved in the signaling cascade of MMP‐9-mediated cardiac dysfunction, the mechanism(s) are unclear. To test the hypothesis that induction of dicer and differential expression of microRNAs (miRNAs) contribute, in part, to the down regulation of sarcoplasmic reticulum calcium ATPase isoform 2a (serca-2a) in MMP-9 and PAR-1-mediated myocytes dysfunction, ventricular cardiomyocytes were isolated from C57BL/6J mice and treated with 3 ng/ml of MMP-9, 12 ng/ml of TIMP-4, and 10 and 100 μM of PAR-1 antagonist with MMP-9. Specific role of MMP-9 was determined by using MMP-9 knock out (MMP-9KO) and their corresponding control (FVB) mice. Ion Optics video-edge detection system and Fura 2-AM loading were used for determining the contractility and calcium release from cardiomyocytes. Quantitative and semi-quantitative PCR were used to determine the expression of dicer, TIMP-4 and serca-2a. miRNA microarrays were used for assessing the expression of different miRNAs between MMP-9KO and FVB cardiomyocytes. The results suggest that MMP‐9 treatment attenuates the voltage‐induced contraction of primary cardiomyocytes while TIMP‐4, an inhibitor of MMP‐9, reverses the inhibition. MMP‐9 treatment is also associated with reduced Ca²⁺ transients. This effect is blocked by a PAR‐1 antagonist, suggesting that PAR‐1 mediates this effect. The effect is not as great at high concentrations (100 μM) perhaps due to mild toxicity. The PAR‐1 antagonist effect did not affect calcium transients unlike TIMP‐4. Interestingly, we show that MMP‐KO myocytes contract more rapidly and release more Ca²⁺ than FVB. The relevant RNA species serca-2a is induced and dicer is inhibited. There is selective inhibition of miR-376b and over-expression of miR-1, miR-26a, miR-30d, and miR-181c in MMP‐9KO that are implicated in regulation of G-PCR and calcium handling.     

Tumor necrosis factor-alpha alters calcium handling and increases arrhythmogenesis of pulmonary vein cardiomyocytes

Inflammation and abnormal calcium homeostasis play important roles in atrial fibrillation. Tumor necrosis factor-alpha (TNFalpha), a proinflammatory cytokine, can induce cardiac arrhythmias. Pulmonary veins (PVs) are critical in initiating paroxysmal atrial fibrillation. This study was designed to investigate whether TNFalpha may change the calcium handling and arrhythmogenic activity of PV cardiomyocytes. We used whole-cell patch clamp and indo-1 fluorimetric ratio technique to investigate the action potentials, ionic currents and intracellular calcium in isolated rabbit single PV cardiomyocytes with and without (control) incubation with TNFalpha (25 ng/ml) for 7-10 h. The expression of sarcoplasmic reticulum ATPase in the control and TNFalpha-treated PV cardiomyocytes was evaluated by confocal micrographs and Western blot. We found that the spontaneous beating rates were similar between the control (n=45) and TNFalpha-treated (n=28) PV cardiomyocytes. Compared with the control PV cardiomyocytes, the TNFalpha-treated PV cardiomyocytes had significantly a larger amplitude of the delayed afterdepolarizations (6.0+/-1.7 vs. 2.6+/-0.8 mV, P<0.05), smaller L-type calcium currents, larger transient inward currents, larger Na(+)-Ca(2+) exchanger currents, a smaller intracellular calcium transient, smaller sarcoplasmic reticulum calcium content, larger diastolic intracellular calcium, a longer decay portion of the calcium transient (Tau), and a decreased sarcoplasmic reticulum ATPase expression. In conclusion, TNFalpha can increase the PV arrhythmogenicity and induce an abnormal calcium homeostasis, thereby causing inflammation-related atrial fibrillation.     

Beta-adrenergic agonists and cyclic AMP decrease intracellular resting free-calcium concentration in ileum smooth muscle

Intracellular free-calcium levels were measured in strips of longitudinal smooth muscle from guinea-pig ileum; fura-2 was used as a calcium monitor. At rest the calcium concentration was about 180 nM, and this rose to 300-400 nM following electrical stimulation and during spontaneous calcium transients (all measurements at 23-25 degrees C). Isoprenaline suppressed the spontaneous calcium transients, and reduced the resting calcium level to about 130 nM. This fall in resting calcium concentration was seen even in muscle strips which did not have spontaneous activity. Elevation of intracellular cyclic AMP levels, produced by forskolin or dibutyryl cyclic AMP, mimicked the actions of isoprenaline. We conclude that the relaxant effects of beta-adrenergic agonists of visceral smooth muscle may be explained partly by a fall in intracellular resting free-calcium level, mediated via an increase in cyclic AMP.     

Maitotoxin stimulates phosphoinositide breakdown in neuroblastoma hybrid NCB-20 cells

1. Maitotoxin (MTX) was an extraordinarily potent stimulant of phosphoinositide breakdown in the neuroblastoma hybrid NCB-20 cells. 2. Maximal responses were obtained at 0.25-0.5 ng MTX/ml, and resulted in increased formation of [3H]inositol mono-, bis-, and trisphosphates. Increased formation of [3H]inositol bis- and trisphosphate was observed as early as 15 sec after the addition of MTX. 3. MTX-induced phosphoinositide breakdown in NCB-20 cells was not antagonized by organic (nifedipine, methoxyverapamil) or inorganic (Mn2+, Co2+, Cd2+) calcium channel blockers. However, the response on phosphoinositide breakdown was completely eliminated in the absence of extracellular calcium. 4. The results suggest that MTX either directly stimulates phosphoinositide breakdown in a calcium-dependent manner or acts indirectly through calcium channels insensitive to organic/inorganic calcium channel blockers.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Differential calcium regulation of proinflammatory activities in human neutrophils exposed to the neuropeptide pituitary adenylate cyclase-activating protein

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide/PACAP receptor-1 to induce phospholipase C/calcium and MAPK-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this study, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A, protein kinase C, and PI3K pathways, to pertussis toxin, genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to 1) thapsigargin, 2) Xestospongin C, and 3) the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane up-regulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.     

Cardiovascular effects of BRX-005 comparison to bimoclomol

Concentration-dependent effects of BRX-005, the novel heat shock protein coinducer, cardioprotective and vasoprotective agent, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea pig hearts loaded with the fluorescent calcium indicator dye Fura-2. BRX-005 increased peak left ventricular pressure, the rate of force development and relaxation significantly in a concentration-dependent manner. The amplitude of [Ca2+]i transients was left unaltered by the drug. In contrast to BRX-005, bimoclomol increased both contractility and the amplitude of [Ca2+]i transients. In canine ventricular cardiomyocytes high concentrations of BRX-005 had no effect on depolarization, whereas bimoclomol suppressed action potential upstroke markedly. In guinea pig pulmonary artery preparations precontracted with phenylephrine, BRX-005 induced concentration-dependent relaxation. This effect of BRX-005 was independent of the integrity of endothelium indicating that vasorelaxant effect of the drug develops directly on vascular smooth muscle.     

Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes

High-mobility group box 1 (HMGB1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca2+ transient within 5 min (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward l-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-epsilon translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury.     

Localization of calcitonin gene-related peptide in cardiomyocytes: comparison of neonatal and dedifferentiating cells to adult myocytes

The purpose of this study was to localize sites of calcitonin gene-related peptide binding in neonatal, freshly isolated and dedifferentiated adult cardiac myocytes in order to help us elucidate the mechanisms of action of this neuropeptides. Previous work has shown that treatment with calcitonin gene-related peptide results in dramatic changes in calcium transients, so we carried out multi-channel acquisitions of fluorescently labeled images to reveal where calcitonin gene-related protein and the L-type calcium channel were localized. Calcitonin gene-related protein was sparse and randomly distributed in rod-like adult cardiomyocytes, found in abundance in areas of the cell where striations were apparent and not where adhesion proteins predominated in dedifferentiating adult myocytes, and in a large perinuclear concentration, with some spreading into the cytoplasm in neonatal cells. Subsequent modeling demonstrated that calcitonin gene-related peptide and the L-type calcium channel protein were closely associated in each of the three myocyte types, suggesting that while the peptide has dramatic and different effects on intracellular calcium levels of the various cardiomyocytes, the action is probably via diverse mechanisms as a result of effects on different channels or pump proteins due to alterations in intracellular calcium concentrations.     

Effects of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium and mitogenesis

Although increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Cai increases. In order to examine in more detail whether Cai increases were related to mitogenesis, we used digital image analysis of intracellular Fura-2 fluorescence to measure Cai in individual BALB/c 3T3 cells stimulated with either platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Cai increases than FGF did, but that both growth factors induced an initial rapid increase in Cai (less than 2 min) followed by a later sustained increase (greater than 20 min). Only the prolonged Cai increase required extracellular calcium. Following PDGF treatment (1-8 units/ml), the percentage of cells with a large peak Cai increase (greater than twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet-poor-plasma). In contrast, purified bovine basic FGF (200-800 pg/ml) and recombinant human acidic FGF (10-300 ng/ml) produced peak Cai increases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Cai transients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Cai increases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stimulated by FGF.