Potential P-glycoprotein pharmacokinetic interaction of telaprevir with morphine or methadone.

Telaprevir (TVR) effects on P-glycloprotein and cytochrome P450 (CYP) may significantly elevate serum levels of morphine and methadone. Recent literature points to major interactions when combining TVR with warfarin or rifampin. Opioid interactions are especially dangerous in hepatitis C patients, as coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs in 50-90% of HIV-infected drug users that are prescribed opioids for chronic pain and/or methadone for maintenance. TVR has been shown to significantly inhibit the active transport enzyme pGP and may therefore increase intestinal morphine absorption. TVR also inhibits hepatic CYP3A4 that are responsible for metabolizing methadone. Patients requiring opioid analgesics must be carefully monitored because of potential for elevated opioid levels and overdose risk. Current recommendations minimize potential drug interactions between telaprevir and opioids, especially methadone, based on a single 7-day trial. We outline the various pharmacokinetic mechanisms involved when combining TVR with methadone or morphine and recommend that current data are not sufficiently robust to minimize the potentially significant interaction with opioids, especially methadone. Clinicians must be mindful of these understated interactions, know that the opioid dose may need to be significantly increased or reduced, and use caution during upward titration of opioids affected by these enzyme systems.

DCM troponin C mutant Gly159Asp blunts the response to troponin phosphorylation.

Dilated cardiomyopathy (DCM) can be caused by a Gly159Asp mutation in cardiac troponin C (cTnC). Our previous work found that partial replacement of endogenous troponin in skinned muscle fibres with human cardiac troponin containing Gly159Asp cTnC had no significant effect on maximum force generation, Ca(2+)-sensitivity or cooperativity, but halved the activation rate. In order to examine whether the mutant affected contractility when troponin was phosphorylated, Gly159Asp cTnC was introduced in the presence of a phosphomimic of protein kinase A phosphorylation of troponin I (Ser23Asp,Ser24Asp). The increased force production of the muscle fibres caused by this phosphomimic was significantly depressed. Furthermore, in the presence of the protein kinase C phosphomimic of troponin T (Thr203Glu), Gly159Asp mutant significantly reversed the decrease in Ca(2+)-sensitivity. We conclude that this DCM mutant significantly blunts the contractile response to phosphorylation and this novel mechanism may contribute to its pathogenic effect.

Mutations in fast skeletal troponin I, troponin T, and beta-tropomyosin that cause distal arthrogryposis all increase contractile function.

Distal arthrogryposes (DAs) are a group of disorders characterized by congenital contractures of distal limbs without overt neurological or muscle disease. Unexpectedly, mutations in genes encoding the fast skeletal muscle regulatory proteins troponin T (TnT), troponin I (TnI), and beta-tropomyosin (beta-TM) have been shown to cause autosomal dominant DA. We tested how these mutations affect contractile function by comparing wild-type (WT) and mutant proteins in actomyosin ATPase assays and in troponin-replaced rabbit psoas fibers. We have analyzed all four reported mutants: Arg63His TnT, Arg91Gly beta-TM, Arg174Gln TnI, and a TnI truncation mutant (Arg156ter). Thin filaments, reconstituted using actin and WT troponin and beta-TM, activated myosin subfragment-1 ATPase in a calcium-dependent, cooperative manner. Thin filaments containing either a troponin or beta-TM DA mutant produced significantly enhanced ATPase rates at all calcium concentrations without alternating calcium-sensitivity or cooperativity. In troponin-exchanged skinned fibers, each mutant caused a significant increase in Ca2+ sensitivity, and Arg156ter TnI generated significantly higher maximum force. Arg91Gly beta-TM was found to have a lower actin affinity than WT and form a less stable coiled coil. We propose the mutations cause increased contractility of developing fast-twitch skeletal muscles, thus causing muscle contractures and the development of the observed limb deformities.

Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033.

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 microM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa-tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release-restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.

Changes in myosin S1 orientation and force induced by a temperature increase.

Force generation in myosin-based motile systems is thought to result from an angular displacement of the myosin subfragment 1 (S1) tail domain with respect to the actin filament axis. In muscle, raised temperature increases the force generated by S1, implying a greater change in tail domain angular displacement. We used time-resolved x-ray diffraction to investigate the structural corollary of this force increase by measuring M3 meridional reflection intensity during sinusoidal length oscillations. This technique allows definition of S1 orientation with respect to the myofilament axis. M3 intensity changes were approximately sinusoid at low temperatures but became increasingly distorted as temperature was elevated, with the formation of a double intensity peak at maximum shortening. This increased distortion could be accounted for by assuming a shift in orientation of the tail domain of actin-bound S1 toward the orientation at which M3 intensity is maximal, which is consistent with a tail domain rotation model of force generation in which the tail approaches a more perpendicular projection from the thin filament axis at higher temperatures. In power stroke simulations, the angle between S1 tail mean position during oscillations and the position at maximum intensity decreased by 4.7 degrees, corresponding to a mean tail displacement toward the perpendicular of 0.73 nm for a temperature-induced force increase of 0.28 P(0) from 4 to 22 degrees C. Our findings suggest that at least 62% of crossbridge compliance is associated with the tail domain.

Endothelin Opens Potassium Channels in Glial Cells.

Endothelin-1 (ET-1), an autocrine hormone synthesized by astrocytes, and endothelin-3 (ET-3), a highly homologous peptide produced by neurons, have both been shown previously to cause proliferation of these astrocytes in culture [Supattapone et al. (1989) Biochem. Biophys. Res. Commun., 165, 1115 - 1122; MacCumber et al. (1990) Proc. Natl. Acad. Sci. USA, 87, 2359 - 2363]. We now demonstrate, using 86Rb+ influx assays and single channel patch-clamp recording, that both endothelins-ET-3 and ET-1-can also open a charybdotoxin-sensitive, calcium-activated K+ channel of 15 - 40 pS in glial cells. The opening of this channel may be important for the regulation of [K+] in the brain microenvironment. Thus, the endothelins may be a general mediator of astroglial response to neuronal injury.