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At least 883 records · Page 49Linked to original sources
PubMed1997

Effects of hypoxia on [Ca2+]i, pHi and myosin light chain phosphorylation in guinea-pig taenia caeci.

1. Hypoxia (achieved by bubbling with N2 instead of O2) reduces the force of a KCl (40 mM)-induced contracture to approximately 10% of the control value in guinea-pig taenia caeci. The underlying mechanism of this relaxation in response to hypoxia was investigated by measuring the major cell signalling parameters, intracellular Ca2+ concentration ([Ca2+]i) and myosin regulatory light chain (LC20) phosphorylation (MLC-P1), as well as intracellular pH (pHi), a factor often suggested to mediate hypoxic relaxation of muscle. 2. [Ca2+]i, measured using the ratiometric fluorescent dye fura-2, increased when 40 mM KCl was added to physiological saline solution (PSS) (peak value assigned 100%), and the steady state after 15 min was 92.8%. There were no detectable decreases in [Ca2+]i during hypoxia. 3. MLC-Pi, measured using isoelectric focusing-polyacrylamide gel electrophoresis and identified using Western blotting, increased from 9% of the total LC20 in Ca(2+)-free PSS to a peak value of 51% in 40 mM KCl-PSS. The steady-state value in hypoxia of 43% was not significantly different from that in control oxygenated conditions at the same point in time. 4. pHi, measured using the ratiometric fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF), under quiescent conditions (Ca(2+)-free PSS) was 7.23 and increased to 7.36 with 40 mM KCl. After imposition of hypoxia pHi remained unchanged despite the known increase in both lactate content and production. 5. As [Ca2+]i and MLC-Pi, key factors in activation, were not decreased by hypoxia and changes in pHi were minor, hypoxic relaxation in guinea-pig taenia caeci appears to be directly related to energy limitation rather than any oxygen-sensing mechanism.

Animals↗
PubMed1988

Non-linearities in response properties of insect visual cells: an analysis in time and frequency domain.

Intracellularly recorded voltage responses of the visual cells of the blowfly (Calliphora erythrocephala) were analysed in the time and frequency domains. The photoreceptors were stimulated with pulse (impulse), sine, sine-sweep and pseudorandomly (white noise) modulated green light. The blowfly photoreceptor responses, as analysed from the linear transfer functions, seem to arise from a system similar to that of cascaded low-pass filters, with a corner frequency at about 63 Hz (SD +/- 12 Hz). The system is likely to have at least five poles, including one linear second order term, and a pure delay element. Arising from the non-linearities a second harmonic can be seen in the power spectra of responses elicited by sine modulated light. This non-linearity is at least partly explained by the self-shunting property of the membrane voltage response. Light adaptation increases the non-linearities in frequencies lower than 20 Hz, as seen in the decrease of the coherence function with the signal-to-noise ratio remaining constant. Light adaptation also accelerates the transduction process and it appears in the linear transfer function in a form typical to negative feedback. With low stimulus frequencies it causes a 'phase lead'-type non-linearity. In addition, the sine-sweep responses show quite different frequency characteristics in respect of depolarization and repolarization. Lateral inhibition between photoreceptor responses recorded from retinular cell axons in the lamina appears as a drop in gain and as an increasing phase-lag in frequencies from 30 Hz upwards in linear transfer functions. The source of this capacitive-like coupling can be considered to be in the high resistance barriers compartmentalizing the second optic ganglion into discrete anatomical units.

Adaptation, Ocular↗
PubMed2002

The adaptive potential of skeletal muscle fibers.

Mammalian skeletal muscle fibers display a great adaptive potential. This potential results from the ability of muscle fibers to adjust their molecular, functional, and metabolic properties in response to altered functional demands, such as changes in neuromuscular activity or mechanical loading. Adaptive changes in the expression of myofibrillar and other protein isoforms result in fiber type transitions. These transitions occur in a sequential order and encompass a spectrum of pure and hybrid fibers. Depending on the quality, intensity, and duration of the alterations in functional demand, muscle fibers may undergo functional transitions in the direction of slow or fast, as well as metabolic transitions in the direction of aerobic-oxidative or glycolytic. The maximum range of possible transitions in either direction depends on the fiber phenotype and is determined by its initial location in the fiber spectrum.

Adaptation, Physiological↗
PubMed2001

Role of cAMP-dependent protein kinase A activity in endothelial cell cytoskeleton rearrangement.

To examine signaling mechanisms relevant to cAMP/protein kinase A (PKA)-dependent endothelial cell barrier regulation, we investigated the impact of the cAMP/PKA inhibitors Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and PKA inhibitor (PKI) on bovine pulmonary artery and bovine lung microvascular endothelial cell cytoskeleton reorganization. Rp-cAMPS as well as PKI significantly increased the formation of actin stress fibers and intercellular gaps but did not alter myosin light chain (MLC) phosphorylation, suggesting that the Rp-cAMPS-induced contractile phenotype evolves in an MLC-independent fashion. We next examined the role of extracellular signal-regulated kinases (ERKs) in Rp-cAMPS- and PKI-induced actin rearrangement. The activities of both ERK1/2 and its upstream activator Raf-1 were transiently enhanced by Rp-cAMPS and linked to the phosphorylation of the well-known ERK cytoskeletal target caldesmon. Inhibition of the Raf-1 target ERK kinase (MEK) either attenuated or abolished Rp-cAMPS- and PKI-induced ERK activation, caldesmon phosphorylation, and stress fiber formation. In summary, our data elucidate the involvement of the p42/44 ERK pathway in cytoskeletal rearrangement evoked by reductions in PKA activity and suggest the involvement of significant cross talk between cAMP- and ERK-dependent signaling pathways in endothelial cell cytoskeletal organization and barrier regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement.

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.

Adenosine Triphosphate↗
PubMed1997

The role of laminin-5 and its receptors in mammary epithelial cell branching morphogenesis.

In vivo, normal mammary epithelial cells utilize hemidesmosome attachment devices to adhere to stroma. However, analyses of a potential role for hemidesmosomes and their components in mammary epithelial tissue morphogenesis have never been attempted. MCF-10A cells are a spontaneously immortalized line derived from mammary epithelium and possess a number of characteristics of normal mammary epithelial cells including expression of hemidesmosomal associated proteins such as the two bullous pemphigoid antigens, alpha 6 beta 4 integrin and its ligand laminin-5. More importantly, MCF-10A cells readily assemble mature hemidesmosomes when plated onto uncoated substrates. When maintained on matrigel, like their normal breast epithelial cell counterparts, MCF-10A cells undergo a branching morphogenesis and assemble hemidesmosomes at sites of cell-matrigel interaction. Function blocking antibodies specific for human laminin-5 and the alpha subunits of its two known receptors (alpha 3 beta 1 and alpha 6 beta 4 integrin) not only inhibit hemidesmosome assembly by MCF-10A cells but also impede branching morphogenesis induced by matrigel. Our results imply that the hemidesmosome, in particular those subunits comprising its laminin-5/integrin 'backbone', play an important role in morphogenetic events. We discuss these results in light of recent evidence that hemidesmosomes are sites involved in signal transduction.

Antibodies↗
PubMed2005

PRR5 (PSEUDO-RESPONSE REGULATOR 5) plays antagonistic roles to CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) in Arabidopsis thaliana.

In general, the clock (or oscillator) is central to circadian rhythms in many organisms. In the model higher plant Arabidopsis thaliana, the best candidates for clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL), which are homologous Myb-related transcription factors. It is also believed that TOC1 (TIMING OF CAB EXPRESSION 1) is another component of the central oscillator. In this connection, we have been characterizing a small family of proteins, designated ARABIDOPSIS PSEUDO-RESPONSE REGULATOR (PRR1, PRR3, PRR5, PRR7, and PRR9), based on the fact that one of the members (PRR1) is identical to TOC1. Nevertheless, it is not yet certain whether other PRR family members are also implicated in clock function per se. To address this issue, in this study we examined a functional interaction between the CCA1 clock component and one of the PRR family members, PRR5, by employing transgenic lines overexpressing both the CCA1 and PRR5 genes. Evidence will be provided that PRR5 plays an antagonistic role(s) to the putative CCA1 clock component.

Arabidopsis↗
PubMed2006

The transient accumulation of the signaling state of photoactive yellow protein is controlled by the external pH.

The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I(2)'. The pH dependence of the equilibrium between the transient photocycle intermediates I(2) and I(2)' was investigated. The formation of I(2)' from I(2) is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I(1), I(2), and I(2)'. Their spectra were determined using the extrapolated difference method. I(2) and I(2)' have electronic absorption spectra, with maxima at 370 +/- 5 and 350 +/- 5 nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I(1), I(2), and I(2)' intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I(2)' ( approximately 2 ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I(2) and I(2)' is pH dependent with a pK(a) of 6.4 and with I(2)' the main species above this pK(a). Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355 nm at a delay of 20 ms confirm this pK(a) value. I(2) and I(2)' are photoreversed with reversal times of approximately 55 micros and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pK(a) of approximately 6.1. Photoreversal from I(2)' dominates above the pK(a). The transient accumulation of I(2)', the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k(3) for the recovery to the initial dark state also has a pK(a) of approximately 6.3. This equality of the equilibrium and kinetic pK(a) values is not accidental and suggests that k(3) is proportional to [I(2)'].

Bacterial Proteins↗
PubMed2003

Color-specific conditioning effects due to both orange and blue stimuli are observed in a Halobacterium salinarum strain devoid of putative methylatable sites on HtrI.

Behavioral responses of Halobacterium salinarum appear as changes in the frequency of motion reversals. Turning on orange light decreases the reversal frequency, whereas blue light induces reversals. Light pulses normally induce the same response as step-up stimuli. However, anomalous behavioral reactions, including inverse responses, are seen when stimuli are applied in sequence. The occurrence of a prior stimulus is conditioning for successive stimulation on a time scale of the same order of adaptational processes. These prolonged conditioning effects are color-specific. The only adaptation process identified so far is methylation of the transducers, and this could be somehow color-specific. Therefore we tested for the behavioral anomalies in a mutant in which all methylation sites on the transducer have been eliminated. The results show that behavioral anomalies are unaffected by the absence of methylation processes on the transducer.

Adaptation, Physiological↗
PubMed1991

Photomovement in Dunaliella salina: fluence rate-response curves and action spectra.

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450-460 nm. These action spectra are not compatible with the hypothesis that flavo-proteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak at 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.

Cell Movement↗
PubMed2004

Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.

Animals↗
PubMedDate not supplied

[Cyclic nucleotides and membrane electrogenesis in cell differentiation in Neurospora crassa].

Significance of membrane electrogenic system and cyclic nucleotides for developmental regulation in lower eukaryotes was studied in Neurospora crassa. Despite of their morphological uniformity, hyphal cells were differentiated by their electrophysiological parameters--membrane potential and input resistance. The cells maintained regulated electric communication through septal pores. Such electric communication allowed the apical "cell" to compensate the energy requirements for membrane transport by means of the electrogenic mechanism of proximal zone. The photoinduced gene expression related to differentiation of reproductive structures has been shown to be mediated via cAMP. The changes of electric parameters observed in illuminated hyphal cells were controlled by the same photoregulation mechanism but were not involved in direct control of photoinduced gene expression.

Cell Membrane↗
PubMed1993

Light-induced currents in Xenopus oocytes expressing bovine rhodopsin.

1. We have investigated the functioning of bovine rod opsin, which is efficiently synthesized from RNA made by in vitro transcription, following injection into Xenopus oocytes. We found that oocytes expressing the gene for opsin exhibit light-dependent ionic currents only after pigment generation by incubation with 11-cis-retinal. These currents are similar to the endogenous muscarinic acetylcholine (ACh) response of oocytes, but their amplitude is substantially smaller. 2. In order to optimize the conditions for obtaining light-induced currents in RNA-injected oocytes, the native ACh response was examined under several conditions. It was found that elevated external calcium markedly enhances the muscarinic response and that these currents have a non-linear dependence on membrane voltage, increasing substantially with depolarization. 3. Using the optimal conditions for evoking the largest ACh responses, (28 mM [Ca2+]o, 0 mV, omission of serum and Hepes from the media), the light-evoked currents obtained in RNA-injected oocytes were remarkably enhanced, and responses to multiple light stimuli could be obtained. 4. The light response appeared to desensitize, even after long periods of recovery and pigment regeneration. By contrast, the ACh responses continued to appear normal. These results suggest that desensitization of photoresponses expressed in Xenopus oocytes involve changes at early stages of the pathway, resulting in a reduced ability of rhodopsin to couple to the endogenous signalling system.

Acetylcholine↗