Robert M. Drummond

Quantitative Analysis of Spontaneous Mitochondrial Depolarizations

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.

P2X7 purinoceptor expression in Xenopus oocytes is not sufficient to produce a pore-forming P2Z-like phenotype

The purinergic rP2X7 receptor expressed in a number of heterologous systems not only functions as a cation channel but also gives rise to a P2Z‐like response, i.e. a reversible membrane permeabilization that allows the passage of molecules with molecular masses of ≥300 Da. We investigated the properties of rP2X7 receptors expressed in Xenopus oocytes. In two‐electrode voltage‐clamp experiments, ATP or BzATP caused inward currents that were abolished or greatly diminished when NMDG+ or choline+ replaced Na+ as the principal external cation. In fluorescent dye experiments, BzATP application did not result in entry of the fluorophore YO‐PRO‐12+. Thus, rP2X7 expression in Xenopus oocytes does not by itself give rise to the pore‐forming P2Z phenotype, suggesting that ancillary factors are involved.

Cytokine upregulation of proteinase‐activated‐receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase β in human endothelial cells

Up‐regulation of proteinase‐activated receptor‐2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor.

The Cl− channel blocker niflumic acid releases Ca2+ from an intracellular store in rat pulmonary artery smooth muscle cells

The effect of the Cl− channel blockers niflumic acid (NFA), 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB), 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), and anthracene‐9‐carboxylic acid (A‐9‐C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura‐2 or fluo‐4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine‐induced increase in [Ca2+]i. These Cl− channel blockers also increased the half‐time (t1/2) to peak for the caffeine‐induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A‐9‐C were found to adversely affect fura‐2 fluorescence, fluo‐4 was used to monitor intracellular Ca2+ in studies involving these Cl− channel blockers. Both DIDS and A‐9‐C increased basal fluo‐4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine‐induced Ca2+ transient, it was significantly increased by A‐9‐C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl− channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA‐induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR.

The role of the C-terminal tail in protease-activated receptor-2-mediated Ca2+ signalling, proline-rich tyrosine kinase-2 activation, and mitogen-activated protein kinase activity

C-terminal truncation mutants were made to investigate the role of the C-terminus in coupling proteinase-activated receptor-2 (PAR-2) to various signalling pathways. Membrane expression of the delta15, delta34, delta43, and delta34-43 mutants was similar; however, expression of deltatail was lost, as was agonist-mediated internalisation of deltatail, delta43, and delta34-43. Additionally, trypsin and SLIGKV-stimulated [3H]IP accumulation was abrogated in cells transiently expressing delta43 or delta34-43 truncations, but remained unaffected in cells expressing delta34 or delta15. PAR-2 agonist-stimulated intracellular Ca(2+) mobilisation and PYK-2 activity were also abolished by deltatail, delta43, and delta34-43 mutants. However, trypsin-stimulated stress-activated protein kinases (SAPKs) or extracellular signal-regulated kinase (ERK) activities were unaffected by the delta34-43 mutation, although activity was abrogated following delta43 or deltatail truncations, suggesting that Ca(2+) mobilisation, PYK-2, or receptor internalisation are not requied for activation of SAPKs or ERK. These studies identify a novel sequence within the PAR-2 C-terminus essential for InsP(3) generation and PYK-2 activity but not mitogen-activated protein kinase (MAPK) activation.

An ATP-gated cation channel with some P2Z-like characteristics in gastric smooth muscle cells of toad

1. Whole‐cell and single‐channel currents elicited by extracellular ATP were studied in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus using standard patch clamp and microfluorimetric techniques. 2. This ATP‐gated cation channel shares a number of pharmacological and functional properties with native rat myometrium receptors, certain native P2Z purinoceptors and the recently cloned P2X7 purinoceptor. But, unlike the last two, the ATP‐gated channel does not mediate the formation of large non‐specific pores. Thus, it may represent a novel member of the P2X or P2Z class. 3. Extracellular application of ATP (> or = 150 microM) elicited an inward whole‐cell current at negative holding potentials that was inwardly rectifying and showed no sign of desensitization. Na+, Cs+ and, to a lesser degree, the organic cation choline served as charge carriers, but Cl‐ did not. Ratiometric fura‐2 measurements indicated that the current is carried in part by Ca2+. The EC50 for ATP was 700 microM in solutions with a low divalent cation concentration. 4. ATP (> or = 100 microM) at the extracellular surface of cell‐attached or excised patches elicited inwardly rectifying single‐channel currents with a 22 pS conductance. Cl‐ did not serve as a charge carrier but both Na+ and Cs+ did, as did choline to a lesser extent. The mean open time of the channel was quite long, with a range in hundreds of milliseconds at a holding potential of ‐70 mV. 5. Mg2+ and Ca2+ decreased the magnitude of the ATP‐induced whole‐cell currents. Mg2+ decreased both the amplitude and the activity of ATP‐activated single‐channel currents. 6. ADP, UTP, P1, P5‐di‐adenosine pentaphosphate (AP5A), adenosine and alpha, beta‐methylene ATP (alpha, beta‐Me‐ATP) did not induce significant whole‐cell current. ATP‐gamma‐S and 2‐methylthio ATP (2‐Me‐S‐ATP) were significantly less effective than ATP in inducing whole‐cell currents, whereas benzoylbenzoyl ATP (BzATP) was more effective. BzATP, alpha, beta‐Me‐ATP, ATP‐gamma‐S and 2‐Me‐S‐ATP induced single‐channel currents, but a higher concentration of alpha, beta‐Me‐ATP was required. 7. BzATP did not induce the formation of large non‐specific pores, as assayed using mag‐fura‐2 as a high molecular mass probe.

Pharmacological profile of store-operated Ca2+ entry in intrapulmonary artery smooth muscle cells

Store-operated Ca(2+) entry (SOCE) plays an important role in the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs). The aim of this study was to characterise the pharmacological properties of the SOCE pathway in freshly isolated PASMCs from rat lung and to determine whether this Ca(2+) entry pathway is sensitive to nitric oxide donor drugs. Following depletion of Ca(2+) from the sarcoplasmic reticulum, by treating cells with thapsigargin, re-addition of Ca(2+) produced an increase in cytosolic fluo-4 fluorescence that was sustained for the period that extracellular Ca(2+) was present. Thapsigargin also increased the rate of quench of fura-2 fluorescence, confirming that SOCE was activated. The SOCE pathway was not affected by nifedipine or verapamil; however, it was inhibited by the divalent cations Ni(2+) (10 microM) and Cd(2+) (10 microM) by 47+/-5% and 49+/-5% respectively. SOCE was also inhibited 42+/-5% by 2-aminoethoxydiphenyl borate (2-APB; 75 microM) and 58+/-4% by Gd(3+) (10 microM), although La(3+) (100 microM) had little effect. None of the NO donors examined, including sodium nitroprusside, glyceryl trinitrate, and 2-(N,N-diethylamino)-diazenolate-2-oxide had any effect on SOCE. Thus, the pulmonary vasorelaxation produced by NO does not involve direct inhibition of SOCE in PASMCs. Western blot and immunocytochemistry using antibodies directed against specific TRPC subunits detected the presence of TRPC1, 3, and 6 in pulmonary artery and the pharmacological profile of SOCE in PASMCs favours a role for TRPC1 in mediating the underlying channels that are activated by store depletion.

Regulation of store-operated Ca2+ entry in pulmonary artery smooth muscle cells

Store-operated Ca2+ entry (SOCE) is an important mechanism for Ca2+ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3microM) potentiated SOCE by 47+/-2%, while the tyrosine kinase inhibitors genistein (100microM) and tyrphostin 23 (100microM) caused a significant reduction in SOCE of 55+/-9% and 43+/-7%, respectively. It has been proposed that Ca2+-insensitive phospholipase A(2) (iPLA(2)) is involved in the activation of SOCE in many different cell types. The iPLA(2) inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10microM) and W-7 (10microM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca2+ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca2+ influx was blocked by Gd3+ (10microM), but unaffected by 2-APB (75microM), indicating a pharmacological profile distinct from the classical SOCE pathway.

Investigation of the selectivity of α, β‐methylene ATP in inhibiting vascular responses of the rat in vivo and in vitro

1 The proposal that α,β‐methylene adenosine 5′‐triphosphate (mATP) inhibits pressor responses in the pithed rat by selective desensitization of P2x‐purinoceptors was examined by comparing the selectivity of its inhibitory effect on vascular responses in vitro and in vivo. 2 In isolated ring preparations of rat femoral and tail artery, which had been denuded of endothelium, mATP markedly reduced the contractile response to exogenous ATP but had no effect on the response of the arteries to exogenous noradrenaline (NA). 3 In the pithed rat a substantial proportion of the pressor response to sympathetic nerve stimulation was resistant to α‐adrenoceptor blockade, suggesting a non‐adrenergic component to the sympathetic vasoconstriction. 4 In the pithed rat, repeated administration of desensitizing doses of mATP attenuated the pressor response to sympathetic nerve stimulation by approximately 80%, suggesting that a component of the sympathetic vasoconstriction is mediated by ATP acting on vascular P2x‐purinoceptors. However, the same mATP treatment also attenuated, to a similar degree, the pressor responses to intravenous NA, angiotensin II and vasopressin, indicating that the desensitization procedure was non‐selective. 5 These results demonstrate that while mATP can be used to desensitize selectively P2x‐purinoceptors in vitro, its attenuation of the sympathetic nerve‐mediated pressor response in vivo is non‐selective.

Spontaneous and electrically evoked Ca2+ transients in cardiomyocytes of the rat pulmonary vein

The pulmonary vein is surrounded by an external sleeve of cardiomyocytes that are widely recognised to play an important role in atrial fibrillation. While intracellular Ca(2+) is thought to influence the electrical activity of cardiomyocytes, there have been relatively few studies examining Ca(2+) signalling in these cells. Therefore, using fluo-4 and fluorescence imaging microscopy, we have investigated Ca(2+) signalling in an intact section of the rat pulmonary vein. Under resting conditions cardiomyocytes displayed spontaneous Ca(2+) transients, which were variable in amplitude and had a frequency of 1.6±0.03Hz. The Ca(2+) transients were asynchronous amongst neighbouring cardiomyocytes and tended to propagate throughout the cell as a wave. Removing extracellular Ca(2+) produced a slight reduction in the amplitude and frequency of the spontaneous Ca(2+) transients; however, ryanodine (20μM) had a much greater effect on the amplitude and reduced the frequency by 94±2%. Blocking IP(3) receptors with 2-aminoethoxydiphenyl borate (20μM) also reduced the amplitude and frequency (by 73±11%) of these events, indicating the importance of Ca(2+) release from the SR. Electrical field stimulation of the pulmonary vein produced Ca(2+) transients in cardiomyocytes that were significantly reduced by either voltage-gated Ca(2+) channel blockers or ryanodine.