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.
We have previously described differences in the oscillatory responses of cytosolic free Ca2+ concentration ([Ca2+]i) in hepatocytes to ADP and ATP, which we have interpreted as evidence that these two nucleotides are acting at distinct receptors. We show here that ADP- and ATP-induced oscillations are differentially sensitive to application of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB). ADP-induced [Ca2+]i oscillations are abolished by low concentrations of PDB (5-10 nM), whereas ATP-induced oscillations of long duration are refractory to PDB, even at greatly elevated concentrations (100 nM). The data illustrate a further difference in the actions of ADP and ATP, strengthening the argument that these agonists are not acting at the same receptor on rat hepatocytes.
Single rat hepatocytes microinjected with aequorin generate oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. The duration of these transients has been shown to be characteristic of the stimulating agonist, so that transients of very different duration can be induced in the same individual hepatocyte by different agonists. In a previous study we have shown that ADP and ATP, which are believed to act through a single P2y-purinoceptor species, elicit very different [Ca2+]i responses in most of the hepatocytes. We have interpreted this as evidence for two Ca(2+)-mobilizing purinoceptors. The methylated derivative of ATP, adenosine 5'-[alpha beta-methylene]-triphosphate (pp[CH2]pA), is only a weak P2y-purinoceptor agonist. When 100 microM pp[CH2]pA was supplied to aequorin-injected hepatocytes, there was no effect on [Ca2+]i. However, 25 microM pp[CH2]pA co-supplied with ATP causes a potentiation of the [Ca2+]i response in most of the hepatocytes. The effect was specific for ATP-induced transients; [Ca2+]i transients induced by other agonists, and importantly by ADP, were not affected by addition of pp[CH2]pA. This further illustrates differences in the actions of ADP and ATP, strengthening the argument for separate receptors for these nucleotides.
Diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) induce distinctive patterns of [Ca2+]i oscillations in single rat hepatocytes. We show here that [Ca2+]i oscillations induced by Ap3A and ADP are indistinguishable and that [Ca2+]i oscillations induced by Ap4A closely resemble those induced by ATP. These similarities embrace the following: (1) ADP and Ap3A invariably induce [Ca2+]i transients of short duration (approx. 9 s). Ap4A, like ATP, can induce, depending upon the individual cell, either transients of short duration (approx. 9 s), transients of much longer duration or a mixture of short and long transients within a single response. We show here that the pattern of oscillations induced by Ap4A is similar to that induced by ATP in the same hepatocyte. (2) Elevated intracellular cyclic AMP concentration modulates Ap3A-induced transients, like ADP-induced transients, through an increase in both the peak [Ca2+]i and the frequency of the transients. In contrast, Ap4A-induced transients, like ATP-induced transients, develop an increased duration or a sustained rise in [Ca2+]i, with no rise in peak [Ca2+]i. (3) Ap3A-induced transients, like ADP-induced transients, are abolished by low concentrations of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB; 5-10 nM), whereas long Ap4A-induced transients, like long ATP-induced transients, are refractory to high concentrations of PDB (100 nM). We propose that the [Ca2+]i oscillations induced in rat hepatocytes by Ap3A are mediated by the same purinoceptor that mediates the effects of ADP, whereas the oscillations induced by Ap4A are mediated by the same purinoceptor(s) that mediate the effects of ATP.
A monoclonal antibody (PH 7), which recognizes the phosphorylated form of phenylalanine hydroxylase from human liver, has been used for the analysis of the enzyme in crude cell extracts from rat. In immunoblot analyses of rat liver cell extracts, the extent of binding of PH 7 closely correlates with the phosphorylation state of phenylalanine hydroxylase, as judged by [32P]Pi incorporation. These observations have made possible the rapid non-radioactive quantification of hormonal effects on phenylalanine hydroxylase phosphorylation state. In particular, the glucagon-dependent phosphorylation of phenylalanine hydroxylase in liver cells was investigated. Epidermal growth factor was shown to modulate this process. In addition, this technique was used to demonstrate, for the first time, that dibutyryl cyclic AMP, unlike the Ca2" ionophore A23187, stimulates the phosphorylation of phenylalanine hydroxylase in isolated kidney tubules from rat.
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