Biomedical subjects
M A Denborough
Publications and source records attributed to M A Denborough.
Molecular cloning of the cDNA encoding human skeletal muscle triadin and its localisation to chromosome 6q22-6q23.
We have cloned and sequenced the cDNA encoding triadin, a junctional terminal cisternae protein from human skeletal muscle. The cDNA, 2941 base pairs in length, encodes a protein of 729 amino acids with a predicted molecular mass of 81,545 Da. Hydropathy analysis indicates that triadin of human skeletal muscle has the same topology in the myoplasmic, transmembrane and sarcoplasmic reticulum luminal domains as that of triadin from rabbit skeletal muscle. The number and relative position of potential modulation sites are also conserved between the human and rabbit proteins. The cDNA sequence of the predicted sarcoplasmic reticulum luminal domain of human triadin diverged from that of rabbit, with an observed similarity of 82%, translating to an identity of 77% in amino acid sequence. Two insertions of 9 and 12 residues in the amino acid sequence were observed in the predicted luminal domain of triadin, although the structural and functional consequences of such insertions are expected to be minimal. Using fluorescence in situ hybridisation, we have assigned the gene encoding human triadin to the long arm of chromosome 6 in the region 6q22-6q23. Our structural analysis of human triadin supports a central role for this protein in the mechanism of skeletal muscle excitation/contraction coupling.
Exclusion of defects in the skeletal muscle specific regions of the DHPR alpha 1 subunit as frequent causes of malignant hyperthermia.
The molecular defect predisposing to the majority of malignant hyperthermia (MH) cases is unknown, although various point mutations in the ryanodine receptor gene (RYR1) have been associated with susceptibility in a small proportion of cases. We report here that one of these, the Arg163Cys substitution, does not cosegregate with MH susceptibility. Comparison of cDNA sequences encoding the skeletal muscle specific components of the dihydropyridine receptor alpha 1 subunit between MH susceptible (MHS) and MH non-susceptible (MHN) patients was made in subjects without the reported MH linked RYR1 mutations. There were no differences within the sequence encoding the II-III loop or the IS3/IS3-IS4 segment, excluding defects in these functional segments of the alpha 1 subunit as frequent causes of MH.
The metabolism of D-myo-inositol 1,4,5-trisphosphate and D-myo-inositol 1,3,4,5-tetrakisphosphate by porcine skeletal muscle.
In soluble and particulate extracts from muscle D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] are metabolised stepwise to inositol. Ins(1,4,5)P3 is rapidly dephosphorylated to D-myo-inositol 1,4-bisphosphate then to D-myo-inositol 4-phosphate and finally inositol. In soluble extracts Ins(1,3,4,5)P4 is dephosphorylated to D-myo-inositol 1,3,4-trisphosphate then sequentially to D-myo-inositol 3,4-bisphosphate, D-myo-inositol 3-phosphate and inositol, while in particulate extracts D-myo-inositol 1,3-bisphosphate is the predominant inositol bisphosphate formed. Dephosphorylation of these inositol polyphosphates is Mg2+ dependent and inhibited by D-2,3-bisphosphoglyceric acid. Ins(1,4,5)P3 is also phosphorylated to form Ins(1,3,4,5)P4 in soluble extracts by Ins(1,4,5)P3 3-kinase. Ins(1,4,5)P3 3-kinase activity is Mg2+ and ATP dependent and is stimulated by Ca2+ and calmodulin. Particulate (sarcotubular) inositol polyphosphate 5-phosphatase (5-phosphatase) is found in membranes which are intimately involved in excitation-contraction coupling and the generation of the primary Ca2+ signal of muscle cells. Particulate 5-phosphatase had the highest specific activity in the transverse-tubule membrane, when compared to the terminal cisternae and longitudinal-tubule membranes of the sarcoplasmic reticulum. Particulate Ins(1,3,4,5)P4-3-phosphatase activity was also detected after fractionation of solubilised sarcotubular membranes by DEAE-Sephacel. Particulate 5-phosphatase activity was purified 25,600-fold to a specific activity of 25.6 mumol Ins(1,4,5)P3 hydrolysed.min-1.mg protein-1, after DEAE-Sephacel and novel affinity chromatography using D-2,3-bisphosphoglycerate/agarose and Sepharose-4B-immobilised Ins(1,4,5)P3-analog matrices. Purified particulate 5-phosphatase had apparent Km of 46.3 microM and 1.9 microM and Vmax of 115 and 0.046 mumol substrate hydrolysed.min-1.mg protein-1, for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. In contrast, purified soluble type I 5-phosphatase had apparent Km of 8.9 microM and 1.1 microM and Vmax of 3.55 and 0.13 mumol substrate hydrolysed.min-1.mg protein-1, for Ins(1,4,5P3 and Ins(1,3,4,5)P4, respectively. As in other cells, muscle 5-phosphatases have a lower affinity, but a higher capacity to metabolise Ins(1,4,5)P3 than Ins(1,3,4,5)P4. Soluble type I 5-phosphatase may have a functional role in the metabolism of both inositol polyphosphates, while particulate 5-phosphatase may primarily metabolise Ins(1,4,5)P3. Purified Ins(1,4,5)P3 3-kinase had an apparent Km of 0.42 microM and a Vmax of 4.12 nmol Ins(1,4,5)P3 phosphorylated.min-1.mg protein-1. The profile of inositol polyphosphate metabolism in muscle is similar to that reported in other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
Purification and characterization of D-myo-inositol (1,4,5)/(1,3,4,5)- polyphosphate 5-phosphatase from skeletal muscle.
In this investigation we report the presence of two forms of inositol (1,4,5)P3/(1,3,4,5)P4-polyphosphate 5-phosphatase activity (types I and II) which were observed in soluble extracts of skeletal muscle after fractionation by DEAE-Sephacel chromatography. Hydrolysis of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and D-myo-inositol 1,3,4,5-tetrakisphosphate by both phosphatases was 5-phosphate-specific, Mg2+ ion-dependent and inhibited by D-2,3-bisphosphoglycerate. Soluble type I 5-phosphatase activity was purified 27,300-fold to a specific activity of 2.54 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg protein after a combination of DEAE-Sephacel, Blue Sepharose, heparin-agarose and structural analogue affinity chromatography. Purified type I 5-phosphatase had an apparent mean Km of 8.9 and 1.1 microM and Vmax of 3.55 and 0.13 mumol of substrate hydrolyzed/min/mg protein for Ins(1,4,5)P3 and Ins(1,3,4,5)P4, respectively. Investigations on soluble type II 5-phosphatase after DEAE-Sephacel chromatography indicated an apparent Km of 71.4 microM Ins(1,4,5)P3 and an apparent molecular mass of 81 kDa. Soluble type I phosphatase has an apparent molecular mass of 48 kDa and an isoelectric point of 5.8. Soluble type I 5-phosphatase has kinetic constants which suggest a role in the regulation of inositol polyphosphates at physiological concentrations. These results support a role for Ins(1,4,5)P3 in the regulation of Ca2+ homeostasis in skeletal muscle.
Kinetic analysis of novel inhibitors of inositol polyphosphate metabolism.
The ability of the novel D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3] analogues, L-chiro-inositol 1,4,6-trisphosphate [L-chr Ins(1,4,6)P3] and the corresponding trisphosphorothioate compound [L-chr Ins(1,4,6)PS3] to inhibit soluble inositol (1,4,5)P3/(1,3,4,5)P4-polyphosphate 5-phosphatase, potently and selectively, has been investigated. L-chr Ins(1,4,6)P3 competitively inhibited 5-phosphate specific dephosphorylation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] with apparent Ki values of 6.35 and 1.76 microM, respectively. L-chr Ins(1,4,6)PS3 competitively inhibited hydrolysis of Ins(1,4,5)P3 and noncompetitively inhibited dephosphorylation of Ins(1,3,4,5)P4 with apparent Ki values of 0.67 and 0.44 microM, respectively. L-chr Ins(1,4,6)PS3 did not affect Ins(1,4,5)P3 3-kinase activity. In the present investigation L-chr Ins(1,4,6)P3 and L-chr Ins(1,4,6)PS3 have been shown to be the most potent and selective inhibitors of inositol polyphosphate metabolism yet described.
Fatal inherited rhabdomyolysis and malignant hyperthermia.
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Detection and partial purification of inositol 1,4,5-trisphosphate 3-kinase from porcine skeletal muscle.
A myoplasmic 3-kinase was detected in porcine skeletal muscle that phosphorylated [3H]Ins(1,4,5)P3 [D-myo-inositol(1,4,5)trisphosphate] to [3H]Ins(1,3,4,5)P4 [D-myo-inositol(1,3,4,5)tetrakisphosphate]. The Ins(1,4,5)P3 3-kinase activity was ATP- and Mg(2+)-dependent, and was activated by Ca2+ and calmodulin. Ins(1,4,5)P3 3-kinase activity was purified 2632-fold from soluble extracts of skeletal muscle by a combination of DEAE-Sephacel, heparin-Agarose and Ins(1,4,5)P3 structural-analogue affinity chromatography. The highest specific activity obtained was 10.6 nmol of Ins(1,4,5)P3 phosphorylated/min/mg protein. The partially purified enzyme had a mean Km and Vmax of 0.46 microM and 3.15 nmol/min/mg protein for Ins(1,4,5)P3 metabolism, respectively. After analytical gel filtration two forms of soluble Ins(1,4,5)P3 3-kinase were observed with M(r) of 39,000 and 62,000. As in other cell types, muscle Ins(1,4,5)P3 3-kinase was soluble, and had a higher affinity but a lower capacity to metabolize Ins(1,4,5)P3 in comparison to Ins(1,4,5)P3 5-phosphatase.
A clinical grading scale to predict malignant hyperthermia susceptibility.
BACKGROUND: The diagnosis of an acute malignant hyperthermia reaction by clinical criteria can be difficult because of the nonspecific nature and variable incidence of many of the clinical signs and laboratory findings. Development of a standardized means for estimating the qualitative likelihood of malignant hyperthermia in a given patient without the use of specialized diagnostic testing would be useful for patient management and would promote research into improved means for diagnosing this disease. METHODS: Using the Delphi method and an international panel of 11 experts on malignant hyperthermia, a multifactor malignant hyperthermia clinical grading scale comprising standardized clinical diagnostic criteria was developed for classification of existing records and for application to new patients. RESULTS: This scale ranks the qualitative likelihood that an adverse anesthetic event represents malignant hyperthermia (malignant hyperthermia event rank) and that, with further investigation of family history, an individual patient will be diagnosed as malignant hyperthermia susceptible (malignant hyperthermia susceptibility rank). The assigned rank represents a lower bound on the likelihood of malignant hyperthermia. The clinical grading scale requires the anesthesiologist to judge whether specific clinical signs are appropriate for the patient's medical condition, anesthetic technique, and surgical procedure. CONCLUSIONS: The malignant hyperthermia clinical grading scale is recommended for use as an aid to the objective definition of this disease. It use may improve malignant hyperthermia research by allowing comparisons among well-defined groups of patients. This clinical grading system provides a new and comprehensive clinical case definition for the malignant hyperthermia syndrome.
Inositol 1,4,5-trisphosphate metabolism in red blood cells from individuals susceptible to malignant hyperpyrexia.
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The effect of calcium channel antagonists and BAY K 8644 on calcium fluxes of malignant hyperpyrexia-susceptible muscle.
1. The calcium channel antagonists verapamil (100 microM) and nifedipine (100 microM) inhibited twitch response and KCl induced hypercontractility in malignant hyperpyrexia (MH)-susceptible porcine skeletal muscle. These calcium channel antagonists did not effect hypercontractility induced by 3% halothane or 2 mM caffeine. 2. The calcium channel agonist BAY K 8644 (50 microM) induced contracture in MH-susceptible muscle but did not potentiate contracture response induced by 2 mM caffeine or 3% halothane. BAY K 8644 did not increase the resting tension of control muscle or increase the sensitivity of control muscle to 4 mM caffeine, 3% halothane or 80 mM KCl. 3. The sarcoplasmic reticulum (SR) from MH-susceptible and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 4. Verapamil and diltiazem [which has been previously shown to inhibit the hypercontractility of MH-susceptible porcine muscle to caffeine, halothane and KCl (Foster and Denborough, 1989 Br. J. Anaesth. 62, 566-572)] did not effect Ca2+ uptake or Ca(2+)-dependent ATPase activities of SR longitudinal tubule membranes, from MH-susceptible or control muscle. These calcium channel antagonists did not effect Ca2+ release from terminal cisternae preparations. 5. The skeletal muscle relaxant dantrolene inhibited Ca2+ efflux and equilibrium-Ca2+ exchange associated with the terminal cisternae membrane of MH-susceptible and control skeletal muscle. 6. Calcium channel antagonists modify Ca2+ fluxes in MH-susceptible and control muscle by acting at a site distal to the SR. Calcium channel antagonists may inhibit contractile response by modifying events of excitation-contraction coupling associated with the voltage sensor molecule (dihydropyridine-receptor) of the transverse-tubule membrane, whereas dantrolene directly acts on the terminal cisternae membrane to inhibit Ca2+ efflux and equilibrium Ca2+ exchange. Different calcium channel antagonists seem to modify the voltage-sensor mechanism in different ways in MH-susceptible muscle. 7. An abnormality in the coupling mechanism of the voltage sensor-SR calcium release channel may exist in MH-susceptible muscle. This dysfunction may be an adaptation to the elevated levels of myoplasmic Ca2+ and/or the molecular defect described in the Ca2+ release channel of the SR of MH-susceptible porcine muscle. In view of these results it is unlikely that nifedipine or verapamil would be of therapeutic value for the treatment of MH.
Propofol anaesthesia in malignant hyperpyrexia susceptible swine.
1. Malignant hyperpyrexia (MH) is an inherited muscle abnormality that presents clinically as a syndrome of life-threatening complications during general anaesthesia. 2. Propofol is a new sedative hypnotic used for the induction and maintenance of anaesthesia. 3. Propofol did not induce MH in five susceptible pigs. Propofol did not induce contracture in isolated MH susceptible muscle but did modify halothane, caffeine and KCl contractures.
The effect of azumolene on hypercontractility and sarcoplasmic reticulum Ca(2+)-dependent ATPase activity of malignant hyperpyrexia-susceptible porcine skeletal muscle.
1. Azumolene sodium is a new water-soluble derivative of dantrolene sodium that also acts as a skeletal-muscle relaxant. 2. Azumolene (6 mumol/L) inhibited the hypercontractility induced separately by 3% halothane, 2 mmol/L caffeine and 80 mmol/L potassium chloride in isolated malignant hyperpyrexia (MH)-susceptible muscle. Azumolene was equipotent with dantrolene in inhibiting the abnormal responses. 3. Like dantrolene, azumolene (6 mumol/L) not only prevented but reversed the abnormal contractures induced by halothane and caffeine. Contracture responses to caffeine were also modified by azumolene in control preparations. 4. In the presence of maximal effective concentrations of dantrolene, azumolene failed to further relax caffeine-induced contractures, and the converse was also true. This was observed in both MH-susceptible and control preparations. 5. Sarcoplasmic reticulum Ca(2+)-dependent ATPase activity from MH-susceptible and control muscle was not affected by azumolene. 6. Like dantrolene, azumolene may inhibit Ca2+ release directly from the sarcoplasmic reticulum and be of therapeutic value for the treatment of MH.
Inositol 1,4,5-trisphosphate phosphatase deficiency and malignant hyperpyrexia in swine.
The sarcoplasmic reticulum from muscle of swine which are susceptible to malignant hyperpyrexia is deficient in inositol 1,4,5-trisphosphate phosphatase (InsP35-ase) activity, which leads to high intracellular concentrations of inositol 1,4,5-trisphosphate (InsP3) and of calcium ions. Halothane inhibits InsP35-ase and further increases myoplasmic InsP3 and calcium ion concentrations, and produces the clinical features of malignant hyperpyrexia.
Calcium currents and asymmetric charge movement in malignant hyperpyrexia.
In both control pigs and pigs susceptible to malignant hyperpyrexia (MH), the size of the calcium current (ICa) and the amount of asymmetric charge movement varied considerably between different gracilis muscle fibers but appeared to vary in parallel. The mean amount of both ICa and charge movement were slightly but significantly smaller in MH-susceptible (MHS) muscle compared with normal muscle. Halothane (1% v/v) reduced both parameters by almost 50%. One interpretation of the data is that the signal, which couples depolarization to calcium release from the sarcoplasmic reticulum, is abnormal in MHS muscle.
31P-NMR spectroscopy: the metabolic profile of malignant hyperpyrexic porcine skeletal muscle.
31Phosphorus-NMR spectroscopy may have the potential to help in the noninvasive diagnosis of malignant hyperpyrexia (MH). Changes in the phosphate-metabolite profile of MH-susceptible (MHS) skeletal muscle occur more readily under conditions of anoxia than in control muscle. Induction of anoxia caused a rapid fall in intracellular phosphocreatine, an elevation of inorganic phosphate, and finally a diminution of ATP in MHS muscle. The onset of metabolic change was slower in control tissue. Increased oxygen consumption may occur in anoxic MHS muscle, which leads to accelerated glycolysis and a rapid fall in the intracellular high-energy phosphates. In MHS muscle an abnormality may exist in carbohydrate metabolism linked with poor resynthesis of the high-energy phosphates, which may be precipitated under anaerobic conditions. Accelerated muscle metabolism is also observed in the presence of 2 mM caffeine and 3% halothane in MHS muscle. Changes in the concentrations of metabolites could be mapped noninvasively under anoxic conditions using topical 31P-NMR.
Characterization of the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum from malignant hyperpyrexia susceptible porcine skeletal muscle.
1. The sarcoplasmic reticulum (SR) from malignant hyperpyrexia susceptible (MHS) and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 2. The two membrane preparations were highly purified and had distinctive features which were associated with their origins in the SR membraneous network. 3. Calsequestrin and calcium were enriched in the terminal cisternae fraction (HSR), in comparison to longitudinal tubule preparations (LSR). 4. The HSR membrane also had a greater total capacity to store Ca2+ and Ca2+ release was more rapid than from LSR preparations. 5. No distinction could be made between the membrane morphology, Ca2+ -fluxes or Ca2+ -dependent ATPase activities, associated with these functionally distinct regions of MHS and control preparations.
Effect of diltiazem on porcine malignant hyperpyrexia induced by suxamethonium and halothane.
We have studied the ability of the calcium channel antagonist diltiazem to inhibit and reverse the porcine malignant hyperpyrexia (MH) syndrome. Pretreatment with diltiazem modified an MH response. Treatment with diltiazem was partially effective against a mild (or early) MH response. Diltiazem should not be considered to be an effective therapeutic agent for MH and should not displace the use of dantrolene.