Steven J. Ennion

Characterization of human skeletal muscle fibres according to the myosin heavy chains they express

SummaryUsing a method of single muscle fibre analysis, we investigated the presence of RNA transcripts for various isoforms of the myosin heavy chain (MyoHC) gene in histochemically, immunohistochemically and electrophoretically characterized individual muscle fibres (n=65) from adult human vastus lateralis muscle. A cDNA clone isolated in this study was shown to contain the 3′ end of a previously uncharacterized human MyoHC gene which is expressed specifically in human fast IIA muscle fibres and we conclude that this clone contains part of the human fast IIA MyoHC gene. In all the fibres histochemically, immunohistochemically and electrophoretically characterized as containing the previously classified IIB MyoHC (n=23), it was shown that the human equivalent to the rat type IIX MyoHC gene is expressed. This observation was taken to suggest that the previously classified IIB muscle fibres in human muscle express a MyoHC isoform equivalent to the rat IIX, not the IIB, and would therefore be more accurately classified as IIX fibres.

The role of positively charged amino acids in ATP recognition by human P2X1 receptors.

P2X receptors for ATP are a family of ligand-gated cation channels. There are 11 conserved positive charges in the extracellular loop of P2X receptors. We have generated point mutants of these conserved residues (either Lys → Arg, Lys → Ala, Arg → Lys, or Arg → Ala) in the human P2X1receptor to determine their contribution to the binding of negatively charged ATP. ATP evoked concentration-dependent (EC50 ∼ 0.8 μm) desensitizing responses at wild-type (WT) P2X1 receptors expressed inXenopus oocytes. Suramin produced a parallel rightward shift in the concentration response curve with an estimated pKB of 6.7. Substitution of amino acids at positions Lys-53, Lys-190, Lys-215, Lys-325, Arg-202, Arg-305, and Arg-314 either had no effect or only a small change in ATP potency, time course, and/or suramin sensitivity. Modest changes in ATP potency were observed for mutants at K70R and R292K/A (20- and 100-fold decrease, respectively). Mutations at residues K68A and K309A reduced the potency of ATP by >1400-fold and prolonged the time course of the P2X1 receptor current but had no effect on suramin antagonism. Lys-68, Lys-70, Arg-292, and Lys-309 are close to the predicted transmembrane domains of the receptor and suggest that the ATP binding pocket may form close to the channel vestibule.

ADP is not an agonist at P2X1 receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets

ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X1, P2Y1 and P2TAC. Given the low potency of ADP at P2X1 receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re‐examined the activation of P2X1 receptors by ADP following HPLC and enzymatic purification. Native P2X1 receptor currents in megakaryocytes were activated by α,β‐meATP (10 μM) and commercial samples of ADP (10 μM), but not by purified ADP (10–100 μM). Purified ADP (up to 1 mM) was also inactive at recombinant human P2X1 receptors expressed in Xenopus oocytes. Purification did not modify the ability of ADP to activate P2Y receptors coupled to Ca2+ mobilization in rat megakaryocytes. In human platelets, P2X1 and P2Y receptor‐mediated [Ca2+]i responses were distinguished by their different kinetics at 13°C. In 1 mM Ca2+ saline, α,β‐meATP (10 μM) and commercial ADP (40 μM) activated a rapid [Ca2+]i increase (lag time 0.5 s) through the activation of P2X1 receptors. Hexokinase treatment of ADP shifted the lag time by ∼2 s, indicating loss of the P2X1 receptor‐mediated response. A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X1 receptors, whereas ADP is a selective agonist at metabotropic (P2Y1 and P2TAC) receptors.

Synaptic P2X receptors.

Over the past two years, ATP has clearly been shown to act as a co-transmitter with GABA, glycine and probably glutamate in the central nervous system. Our understanding of the ATP-gated P2X receptors is progressing rapidly, and the pharmacology, stoichiometry and subunit combinations of heteropolymeric P2X channels has been substantially elucidated.

Agonist-stimulated internalisation of the ligand-gated ion channel P2X1 in rat vas deferens

Using cell surface biotinylation and Western blotting, we investigated the extent to which native P2X1 receptors in rat vas deferens are internalised after exposure to agonist. Exposure to 100 μM α,β‐meATP 30 min prior and during a 10 min biotinylation period resulted in a ∼50% reduction in the amount of biotinylated P2X1 receptor indicating that activation of the receptor by agonist induces receptor internalisation. Furthermore, biotinylation under saturating conditions suggests that once internalised, a rapid recycling of P2X1 receptor back to the cell surface occurs. The physiological implications of these mechanisms in terms of receptor function are discussed.

P2 purinoceptor‐mediated control of rat cerebral (pial) microvasculature; contribution of P2X and P2Y receptors

1 Purine and pyrimidine nucleotides evoke changes in the vascular tone of medium to large cerebral vessels through the activation of P2 purinoceptors. We have applied P2 receptor drugs to rat pial arterioles and measured changes in arteriole diameter (o.d. 40–84 μm at rest), and recorded currents from arteriolar smooth muscle cells using patch‐clamp techniques. 2 Transient vasoconstrictions and rapidly inactivating currents were evoked by α,β‐methylene ATP (0.1–30 μm) and were sensitive to the P2 receptor antagonists suramin and iso‐PPADS. 3 UTP and UDP (0.1–1000 μm) evoked sustained suramin‐sensitive vasoconstrictions. 4 ATP (0.1–1000 μm) and 2‐methylthioATP (2MeSATP, 300 μm) evoked transient vasoconstrictions followed by sustained vasodilatations. ADP application resulted in only vasodilatation (EC50∼4 μm). Vasodilator responses to ATP, 2MeSATP or ADP were unaffected by suramin (100 μm). 5 RT‐PCR analysis indicated that P2X1–7 and P2Y1,2,6 RNA can be amplified from the pial sheet. Our results provide direct evidence for the presence of functional P2X receptors with a phenotype resembling the P2X1 receptor subtype on cerebral resistance arterioles. The pharmacological properties of the pyrimidine‐evoked responses suggest that a combination of P2Y2‐ and P2Y6‐like receptors are responsible for the sustained vasoconstrictions. It is therefore likely that the nucleotides and their associated receptors are involved in a complicated regulatory system to control cerebral blood pressure.

Functional Characterization of Intracellular Dictyostelium discoideum P2X Receptors

Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA–E) are present in this organism. However, their roles in purinergic signaling are unclear, because dP2XA proved to have an intracellular localization on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knock-out Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. β,γ-Imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na+ but insensitive to copper and the P2 receptor antagonists pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl−-permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knock-out Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB, -C, -D, and -E were found to be intracellularly localized to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium, however, were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signaling.

Altered Expression of Gq/11α Protein Shapes mGlu1 and mGlu5 Receptor-Mediated Single Cell Inositol 1,4,5-Trisphosphate and Ca2+ Signaling

The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP3) and Ca2+ signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP3 and Ca2+, which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP3 and Ca2+ oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of Gq/11α protein expression levels in shaping mGlu1/5 receptor-mediated IP3 and Ca2+ signals, using RNA interference (RNAi). RNAi-mediated knockdown of Gq/11α almost abolished the single-cell increase in IP3 caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca2+ oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca2+ oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of Gqα enhanced IP3 signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP3 and Ca2+ oscillations in control cells were largely insensitive to agonist concentration, increasing Gqα expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca2+ signals, up- or down-regulation of Gq/11α expression alters the threshold for the concentration of glutamate at which a measurable Ca2+ signal could be detected. These experiments indicate that altering Gq/11α expression levels differentially affects spatiotemporal aspects of IP3 and Ca2+ signaling mediated by the mGlu1 and mGlu5 receptors.

Purinergic-mediated Ca2+ influx in Dictyostelium discoideum

Summary The presence of five P2X-like genes (p2xA–E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca2+ influx in response to extracellular ATP and ADP (EC50 = 7.5 μM and 6.1 μM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91 ± 0.04 s, required extracellular Ca2+, were inhibited by Gd3+, modified by extracellular pH and were not affected by deletion of either the single Gβ or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu2+ and Zn2+ inhibited purine-evoked Ca2+ influx with IC50 values of 0.9 and 6.3 μM, respectively. 300 μM Zn2+ completely abolished the initial large rapid rise in intracellular Ca2+ revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.

Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish

To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site.