Caffeine inhibits cytosolic calcium oscillations induced by noradrenaline and vasopressin in rat hepatocytes.
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ABSTRACT:
SUBMITTER: Combettes L
PROVIDER: S-EPMC1137049 | biostudies-other | 1994 Aug
REPOSITORIES: biostudies-other
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Project description:UNLABELLED:The process of capacitative or store-operated Ca(2+) entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca(2+) stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca(2+) entry is readily demonstrated with protocols that provide extensive Ca(2+) store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca(2+) influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca(2+) oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca(2+) oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca(2+) entry but only partially reduced the frequency of Ca(2+) oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. CONCLUSION:Our findings establish that store-operated Ca(2+) entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent.
| S-EPMC2808042 | biostudies-literature
Project description:Previously it has been shown that injecting a cytosolic sperm protein factor into mammalian eggs induces sustained repetitive transients of cytosolic free Ca2+ ([Ca2+]i), or [Ca2+]i oscillations [Swann (1990) Development 110, 1295-1302]. These sperm-factor (SF)-induced [Ca2+]i oscillations are similar to those seen at fertilization. Here we demonstrate that injecting the same cytosolic extracts of mammalian sperm into single rat hepatocytes induces a series of [Ca2+]i oscillations, as measured by aequorin luminescence. SF injection into hepatocytes induced [Ca2+]i oscillations that were of longer duration, lower frequency and greater amplitude than those seen with the Ins (1,4,5)P3-generating agonist phenylephrine. The SF-induced [Ca2+]i responses appeared to be due to internal release of Ca2+, since transients could occur in Ca(2+)-free media. Addition of the phorbol ester phorbol 12,13-dibutyrate (PDBu) at low concentrations did not inhibit the SF-induced [Ca2+]i oscillations; high concentrations of PDBu led to a sustained increase in [Ca2+]i concentrations. These data demonstrate that sperm contain a protein factor capable of inducing a characteristic series of [Ca2+]i oscillations in a somatic cell, the hepatocyte. Along with previous observations in dorsal root ganglion neurons, the data suggest a widespread efficacy of the factor in triggering Ca2+ oscillations.
| S-EPMC1216917 | biostudies-other
Project description:Spontaneous low-frequency oscillations (LFOs) of blood-oxygen-level-dependent (BOLD) signals are used to map brain functional connectivity with functional MRI, but their source is not well understood. Here we used optical imaging to assess whether LFOs from vascular signals covary with oscillatory intracellular calcium (Ca(2+)i) and with local field potentials in the rat's somatosensory cortex. We observed that the frequency of Ca(2+)i oscillations in tissue (∼0.07 Hz) was similar to the LFOs of deoxyhemoglobin (HbR) and oxyhemoglobin (HbO2) in both large blood vessels and capillaries. The HbR and HbO2 fluctuations within tissue correlated with Ca(2+)i oscillations with a lag time of ∼5-6 s. The Ca(2+)i and hemoglobin oscillations were insensitive to hypercapnia. In contrast, cerebral-blood-flow velocity (CBFv) in arteries and veins fluctuated at a higher frequency (∼0.12 Hz) and was sensitive to hypercapnia. However, in parenchymal tissue, CBFv oscillated with peaks at both ∼0.06 Hz and ∼0.12 Hz. Although the higher-frequency CBFv oscillation (∼0.12 Hz) was decreased by hypercapnia, its lower-frequency component (∼0.06 Hz) was not. The sensitivity of the higher CBFV oscillations to hypercapnia, which triggers blood vessel vasodilation, suggests its dependence on vascular effects that are distinct from the LFOs detected in HbR, HbO2, Ca(2+)i, and the lower-frequency tissue CBFv, which were insensitive to hypercapnia. Hemodynamic LFOs correlated both with Ca(2+)i and neuronal firing (local field potentials), indicating that they directly reflect neuronal activity (perhaps also glial). These findings show that HbR fluctuations (basis of BOLD oscillations) are linked to oscillatory cellular activity and detectable throughout the vascular tree (arteries, capillaries, and veins).
| S-EPMC4217406 | biostudies-literature
Project description:In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterized. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).
| S-EPMC6152895 | biostudies-literature
Project description:Single rat hepatocytes microinjected with the photoprotein aequorin generate oscillations in the cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. We show here that, in single rat hepatocytes, bovine growth hormone (bGH) is able to induce [Ca2+]i oscillations which display similarities with oscillations induced by phenylephrine. Thus the rate of rise of intracellular Ca2+ in each oscillation closely resembles that induced by Ins(1,4,5)P3-mediated agonists. However, the duration of bGH-induced oscillations increases with agonist concentration, in contrast to phenylephrine-induced oscillations, which undergo an increase in frequency as the agonist concentration is raised, without any increase in the duration of individual oscillations.
| S-EPMC1216938 | biostudies-other
Project description:Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES-expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES-expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells.Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle.
| S-EPMC3572840 | biostudies-literature
Project description:BACKGROUND: Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. METHODS: Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. RESULTS: Positive RT-PCR results were obtained for nAChR subunits ?3, ?5, ?9, ?10, ?1, and ?2, with most stable expression being noted for subunits ?9, ?10, ?1, and ?2. Notably, mRNA coding for subunit ?7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 ?M), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by ?-bungarotoxin and did not depend on the presence of extracellular calcium. CONCLUSIONS: Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.
| S-EPMC2955664 | biostudies-literature
Project description:We show here, by aequorin measurements in single isolated rat hepatocytes, that elevation of cyclic AMP, by dibutyryl cyclic AMP, forskolin or glucagon, has different effects on oscillations in cytosolic concentration of free Ca2+ ('free Ca') induced by phenylephrine or vasopressin. Elevated cyclic AMP does not itself induce free Ca oscillations, but enhances both the peak free Ca and the frequency of spikes induced by phenylephrine. In contrast, elevated cyclic AMP has no effect on peak free Ca of vasopressin-induced spikes, but markedly prolongs the falling phase, with the result that spiking frequency (peak to peak) falls, although the period between spikes of resting free Ca is usually decreased. The data provide another example of receptor-specific information being retained in the oscillator mechanism, with implications for models of the hepatocyte calcium oscillator.
| S-EPMC1132496 | biostudies-other
Project description:The action of alpha 1-adrenergic agonists (noradrenaline in the presence of propranolol), vasopressin and angiotensin on the intracellular free Ca2+ concentration, [Ca2+]i, was determined by using the fluorescent dye quin2 in isolated rat liver cells. In the presence of external Ca2+ (1.8 mM), 1 microM-noradrenaline induced an increase in [Ca2+]i up to about 800 nM without apparent delay, whereas 10 nM-vasopressin and 1 nM-angiotensin increased [Ca2+]i to values higher than 1500 nM with a lag period of about 6s. The successive addition of the hormones and of their specific antagonists indicated that the actions of the three Ca2+-mobilizing hormones occurred without apparent desensitization (over 6 min) and via independent receptors. The relative contributions of internal and external Ca2+ pools to the cell response were determined by studying the hormone-mediated [Ca2+]i increase and glycogen phosphorylase activation in low-Ca2+ media (22 microM). In this medium: (1) [Ca2+]i was lowered and the hormones initiated a transient instead of a sustained increase in [Ca2+]i; subsequent addition (2 min) of a second hormone promoted a lesser increase in [Ca2+]i; in contrast, the subsequent addition (2 min) of Ca2+ (1.8 mM) caused [Ca2+]i to increase to a value close to that initiated by the hormone in control conditions, the amplitude of the latter response being dependent on the concentration of Ca2+ added to the medium; (2) returning to normal Ca2+ (1.8 mM) restored the resting [Ca2+]i and allowed the hormone added 2 min later to promote a large increase in [Ca2+]i whose final amplitude was also dependent on the concentration of Ca2+ added beforehand. Similar results were found when the same protocol was applied to the glycogen phosphorylase activation. It is concluded that Ca2+ influx is required for a maximal and sustained response and to reload the hormone-sensitive stores.
| S-EPMC1145024 | biostudies-other
Project description:Single aequorin-injected hepatocytes respond to agonists acting via the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]free). The duration of [Ca2+]free transients is characteristic of the stimulating agonist. We have previously reported that ADP and ATP, which are believed to act through a single P(2y)-purinoceptor species, induce very different oscillatory [Ca2+]free responses in the majority of hepatocytes. We have interpreted these data as evidence for two separate Ca(2+)-mobilizing purinoceptors for these nucleotides. We show here that the elevation of intracellular cyclic AMP concentration, by the co-application of either dibutyryl cyclic AMP or 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)butyryl]- forskolin (L858051), exerts different modulatory effects on [Ca2+]free oscillations induced by ADP and ATP in single rat hepatocytes. Elevated intracellular cyclic AMP levels enhance the frequency and peak [Ca2+]free of transients induced by ADP. In contrast, the elevation of intracellular cyclic AMP levels in hepatocytes producing [Ca2+]free oscillations in response to ATP stimulates either an increase in the duration of transients or a sustained rise in [Ca2+]free. The data illustrate a further difference between the oscillatory [Ca2+]free responses of hepatocytes to ADP and ATP, thus further arguing against ADP and ATP acting via a single purinoceptor species.
| S-EPMC1137322 | biostudies-other