Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis.

High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 125I-ET-1 with its site was specific, saturable, and time dependent (kobs = 0.019 +/- 0.001 min-1), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4 +/- 9.8 pM and a Bmax of 1084 +/- 118 fmol/mg protein. ET-3 and big-ET-1 (1-38) (human) inhibited 125I-ET-1 binding with IC50 values of 1.85 +/- 1.03 nM and 43 +/- 6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained Ca2+ transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation of glycogenolysis in rat hepatocytes. A 1.8-fold maximal increase in glycogen phosphorylase alpha was observed at 1 pM ET-1, with an EC50 of 0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency of related peptides was similar to that in binding experiments (ET-1 greater than ET-3 greater than big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2+ and a potent glycogenolytic effect. We conclude that ET-1 behaves as a typical Ca2+ mobilizing hormone in liver.

Maitotoxin-induced free Ca changes in single rat hepatocytes.

We report the results using bioluminescent and fluorescent indicators to investigate maitotoxin-induced free Ca changes in single rat hepatocytes. Maitotoxin generated a steadily rising free Ca increase after a long lag period. The free Ca increase was dependent on extracellular calcium and could be antagonised by chelation of extracellular calcium or the inclusion of nickel in the superfusate. Manganese-induced quench of cytoplasmic Fura2 dextran revealed an accelerated rate of calcium entry during the final period of the lag phase, immediately prior to the free Ca increase. Imaging experiments demonstrated a markedly different part of free Ca mobilisation compared with glycogenolytic stimuli. Moreover, the use of a combination of hormonal stimuli and maitotoxin revealed that some cells could exhibit free Ca oscillations despite steadily rising intracellular free Ca level. The significance of these observations in terms of the mechanism of action of maitotoxin and the mechanism of free Ca transient generation is discussed.

Continuous measurements of cytoplasmic ATP in single cardiomyocytes during simulation of the "oxygen paradox".

OBJECTIVE: It is now possible to monitor cytoplasmic ATP in single cardiomyocytes and it has recently been shown that cardiomyocytes exposed for several minutes to metabolic inhibitors undergo an abrupt rigor mediated shortening which coincides with a sudden fall in cytoplasmic ATP, from approximately 150 mumol.litre-1 to a few micromolar or less. The objective of this work was to monitor cytoplasmic ATP during simulated reoxygenation of a poisoned cardiomyocyte. METHODS: Firefly luciferase was injected into a single cell and the light signal generated when luciferin was superfused was monitored. Calibration of the signal is complicated by a transient enhancement of the signal (possibly the result of complex luciferase kinetics), and by uncertainties about cytoplasmic pH. RESULTS: The data indicate that millimolar levels of cytoplasmic ATP are restored within 1-2 min of cyanide removal. CONCLUSIONS: Cytoplasmic free calcium is known to rise after poisoned cells undergo shortening, so it is conceivable that the restoration of cytoplasmic ATP in a cell in which free calcium is at micromolar levels may provide a plausible cellular mechanism for the "oxygen paradox". Reoxygenation induces large amplitude, but slow, oscillations in free calcium which, together with the millimolar levels of ATP indicated here, could provide the stimuli for generating the uncoordinated mechanical forces that are prevalent in the oxygen paradox.

Both activators and inhibitors of protein kinase C promote the inhibition of phenylephrine-induced [Ca2+]i oscillations in single intact rat hepatocytes.

In single isolated rat hepatocytes Ca(2+)-mobilising hormones induce oscillations in cytosolic free Ca2+ ([Ca2+]i) in which the frequency of spiking depends on agonist dose, but the time course of individual spikes depends on the hormone species, rather than agonist concentration. We have previously presented data using sphingosine and staurosporine as evidence of a negative feedback role for protein kinase C (PKC) in the elongation of the falling phase of [Ca2+]i spikes. We show here that the principal effect of three specific PKC inhibitors, namely the bis-indolylmaleimide GF 109203X, the tetracyclic aromatic alkaloid chelerythrine, and a myristoylated PKC pseudosubstrate peptide, that act at different sites on the PKC molecule, is a reduction in, or a complete suppression of, the phenylephrine-induced [Ca2+]i oscillation frequency. These results resemble the effects of activators of PKC and modulators of diacylglycerol (DAG) metabolism. Furthermore, following phorbol ester-induced inhibition of the hepatocyte [Ca2+]i oscillator, the addition of all three of these PKC inhibitors further reduces the [Ca2+]i oscillation frequency, with high concentrations of chelerythrine being the only agent that overcomes this inhibition by phorbol ester. These paradoxical results point to the need for caution in interpreting the effects of protocols involving PKC activators and inhibitors in assessing the feedback control from PKC on cellular [Ca2+]i oscillations.

Agonist-induced oscillations in cytoplasmic free calcium concentration in single rat hepatocytes.

The effects of the alpha 1-adrenergic agonist phenylephrine and the peptide hormones angiotensin II and arg8-vasopressin on cytoplasmic free calcium concentration were investigated in single rat hepatocytes microinjected with the photoprotein aequorin. Hepatocytes responded to physiological concentrations of the glycogenolytic agonists with a series of repetitive Ca transients. In each transient free Ca rose in 2-3s to above 600 nM from a resting level of 200 nM. Transient duration depended on the agonist and ranged from approximately 7s for phenylephrine to approximately 15s for angiotensin. Transient frequency, but not shape or size, depended on agonist concentration. The period ranged from less than 20s to several minutes. We suggest that the frequency of the Ca transients is the principal determinant of the amplitude of the cellular response to calcium-mobilizing agonists.

The hepatocyte calcium oscillator.

Hepatocytes stimulated with calcium-mobilising agonists generate free Ca transients whose frequency is modulated by hormone concentration. Importantly, the time-course of individual free Ca transients is independent of agonist dose but does change with agonist species. A receptor-controlled model in which protein kinase C provides negative feedback directed against the different receptors, or receptor-specific G proteins, has been proposed in order to explain the agonist-specificity of the falling phase of the free Ca spikes. Here we show further evidence, from mixing of hormones and from the effects of elevated cAMP, of receptor-specific information within the spikes.

Effects on the hepatocyte [Ca2+]i oscillator of inhibition of the plasma membrane Ca2+ pump by carboxyeosin or glucagon-(19-29).

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the receptor species activated; the variability results in differences in the rate of fall of [Ca2+]i from its peak. It is conceivable that the plasma membrane Ca(2+)-ATPase (PM Ca2+ pump) may have an important role in the mechanism underlying agonist specificity. It has recently been shown that an esterified form of carboxyeosin, an inhibitor of the red cell PM Ca2+ pump, is suitable for use in whole cell studies. Glucagon-(19-29) (mini-glucagon) inhibits the Ca2+ pump in liver plasma membranes, mediated by Gs. We show here that carboxyeosin and mini-glucagon inhibit Ca2+ efflux from populations of intact rat hepatocytes. We show that carboxyeosin and mini-glucagon enhance the frequency of oscillations induced by Ca(2+)-mobilizing agonists in single hepatocytes, but do not affect the duration of individual transients. Furthermore, we demonstrate that inhibition of the hepatocyte PM Ca2+ pump enables the continued generation of [Ca2+]i oscillations for a prolonged period following the removal of extracellular Ca2+.

Thimerosal enhances agonist-specific differences between [Ca2+]i oscillations induced by phenylephrine and ATP in single rat hepatocytes.

Single rat hepatocytes, microinjected with the Ca(2+)-sensitive photoprotein aequorin, respond to agonists acting through the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]i). The duration of [Ca2+]i transients generated is characteristic of the stimulating agonist; the differences lie in the rate of fall of [Ca2+]i from its peak. We considered that differential sensitivity of the InsP3 receptor may underlie agonist specificity. The thiol reagent, thimerosal, is known to increase the sensitivity of the Ca2+ stores to InsP3 by increasing the affinity of the InsP3 receptor for InsP3 in rat hepatocytes. We show here that a low dose of thimerosal (1 microM), insufficient alone to elevate [Ca2+]i, potentiates [Ca2+]i oscillations induced by phenylephrine or ATP in single, aequorin-injected, rat hepatocytes. Moreover, thimerosal enhances both the frequency and amplitude of phenylephrine-induced oscillations, whereas, in contrast, ATP-induced oscillations undergo an increase in the duration of the falling phase of individual [Ca2+]i transients. Thimerosal, therefore, enhances, rather than eliminates, agonist-specific differences in the hepatocyte [Ca2+]i oscillator.

Modulation of free Ca oscillations in single hepatocytes by changes in extracellular K+, Na+ and Ca2+.

Single rat hepatocytes, microinjected with the calcium-sensitive photoprotein aequorin, when stimulated with either phenylephrine or arg8-vasopressin exhibit agonist-specific oscillations in cytosolic free calcium levels (free Ca). In the majority of the cells examined adding excess potassium chloride, sodium chloride or choline chloride abolished transient behaviour. However, in cells that continued to oscillate the transient parameters were subtly modified by these treatments. In experiments using phenylephrine as the agonist, adding excess potassium chloride to the superfusate significantly reduced transient length, increased the rate of transient rise and reduced the smoothed peak free Ca level without significantly altering the intertransient resting free Ca level or the falling time constant. The possible mechanisms by which these alterations may occur are discussed.

Inositol-phosphoglycan inhibits calcium oscillations in hepatocytes by reducing calcium entry.

Inositol-phosphoglycan (IPG) is a putative mediator of insulin action that has been shown to affect numerous biochemical processes. IPG, prepared from liver membranes, promptly inhibited phenylephrine- or vasopressin-induced [Ca2+]i oscillations when perfused over Fura-2-dextran injected rat hepatocytes. An antibody to IPG suppressed the inhibitory effect of insulin on the [Ca2+]i oscillations. Measurement of the rate of quench of cytoplasmic Fura-2 by extracellular Mn2+ showed that Ca2+ entry occurred continuously in the unstimulated cell and was not affected by phenylephrine or vasopressin. IPG, specifically, almost completely abolished the Mn2+ quench rate. Elevated extracellular [Ca2+] reversed the inhibitory effect of IPG on [Ca2+]i oscillations. We conclude that IPG inhibits the hepatocyte Ca2+ oscillatory by reducing the continuous Ca2+ influx that is required to sustain oscillations in [Ca2+]i.

Effect of perturbing diacylglycerol metabolism on cytosolic free Ca2+ oscillations induced in single hepatocytes.

Single rat hepatocytes microinjected with aequorin show free Ca oscillations when stimulated with Ca(2+)-mobilizing hormones. We show here that an inhibitor of diacylglycerol kinase (R 59 022) and an analogue of native diacylglycerol (diC8) inhibit free Ca oscillations induced by phenylephrine and vasopressin. These results agree with a negative feedback effect of protein kinase C on free Ca oscillations.

Calcium transients in single adrenal chromaffin cells detected with aequorin.

The effect of 55 mM K+ and nicotine on intracellular free calcium was monitored in bovine adrenal chromaffin cells microinjected with aequorin. In contrast to results with quin 2, which suggested that stimulation of chromaffin cells resulted in sustained rises in free calcium, aequorin measurements showed that 55 mM K+ and nicotine resulted in a transient (60-90 s) elevation of free calcium. The peak free calcium and duration of the transient elicited by nicotine were dose-dependent. The concentration of nicotine (10 microM) giving a maximal secretory response gave a peak rise in free calcium of up to 1 microM. 55 mM K+ which only releases 30% of the catecholamine released by 10 microM nicotine generated a calcium transient indistinguishable from that due to 10 microM nicotine. These results support the idea that nicotine agonists generate an alternative second messenger in addition to the rise in free calcium.

Adenine dinucleotide-mediated cytosolic free Ca2+ oscillations in single hepatocytes.

Single rat hepatocytes microinjected with aequorin respond to Ca(2+)-mobilizing agonists, including ADP and ATP, with oscillations in cytosolic free Ca2+. We show here that single rat hepatocytes also respond to the adenine dinucleotides Ap3A and Ap4A with Ca2+ oscillations which resemble those induced by ADP and ATP.

Tri-iodothyronine increases intra-cellular calcium levels in single rat myocytes.

We have studied the effects of acute administration of triiodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to less than 1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80 mM KCl, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150 +/- 56 nM (mean +/- SD, n = 14) by 127 +/- 47 nM on depolarization with 80 mM KCl. Application of T3 (1-100 nM) led to an increase (P less than 0.05) in [Ca2+]i (mean amplitude of 152 +/- 35 nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when 100 nM T3 was applied (median and range; 6.8, 0-14 min) than when 1 nM T3 was used (16, 7.0-56.1 min) (P less than 0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.

Actions of ADP, but not ATP, on cytosolic free Ca2+ in single rat hepatocytes mimicked by 2-methylthioATP.

1. Aequorin-injected, single rat hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway, including ADP and ATP. We have previously described differences in the [Ca2+]i responses of aequorin-injected hepatocytes to ADP and ATP. 2. The effects of the phosphorothioate analogue of ATP, 2-methylthioATP (2-meSATP), have been examined on single rat hepatocytes. This analogue is belived to be the most potent agonist at the P2Y1 subclass of purinoceptor. 3. The [Ca2+]i transients induced by 2-meSATP were indistinguishable from those induced by ADP, and in contrast to those induced by ATP. 4. At hig concentrations, 2-meSATP and ADP both induced transients at high frequency. In contrast, hepatocytes responded to high concentrations of ATP with an initial rapid rise in [Ca2+]i, followed by a slowly decaying fall. 5. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both 2-meSATP- and ADP-induced [Ca2+]i transients; the peak height and frequency of transients were enhanced. ATP-induced transients, however, underwent either an increase in duration or conversion into a sustained rise in [Ca2+]i. 6. ATP-induced transients were specifically potentiated by the co-addition of alpha, beta-methyleneATP, whereas 2-meSATP- and ADP-induced transients were unaffected by this treatment. 7. We conclude that 2-meSATP acts at the same receptor as ADP on rat hepatocytes, and that this is distinct from teh receptor(s) mediating the effects of ATP.

Preservatives in the vehicle of naloxone: pharmacological effects.

The pharmacological activity of the preservatives methyl and propyl hydroxybenzoate, until recently components of the vehicle of naloxone (Narcan), was investigated in vitro. This vehicle produced reversible, concentration-dependent relaxation of guinea pig trachea, not mediated via adrenergic or cholinergic receptors, prostanoid activity or phosphodiesterase inhibition. Sensitivity of the tissue to calcium-induced contraction was decreased. In single isolated rat hepatocytes, surface receptor stimulation elicits repetitive transient rises in intracellular free calcium measured with the photoprotein aequorin. The vehicle reversibly inhibited these transients. These observations suggest that the effect of hydroxybenzoates may be mediated via a perturbation of intracellular calcium-related processes.

Measurements of the free calcium concentration of single quiescent human fibroblasts before and after serum addition.

We have developed the technique for injecting aequorin into cells so that it is now possible to measure the free calcium concentration of single mammalian somatic cells. Using this method, the free calcium concentration of serum-starved quiescent human fibroblasts was determined to be 3.3 x 10(-7) M. After feeding the cells with serum a significant increase in free calcium was observed in some cells. The exact cause of such an increase is unclear but it may indicate that a large increase in free calcium is an important event in the stimulation of cell growth.

Cytoplasmic free calcium and amoeboid movement.

Measurements with the Ca2+ -sensitive photoprotein aequorin show that locomotion in the amoeba Chaos carolinense occurs without changes in the aequorin signal and that not more than 0.025% of the cytoplasm can exist at the micromolar threshold concentration for contraction. The results do not support the hypothesis that cytoplasmic streaming is under the control of changes in the cytoplasmic free Ca2+ concentration.

Calcium oscillations: phenomena, mechanisms and significance.

Many hormones that mobilise intracellular calcium via inositol 1,4,5 trisphosphate induce oscillations in cytoplasmic free Ca. Two basic oscillatory patterns occur: quasi-sinusoidal oscillations and repetitive free Ca transients. The mechanisms responsible for generating these oscillations are not clear; calcium-induced calcium release, interplay between two intracellular calcium pools and repetitive generation of InsP3 are discussed. The significance of different oscillatory patterns induced by different agonists in the same cell is emphasised, and mechanisms by which the oscillators may retain-receptor specific information are proposed, such as negative feedback onto receptors or G-proteins by protein kinase C. Reasons why cells generate free Ca oscillations and possible consequences such as oscillations in downstream pathways are explored. The possibility that pathological conditions such as aluminium toxicity are exerted through distortion of oscillatory free Ca signalling is raised.