C. Jane Dixon

Regulation of epidermal homeostasis through P2Y2 receptors

Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. Reverse transcriptase‐polymerase chain reaction (RT–PCR) revealed expression of mRNA for the G‐protein‐coupled, P2Y2 receptor in primary cultured human keratinocytes. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPγS were found to stimulate the proliferation of keratinocytes. Using a real‐time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies

Human osteoblasts responded to the application of extracellular nucleotides, acting at P2‐receptors, with increases in cytosolic free calcium concentration ([Ca2+]i). In populations of human osteoblasts, adenosine 5′‐diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5′‐triphosphate (ATP). ATP and uridine 5′‐triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested. However, from the results of single cell studies with ADP (and 2‐methylthioATP (2‐meSATP)) two groups of cells were delineated: one group responded to ADP (or 2‐meSATP) with a rise in [Ca2+]i indistinguishable from that evoked by ATP; whereas the second group failed completely to respond to ADP (or 2‐meSATP). Therefore heterogeneity of receptor expression exists within this population of human osteoblasts. The limited distribution of the ADP‐responsive receptor underlies the small response to ADP, compared with ATP, recorded in populations of human osteoblasts. This heterogeneity may reflect differences in the differentiation status of individual cells.

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 (P2Y1, P2Y2, P2Y4 and P2Y6). The effects of UTP have been examined on single aequorin‐injected rat hepatocytes. The [Ca2+]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 [Ca2+]i transients. UDP, an agonist at the P2Y6 receptor, failed to induce transients in hepatocytes, indicating that functional P2Y6 receptors coupled to increased [Ca2+]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 P2Y2 rather than P2Y4 receptors. Co‐application of 30 μM 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 P2Y1 and P2Y2 receptors by rat hepatocytes.

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

Single rat hepatocytes microinjected with aequorin respond to Ca2+‐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.

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+.

Evidence that 2-methylthioATP and 2-methylthioADP are both agonists at the rat hepatocyte P2Y1 receptor

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2‐methylthioADP (2‐MeSADP) is a selective agonist for the P2Y1 receptor. The agonist action of 2‐MeSATP at the P2Y1 receptor has recently been questioned. The effects of both 2‐MeSADP and 2‐MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca2+ indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2‐MeSADP and 2‐MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2‐MeSATP with an ATP‐regenerating system to remove contaminating 2‐MeSADP did not abolish its agonist activity. Application of the P2Y1 antagonist, adenosine‐3′‐phosphate‐5′‐phosphate (A3P5P) inhibited the transients induced by both 2‐MeSADP and 2‐MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2‐MeSADP and 2‐MeSATP are agonists at the rat hepatocyte P2Y1 receptor.

A Role for Akt in Epidermal Growth Factor-Stimulated Cell Cycle Progression in Cultured Hepatocytes: Generation of a Hyperproliferative Window after Adenoviral Expression of Constitutively Active Akt

Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.

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.

Aequorin Measurements of Cytosolic Ca 2

Aequorin is a 21 kDa luminescent protein, present in photocytes of the jellyfish Aequorea forskalea, which generates blue-green light upon binding Ca2+ ions. Aequorin can be used to measure cytosolic free Ca2+ ([Ca2+]i) in single cells by injecting the purified protein, and then measuring the light emitted from the cell. This protocol describes the transfer of a single cell into an optically- flat capillary (microslide), microinjection of aequorin into the cell, and detection of the signal. Inside the microslide, the cell is suspended within an agarose gel. Such an arrangement enables access to the cell for microinjection, and facilitates transfer of the cell between apparatus throughout the experimental procedure. During recording of the signal from the aequorin- injected cell, culture medium containing agonists etc. is superfused over the micro slide (see subprotocol 6). Inside the agarose gel, the cell is protected from the flow of medium, but since the diffusion path through the agarose to the cell is only ~50 µm, the cell is readily accessed by the superfused culture medium and agonists.