Bernard P. Hughes

pH-Dependent Interactions of Cd2+ and a Carboxylate Blocker with the Rat ClC-1 Chloride Channel and Its R304E Mutant in the Sf-9 Insect Cell Line

1 Gating of the skeletal muscle chloride channel (ClC‐1) is sensitive to extracellular pH. In this study, whole‐cell recording of currents from wild‐type (WT) ClC‐1 and a mutant, R304E, expressed in the Sf‐9 insect cell line was used to investigate further the nature of the pH‐sensitive residues. 2 Extracellular Cd2+ produced a concentration‐dependent block of WT ClC‐1 with an IC50 of 1.0 ± 0.1 mm and a Hill coefficient of 2.0 ± 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 ± 0.1 and a Hill coefficient for proton binding of 3.0 ± 0.3. Anthracene‐9‐carboxylate (A‐9‐C) block of WT ClC‐1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 ± 0.1 and a Hill coefficient for proton binding of 1.0 ± 0.2. 3 Compared with WT ClC‐1, R304E had a lower affinity for Cd2+ (IC50, 3.0 ± 0.3 mm) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 ± 0.3. In contrast, the A‐9‐C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC‐1. 4 ClC‐1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A‐9‐C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A‐9‐C binding site to affect its characteristics, but it does alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5 The block of ClC‐1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A‐9‐C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC‐1.

Characteristics of skeletal muscle chloride channel CIC-1 and point mutant R304E expressed in Sf-9 insect cells

Using the baculovirus system, the skeletal muscle chloride channel, CIC-1 (rat), and a point mutant replacing arginine 304 with glutamic acid were expressed at high levels in cultured Sf-9 insect cells. Whole-cell patch-clamping revealed large inwardly rectifying currents with maxima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produced inactivating currents made up of a steady state component and two exponentially decaying components with tau 1 = 6.14+/- 0.92 ms, tau 2 = 36.5+/- 3.29 ms (S.D) n = 7 for steps to -120 mV. Currents recorded in the outside-out patch configuration were often unexpectedly large and up to 5% of whole-cell currents obtained in the same cell, suggesting an uneven channel distribution in the plasmalemma of Sf-9 cells. The pharmacology of a number of chloride channel blockers, including anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was investigated and showed for the first time that perrhenate is an effective blocker of C1C-1 and that it has a complex mechanism of action. Further, the potency of A9C was found to be dependent on external chloride concentration. As in studies on muscle cells themselves, blockade was rapidly effective and easily reversible, except when applying the indanyloxyacetate derivative, IAA94/95, which took up to 10 min to act, and, consistent with an intracellular site of action, was difficult to reverse by washing. Mutation of the highly conserved arginine at position 304 to a glutamic acid did not significantly alter the behaviour of the channel.

Functional complementation of truncated human skeletal-muscle chloride channel (hClC-1) using carboxyl tail fragments

Crystal structures of bacterial CLC (voltage-gated chloride channel family) proteins suggest the arrangement of permeation pores and possible gates in the transmembrane region of eukaryotic CLC channels. For the extensive cytoplasmic tails of eukaryotic CLC family members, however, there are no equivalent structural predictions. Truncations of cytoplasmic tails in different places or point mutations result in loss of function or altered gating of several members of the CLC family, suggesting functional importance. In the present study, we show that deletion of the terminal 100 amino acids (N889X) in human ClC-1 (skeletal-muscle chloride channel) has minor consequences, whereas truncation by 110 or more amino acids (from Q879X) destroys channel function. Use of the split channel strategy, co-injecting mRNAs and expressing various complementary constructs in Xenopus oocytes, confirms the importance of the Gln879-Arg888 sequence. A split between the two CBS (cystathionine b-synthase) domains (CBS1 and CBS2) gives normal function (e.g. G721X plus its complement), whereas a partial complementation, eliminating the CBS1 domain, eliminates function. Surprisingly, function is retained even when the region Gly721-Ala862 (between CBS1 and CBS2, and including most of the CBS2 domain) is omitted from the complementation. Furthermore, even shorter peptides from the CBS2-immediate post-CBS2 region are sufficient for functional complementation. We have found that just 26 amino acids from Leu863 to Arg888 are necessary since channel function is restored by co-expressing this peptide with the otherwise inactive truncation, G721X.

The ethics of evidence implementation in health care

Evidence based practice is increasingly mandated by all stakeholders as an integral process of ensuring safe and quality health care. It is recognised that evidence based practice can contribute to minimising misuse, overuse and underuse of health care. Operationalising evidence based practice requires physiotherapists to access relevant evidence, appraise the evidence for its methodological quality, extract information relevant to their practice, and implement it as part of health care service delivery. The final step in this process is to evaluate evidence implementation and reflect what, if any, changes to health care processes and outcomes was achieved. From a theoretical perspective, these steps seem logical and readily achievable. However, practical application in clinical practice settings has encountered numerous barriers. One such barrier, which is commonly encountered and is a contentious area, is the issue of ethics in evidence implementation. Using two hypothetical case studies, we aim to highlight common frustrations encountered by physiotherapists when implementing evidence into practice, ethical ambiguity underpinning evidence implementation and discuss implications in terms of clinical practice and research.

Arachidonic acid and its COX1/2 metabolites inhibit interferon-γ mediated induction of indoleamine-2,3 dioxygenase in THP-1 cells and Human monocytes

Using human acute monocytic leukaemic THP-1 cells and human primary monocytes, this study examined the ability of arachidonic acid (AA) to modulate the activity of the IFNγ signalling cascade and its downstream effector indoleamine 2,3-dioxygenase (IDO). We established that AA inhibited IDO enzyme activity with an IC(50) of 20 μM in THP-1 cells and 12 μM in monocytes, and this was due to reduced expression of INDO1 mRNA and reduced level of IDO protein. Further mechanistic analysis revealed that AA interfered with the transcriptional function of the IFNγ signalling pathway by reducing phosphorylation of signal transducer and activator of transcription (STAT1) on tyrosine 701. The importance of AA metabolism via the COX and LOX pathways was investigated using inhibitors. Indomethacin, but not nordihydroguaiaretic acid, prevented the AA-mediated inhibition of STAT1 phosphorylation and thereby IDO enzymatic activity in THP-1 cells and monocytes. This is the first study to demonstrate that AA inhibits the IFNγ/STAT/IDO pathway, and this function is mediated by COX1/2 produced metabolites of AA. We now have evidence demonstrating that the AA metabolites, prostaglandins A(2) and D(2,) were highly inhibitory towards the IFNγ pathway, while prostaglandin E(2) had no effect. Together, these results indicate that the fatty acid AA has the potential to modulate the immunosuppressive activity of IDO and may form the basis of novel inhibitory compounds.

Analysis of carboxyl tail function in the skeletal muscle Cl- channel hClC-1.

Human ClC-1 (skeletal muscle Cl- channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine beta-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser(720) and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser(720) (but with CBS1 deleted) and a complete C-tail beginning at Leu(598). Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1-CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu(665) and Lys(680).

Inhibition of indoleamine 2,3-dioxygenase activity by fatty acids and prostaglandins: A structure function analysis.

Indoleamine 2,3-dioxygenase-1 (IDO-1) catalyses the first and rate-limiting step in the metabolism of L-tryptophan. Degradation of L-Trp leads to the production of several immunosuppressive metabolites, including N-formyl kynurenine and kynurenine (Kyn). Apart from a normal physiological role, IDO-1 has also been identified to play a crucial role in immune suppression and tumour induced tolerance. Indeed, many primary tumours express high levels of IDO-1 compared to normal cells of the same stroma. IDO-1 is accepted as being an inducible negative regulator of T cell viability, proliferation and activation. As such, IDO-1 has become a target of intense interest for pharmacological inhibition, for the treatment of cancer. We have previously demonstrated that AA and the prostaglandin metabolite, PGD2, repressed the IFNγ mediated activity of IDO-1 in THP-1 cells and human monocytes. In this study, we characterise the structure-function relationship of fatty acids and eicosanoids towards inhibition of IDO-1 activity in THP-1 cells and human monocytes. Using a commercial library of fatty acids, 55% of fatty acids inhibited IDO-1 activity. The activity of individual FAs was affected by chain length, number of double bonds and bond configuration. Interrogation of an AA derived eicosanoid library identified 13 PGs with significant inhibitory activity. A structure-function analysis revealed that the γ position of the cyclopentenone ring, double bond in the α-β position of the cyclopentenone ring, the presence of multiple OH groups in the side arm and the addition of an ethanolamide group, significantly increased the inhibitory activity of the PGs. Based on this data we have identified the structure of two possible compounds that may be even more potent pharmacological repressors of IDO-1.

Evidence that the Ca2+ inflow pathway in hepatocytes stimulated by thapsigargin is similar to that activated by vasopressin.

Experiments were conducted to characterize the thapsigargin-stimulated plasma membrane Ca2+ inflow pathway in hepatocytes. Ca2+ inflow was estimated by measurement of the initial rate of activation of glycogen phosphorylase a following the addition of Ca2+ to cells previously incubated in the absence of added Ca2+. Pretreatment of hepatocytes with thapsigargin caused a substantial stimulation of the rate of Ca2+ activation of glycogen phosphorylase a. This was interpreted to reflect a stimulation of plasma membrane Ca2+ inflow. The effect of thapsigargin on plasma membrane Ca2+ inflow was approximately 65% of the magnitude of the effect caused by vasopressin. When thapsigargin and vasopressin were combined as a stimulus, the degree of stimulation was similar to that caused by vasopressin alone. The thapsigargin-induced stimulation of the rate of Ca2+ activation of glycogen phosphorylase a was inhibited in a concentration-dependent manner by both Zn2+ and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). The concentration of each agent required for half-maximal inhibition was approximately 20 microM. It is concluded from: (i) the apparent lack of additivity in the responses of thapsigargin and vasopressin, and (ii) the sensitivity to inhibitors, that the Ca2+ inflow pathway in hepatocytes stimulated by thapsigargin is likely to be similar to that which is activated by vasopressin.

Prostaglandin D2 is a novel repressor of IFNγ induced indoleamine-2,3-dioxygenase via the DP1 receptor and cAMP pathway.

Expression of elevated levels of Indoleamine 2,3-dioxygenase (IDO) is well established as a mechanism of cancer induced immunosuppression. Pharmacological inhibition of IDO activity is thus a promising alternative in the treatment of cancer. Previously we demonstrated that cyclooxygenase derived metabolites of arachidonic acid inhibited the interferon-gamma mediated induction of IDO in both THP-1 cells and human monocytes. Here we identified that of the five primary prostanoids produced by COX-1/COX-2, only PGD2 displayed significant repressor activity. PGD2 inhibited IDO activity with an IC50 of 7.2µM in THP-1 cells and 5.2µM in monocytes. PGD2 caused a significant decrease in both IDO mRNA and protein. Using receptor specific agonists, PGD2 was found to act via the DP1 receptor, while the CRTH2 receptor was not involved. A DP1 antagonist significantly reduced the activity of PGD2, while CRTH2 agonists were ineffective. PGD2 increased intracellular cAMP levels and exogenous N(6)-cAMP was also found to be highly inhibitory. The effects of PGD2 via cAMP were blocked by Rp-cAMP indicating involvement of PKA. PGD2 also stimulated CREB phosphorylation, a PKA dependent transcription factor. This is the first report demonstrating that PGD2, a prostanoid typically associated with allergy, can inhibit IDO activity via the DP1/cAMP/PKA/CREB pathway. Our findings suggest that PGD2 and its derivatives may form the basis of novel repressors of IFNγ-mediated IDO expression.