Changes in [Ca2+]i induced by several glucose transport-enhancing stimuli in rat epitrochlearis muscle.

The purpose of the present investigation was to establish a method for estimating intracellular Ca(2+) concentrations ([Ca(2+)](i)) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wk-old male Sprague-Dawley rats were loaded with a fluorescent Ca(2+) indicator, fura 2-AM, for 60-90 min at 35 degrees C in oxygenated Krebs-Henseleit buffer. After fura 2 loading and subsequent 20-min incubation, the intensities of 500-nm fluorescence, induced by 340- and 380-nm excitation lights (F(total)340 and F(total)380), were measured. The fluorescences specific to fura-2 (F(fura 2)340 and F(fura 2)380) were calculated by subtracting the non-fura 2-specific component from F(total)340 and F(total)380, respectively. The ratio of F(fura 2)340 to F(fura 2)380 was calculated as R, and the change in the ratio from the baseline value (DeltaR) was used as an index of the change in [Ca(2+)](i). In resting muscle, DeltaR was stable for 60 min. Incubation for 20 min with caffeine (3-10 mM) significantly increased DeltaR in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10-60 min significantly elevated DeltaR, depending on the duration of the incubation. Incubation with 50 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide for 20 min significantly elevated DeltaR (P < 0.05). No significant increases in DeltaR were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca(2+)](i) can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca(2+)](i) that may be involved in contraction- and hypoxia-stimulated glucose transport activity in skeletal muscle.     

Depolarization decreases the [Ca2+]i sensitivity of myosin light-chain kinase in arterial smooth muscle: comparison of aequorin and fura 2 [Ca2+]i estimates

Histamine stimulation of swine arterial smooth muscle is associated with a high [Ca2+]i sensitivity for increases in myosin light-chain phosphorylation. In contrast, KCl depolarization produces a relatively lower [Ca2+]i sensitivity (i.e., similar increases in [Ca2+]i induce less myosin phosphorylation). We evaluated whether 1) artifacts in the methodology for measuring [Ca2+]i or 2) true alterations in the [Ca2+]i sensitivity of myosin light-chain kinase were responsible for these apparent changes in the [Ca2+]i sensitivity of phosphorylation. The [Ca2+]i sensitivity of phosphorylation was higher with histamine stimulation regardless of whether the [Ca2+]i indicator was aequorin (which was loaded intracellularly by reversible hyperpermeabilization) or Fura 2 (which was loaded intracellularly by incubation of the tissues in Fura 2 AM). Aequorin and Fura 2 appeared to detect qualitatively similar stimulus-induced changes in [Ca2+]i with the exception that the initial response to histamine stimulation was different (histamine initially induced a large aequorin light transient and a relatively smaller increase in Fura 2 fluorescence). The [Ca2+]i sensitivity of myosin light-chain kinase extracted from KCl depolarized tissues was lower than the [Ca2+]i sensitivity of myosin light-chain kinase extracted from unstimulated or histamine stimulated tissues. These results suggest that depolarization specifically modifies myosin light-chain kinase to decrease its [Ca2+]i sensitivity. Changes in the [Ca2+]i sensitivity of myosin light-chain phosphorylation are not an artifact of the [Ca2+]i measurement technique.     

血小板源生长因子-AA对高血压血管平滑肌细胞钙信号的影响

目的 :明确自发性高血压大鼠血管平滑肌细胞 (SHR VSMC)增殖与血小板源生长因子 AA(PDGF AA)、PDGF α受体表达的关系及钙信号在其中的作用。方法 :在培养的血管平滑肌细胞模型中 ,采用免疫印迹 (Westernblot)、3 H TdR及3 H Leu掺入、荧光探针标记测定单细胞内钙浓度等方法 ,观察不同来源大鼠 (SHR/WKY)VSMC ,PDGF AA、PDGF α受体和PDGF β受体表达的差异性以及在PDGF AA刺激下 ,VSMC增殖肥大反应、胞内 [Ca2 ]i变化和钙离子阻断剂 (nimodipine)对其的影响。 结果 :与WKY VSMC相比SHR VSMC中PDGF AA、PDGF α受体蛋白表达明显增加 ,而PDGF β受体蛋白表达在SHR VSMC与WKY VSMC无明显变化。在PDGF AA刺激下 ,增殖细胞核抗原 (PCNA)、3 H掺入率及胞内 [Ca2 ]i浓度在SHR VSMC明显增强 ;钙离子阻断剂 (nimodipine)明显抑制PCNA表达及3 H掺入 ,胞内 [Ca2 ]i浓度明显下降。结论 :自发性高血压大鼠VSMCPDGF A链及其α受体的自发性增高 ,可能是导致SHR VSMC异常增殖、肥大 ,从而触发血管反应性和血管构型变化的重要原因之一 ;细胞膜钙通道在调控VSMC的钙内流时起主要作用     

卵细胞受精相关胞质钙离子波、钙离子震荡信号特征及其形成机制

胞质[Ca2 ]i震荡的动力学变化在哺乳动物早期胚胎发育中发挥重要作用。卵母细胞的成熟伴随间断的、快速的[Ca2 ]i震荡的时空表达;在受精过程中精子因子诱导的反复[Ca2 ]i震荡的振幅和持续时间是卵细胞最有效的激活信号,这种信号形成自然连续的受精[Ca2 ]i波,并以长时持续[Ca2 ]i震荡形式在受精卵空间传递并持续数小时,直至受精完成;受精卵内源性的Ca2 释放所引起的[Ca2 ]i震荡形成第一次卵裂信号,启动早期胚胎的发育。精子PLCζ和cPKCs是形成受精卵[Ca2 ]波、[Ca2 ]震荡的重要因素。     

Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration.

In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.     

Intracellular free [Ca2+] in human skeletal muscle with myopathic carnitine deficiency

Carnitine is required for the transport of activated long chain fatty acids through the mitochondrial inner membrane. We measured the intracellular free calcium concentration [( Ca2+]i) by means of a calcium selective microelectrode in skeletal muscle biopsies obtained from nine patients in which myopathic carnitine deficiency (MCD) was diagnosed, and from six subjects with no evidence of neuromuscular disease. Intact intercostal muscle bundles were dissected and then split for electron microscopic studies and electrophysiological measurements. The [Ca2+]i in muscle fibers from MCD patients was 0.46 +/- 0.02 mumol.l-1 (mean +/- SEM) and 0.10 +/- 0.01 mumol.l-1 in control subjects. At the electron microscopic level, the predominant abnormality was the presence of lipid vacuoles between the myofibrils. These results show that in patients with myopathic carnitine deficiency there is a significant increase in the resting myoplasmic calcium concentration which might be related to a malfunction of some mechanisms responsible for the homeostasis of intracellular calcium.     

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).     

Imaging [Ca2+]i dynamics during signal transduction

The elevation of free intracellular Ca2+ activity ([Ca2+]i) is widely recognised as a central event in many signal transduction processes in cellular physiology. Recent advances in optical techniques for measuring [Ca2+]i as well as developments in quantitative low light level fluorescence microscopy have led to the application of these methods to the study of subcellular [Ca2+]i in many biological systems. In the following paper we describe some techniques in our laboratory to provide quantitative high spatio-temporal resolution measurements of [Ca2+]i in individual living cells during the signal transduction of cell surface receptor ligand interactions. In particular, we are studying the changes in [Ca2+]i induced by the micro-aggregation of immunoglobulin E (IgE) receptor complexes on the surface of rat basophilic leukemia (RBL) cells (a tumor mast cell line) by multivalent antigen. We seek to understand the mechanisms which are involved in the detection of these cell surface events which lead to changes in [Ca2+]i as well as the interactions between the various subcellular components which impart the delicate control of [Ca2+]i during cellular stimulation. The limitations and properties of the technology used for these studies will be discussed, and some illustrative examples of the type of [Ca2+]i changes found in this biological system will be given.     

Selected contribution: Effects of aging on cerebrovascular tone and [Ca2+]i.

The lower limits of cerebral blood flow autoregulation shift toward high pressures in aged compared with young rats. Intraluminal pressure stimulates contractile mechanisms in cerebral arteries that might, in part, cause an age-dependent shift in autoregulation. The present project tested two hypotheses. First, cerebral artery tone is greater in isolated arteries from aged compared with mature adult rats. Second, aging decreases the modulatory effect of endothelium-derived nitric oxide (NO) and increases vascular smooth muscle Ca2+ sensitivity. Isolated segments of middle cerebral arteries from male 6-, 12-, 20-, and 24-mo-old Fischer 344 rats were cannulated and loaded with fura-2. Diameter and Ca2+ responses to increasing pressure were measured in HEPES, during NO synthase inhibition [NG-nitro-l-arginine methyl ester (l-NAME)], and after removal of the endothelium. Cerebral artery tone (with endothelium) increased with age. Only at the lowest pressure (20 and 40 mmHg) was intracellular Ca2+ concentration ([Ca2+]i) greater in arteries from 24-mo-old rats compared with the other age groups. l-NAME-sensitive constriction increased significantly in arteries from 6- to 20-mo-old rats but declined significantly thereafter in arteries from 24-mo-old rats. [Ca2+]i was less in arteries from 24-mo-old rats compared with the other groups after treatment with l-NAME. Another endothelial-derived factor, insensitive to l-NAME, also decreased significantly with age. For example, at 60 mmHg, the l-NAME-insensitive constriction decreased from 47 +/- 10, 42 +/- 5, 21 +/- 2, and 3 +/- 1 microm in 6-, 12-, 20-, and 24-mo-old rats, respectively. Our data suggest that aging alters cerebral artery tone and [Ca2+]i responses through endothelial-derived NO synthase-sensitive and -insensitive mechanisms. The combined effect of greater cerebral artery tone with less endothelium-dependent modulation may in part contribute to the age-dependent shift in cerebral blood flow autoregulation.     

Effects of injecting calcium-buffer solution on [Ca2+]i in voltage-clamped snail neurons.

We have investigated why fura-2 and Ca(2+)-sensitive microelectrodes report different values for the intracellular free calcium ion concentration ([Ca(2+)]i or its negative log, pCa(i)) of snail neurons voltage-clamped to -50 or -60 mV. Both techniques were initially calibrated in vitro, using calcium calibration solutions that had ionic concentrations similar to those of snail neuron cytoplasm. Pressure injections of the same solutions at resting and elevated [Ca(2+)]i were used to calibrate both methods in vivo. In fura-2-loaded cells these pressure injections generated changes in [Ca(2+)]i that agreed well with those expected from the in vitro calibration. Thus, using fura-2 calibrated in vitro, the average resting [Ca(2+)]i was found to be 38 nM (pCa(i) 7.42 +/- 0.05). With Ca(2+)-sensitive microelectrodes, the first injection of calibration solutions always caused a negative shift in the recorded microelectrode potential, as if the injection lowered [Ca2+]i. No such effects were seen on the fura-2 ratio. When calibrated in vivo the Ca(2+)-sensitive microelectrode gave an average resting [Ca2+]i of approximately 25 nM (pCa(i) 7.6 +/- 0.1), much lower than when calibrated in vitro. We conclude that [Ca(2+)]i in snail neurons is approximately 40 nM and that Ca(2+)-sensitive microelectrodes usually cause a leak at the point of insertion. The effects of the leak were minimized by injection of a mobile calcium buffer.     

The use of Fura-2 to estimate myoplasmic [Ca2+] in human skeletal muscle

Fura-2 was used to estimate myoplasmic [Ca2+] in intact fibers and fiber segments from normal and diseased human muscles. Small muscle bundles (20-50 fibers) were loaded with the membrane-permeant form of the dye (Fura-2 AM). High-performance liquid chromatography was utilized to study the ability of these cells to hydrolyze Fura-2 AM. Immediately after the 30 min loading period, Fura-2 (the Ca2+ indicator) was the predominant form of the dye in all preparations and the concentration within these fibers remained stable for over 4 1/2 hours. In addition, the resting myoplasmic [Ca2+] in fiber segments from normal subjects and those susceptible to malignant hyperthermia were the same. However, halothane administration (1.5%) induced correlated increases in myoplasmic [Ca2+] and force only in fibers from the susceptible patients. In contrast, caffeine administration causes correlated increases in myoplasmic [Ca2+] and force in both types of muscle, but lower concentrations were needed to do so in the fibers from the susceptible patients. The effects of halothane and caffeine were reversible. We conclude that Fura-2 can be used successfully to estimate resting levels and changes in myoplasmic [Ca2+] in human skeletal muscle.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

谷氨酸促进大鼠海马神经元的内钙升高

谷氨酸能影响大鼠海马神经元胞内钙信号的变化,进而影响海马神经元神经冲动的发放和学习记忆过程。运用荧光测钙技术实时监测了大鼠海马神经元内钙信号的动态变化,同时分析了谷氨酸对其胞内钙信号的影响。试验表明：谷氨酸能够显著提高胞内游离钙离子的浓度;细胞外钙离子的存在、谷氨酸刺激时间及刺激频率的增加都能引起胞内钙信号不同程度的升高;但谷氨酸的过度刺激会引起钙离子浓度的超负荷,从而导致神经元结构和功能的损坏。     

Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (approximately 1.5 microm3) and global (whole cell) changes in [Ca2+]i were measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]i oscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]i oscillations were 'biphasic' with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]i oscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]i oscillations lies in the establishment of mean [Ca2+]i level for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.     

Apical P2XR contribute to [Ca2+]i signaling and Isc in mouse renal MCD

We examined P2X receptor expression and distribution in the mouse collecting duct (CD) and their functional role in Ca(2+) signaling. Both P2X(1) and P2X(4) were detected by RT-PCR and Western blot. Immunohistochemistry demonstrated apical P2X(1) and P2X(4) immunoreactivity in principal cells in the outer medullary CD (OMCD) and inner medullary CD (IMCD). Luminal ATP induced an increase in Ca(2+) signaling in native medullary CD (MCD) as measured by fluorescence imaging. ATP also induced an increase in Ca(2+) signaling in MCD cells grown in primary culture but not in the presence of P2XR antagonist PPNDS. Short circuit current (I(sc)) measurement with mouse IMCD cells showed that P2XR agonist BzATP induced a larger I(sc) than did P2YR agonist UTP in the apical membrane. Our data reveal for the first time that P2X(1) and P2X(4) are cell-specific with prominent immunoreactivity in the apical area of MCD cells. The finding that P2XR blockade inhibits ATP-induced Ca(2+) signaling suggests that activation of P2XR is a key step in Ca(2+)-dependent purinergic signaling. The result that activation of P2XR produces large I(sc) indicates the necessity of P2XR in renal CD ion transport.     

糖皮质激素快速抑制气道平滑肌游离钙升高与磷脂酶C活性的关系

目的:本实验通过对平滑肌细胞行GCs快速预处理,拟证实糖皮质激素对平滑肌细胞内[Ca2+]i浓度升高有快速抑制作用,并初步探讨该现象的可能分子机制。方法:原代培养的大鼠平滑肌细胞,应用Fura-2/AM显微荧光检测技术,检测肌细胞内[Ca2+]i在受到激动剂刺激后的浓度变化;比较不同浓度地塞米松预处理后10min与对照组之间游离钙上升情况的区别。用Western blot方法,分析气道平滑肌细胞内抑制型磷脂酶C(phospho-PLCβ-ser1105)含量的变化。设立RU486及CHX对照组,排除基因组作用在该反应中的影响。结果:GCs温浴10min,能够明显降低乙酰胆碱引起的ASMCs细胞内[Ca2+]i峰值。并能够明显上调ASMCs内抑制型PLC含量。这些反应不受RU486和CHX影响。结论:GCs能够通过非基因组作用快速抑制刺激物引起的气道平滑肌的收缩反应,这一效应的实现可能是通过抑制PLC分子活性,使其下游的[Ca2+]i浓度降低实现的。     

Effect of indomethacin on force responses and sarcoplasmic reticulum function in skinned skeletal muscle fibers and cytosolic [Ca2+] in myotubes

In this study, the effects of phospholipase A2 (PLA2) inhibitors on excitation-contraction coupling (ECC) and sarcoplasmic reticulum (SR) function were examined in skinned extensor digitorum longus (EDL) muscle fibers of the rat. The nonspecific PLA2 inhibitor indomethacin (200 microM) significantly increased the peak (approximately 2-fold, P = 0.02) and the width (approximately 6-fold, P = 0.008) of depolarization-induced force responses (DIFRs) elicited in the fibers (n = 4). Exposure of the skinned EDL fibers to indomethacin (200 microM) (n = 7) and another PLA2 inhibitor quinacrine (200 microM) (n = 5) resulted in the return of large DIFRs after use-dependent rundown. However, aristolochic acid (100 microM), an inhibitor of secretory PLA2, failed to return DIFRs after rundown. Indomethacin did not protect against the loss of DIFRs induced by exposure to elevated myofibrillar [Ca2+]. Indomethacin (200 microM) produced a small but significant increase in the Ca2+ sensitivity of the contractile apparatus of skinned EDL fibers and the maximum force production. Indomethacin (200 microM) also had significant effects on SR function, increasing SR Ca2+ loading in the skinned fibers (117.2 +/- 3.0% of controls, P = 0.0008, n = 8) and inducing intracellular Ca2+ release in isolated intact flexor digitorum brevis (FDB) fibers (n = 7) and C2C12 myotubes (n = 6). These data suggest that intracellular PLA2 may be an important modulator of ECC in skeletal muscle.     

An insulin-sensitive cation channel controls [Na+]i via [Ca2+]o-regulated Na+ and Ca2+ entry.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.     

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.