Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors.

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.

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.

Evidence for two Ca2(+)-mobilizing purinoceptors on rat hepatocytes.

Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration.

Aluminium perturbs oscillatory phosphoinositide-mediated calcium signalling in hormone-stimulated hepatocytes.

Aluminium is known to be toxic to cells from bone, brain and bone marrow but the molecular target(s) affected by Al3+ are not known. We show here that Al3+ disrupts the oscillatory free Ca2+ responses of hepatocytes exposed to the Ca2(+)-mobilizing agonist phenylephrine. Al3+ initially increases the frequency of the oscillations and later induces broad Ca2+ spikes lasting several minutes. These broad spikes persist after removal of both agonist and Al3+ from the medium. In the absence of agonist, Al3+ has no effect on free Ca2+. The data suggest that some component(s) of the receptor-phosphoinositide-Ca2+ signalling pathway might be the site at which Al3+ exerts toxic effects.

Inhibitors of protein kinase C prolong the falling phase of each free-calcium transient in a hormone-stimulated hepatocyte.

Many cells generate oscillations in cytoplasmic free Ca2+ concentration ('free Ca') when stimulated with Ca-mobilizing hormones. The frequency of repetitive free-Ca transients in a rat hepatocyte is a function of hormone concentration and can be depressed by phorbol esters. We show here that the protein kinase C (PKC) inhibitors staurosporine and sphingosine can reverse the effects of phorbol dibutyrate on the frequency of free-Ca transients induced by phenylephrine or vasopressin. An important feature of the hepatocyte free-Ca oscillator is that the transient's time course, particularly the rate of fall of free Ca from peak to resting, depends on the species of agonist, and is measurably different for phenylephrine, vasopressin, angiotensin II or ATP. We show here that the rate of fall of free Ca in transients induced by phenylephrine or vasopressin is markedly decreased after treatment of the cells with a PKC inhibitor. A receptor-controlled oscillator model is discussed, in which PKC provides negative feedback during the falling phase of free-Ca transients.

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.

Fluoroaluminate mimics agonist application in single rat hepatocytes.

Single rat hepatocytes microinjected with the photoprotein aequorin were stimulated with glycogenolytic agonists or low concentrations of fluoroaluminate. Both protocols resulted in the generation of oscillations in cytosolic free Ca2+ levels from a resting value of approx. 200 nM and peaking at over 600 nM. However, oscillations induced by receptor-dependent agonists were more regular in both frequency and time course than those induced by direct activation of G-proteins. The role of G-proteins in the generation of repetitive free Ca2+ oscillations is discussed.

Receptor-mediated endocytosis of asialoglycoproteins and diferric transferrin is independent of second messengers.

The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.

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.

Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.

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.

Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes.

In the stressed animal, the vasoactive hormones vasopressin and angiotensin-II and the neurotransmitter noradrenaline induce liver cells to release glucose from glycogen. The intracellular signal that links the cell-surface receptors for noradrenaline (alpha 1) and vasoactive peptides to activation of glycogenolysis is known to be a rise in the cytoplasmic concentration of free calcium ions (free Ca). The receptors for these agonists induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate, a minor plasmalemma lipid, to produce inositol trisphosphate and diacylglycerol. Inositol trisphosphate has been shown to mobilize intracellular calcium in hepatocytes. We show here, by means of aequorin measurements in single, isolated rat hepatocytes, that the free Ca response to these agonists consists of a series of transients. Each transient rose within 3 s to a peak free Ca of at least 600 nM and had a duration of approximately 7 s. The transients were repeated at intervals of 0.3-4 min, depending on agonist concentration. Between transients, free Ca returned to the resting level of approximately 200 nM. Clearly, the mechanisms controlling free Ca in hepatocytes are more complex than hitherto suspected.