Anne K. Green

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+.

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 believed 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 high 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 α,β‐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 the receptor(s) mediating the effects of ATP.

Phenylalanine hydroxylation in isolated rat kidney tubules

1. Phenylalanine hydroxylation has been demonstrated to occur in isolated rat kidney tubules under physiological conditions. 2. The hydroxylation flux response is hyperbolic with apparent Km and Vmax values of ca 85 microM phenylalanine and 49 nmol tyrosine formed/mg dry wt per hr respectively. 3. Hydroxylation in kidney tubules is substantially less sensitive to effectors of cyclic AMP turnover and Ca2+ mobilization than phenylalanine hydroxylation in isolated liver cells.

The effect of vanadate upon the expression of phenylalanine hydroxylase in streptozotoxin-diabetic rat liver

Induction of diabetes in rats is associated with a significant elevation in the phenylalanine hydroxylating capacity of the liver. This phenomenon reflects an increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine hydroxylase-specific mRNA. These changes can be abolished by insulin-dependent control of diabetes. We show here that the control of diabetes by oral administration of sodium orthovanadate will also nullify the diabetes-related alterations in phenylalanine hydroxylase expression. In addition, diabetes-induced changes in the extent of phosphorylation of phenylalanine hydroxylase are reversed by either insulin or vanadate treatment in vivo. These treatments also abolished the diabetes-related, approx. 30-fold, decrease in glucagon sensitivity of phenylalanine hydroxylation in isolated liver cells.

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.