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Biomedical subjects

R M Simmons

Publications and source records attributed to R M Simmons.

At least 37 records · Page 2Linked to original sources
PubMed1997

Elasticity and unfolding of single molecules of the giant muscle protein titin.

The giant muscle protein titin, also called connectin, is responsible for the elasticity of relaxed striated muscle, as well as acting as the molecular scaffold for thick-filament formation. The titin molecule consists largely of tandem domains of the immunoglobulin and fibronectin-III types, together with specialized binding regions and a putative elastic region, the PEVK domain. We have done mechanical experiments on single molecules of titin to determine their visco-elastic properties, using an optical-tweezers technique. On a fast (0.1s) timescale titin is elastic and force-extension data can be fitted with standard random-coil polymer models, showing that there are two main sources of elasticity: one deriving from the entropy of straightening the molecule; the other consistent with extension of the polypeptide chain in the PEVK region. On a slower timescale and above a certain force threshold, the molecule displays stress-relaxation, which occurs in rapid steps of a few piconewtons, corresponding to yielding of internal structures by about 20 nm. This stress-relaxation probably derives from unfolding of immunoglobulin and fibronectin domains.

Connectin↗
PubMed1997

LY303870, a centrally active neurokinin-1 antagonist with a long duration of action.

The selective neurokinin (NK)-1 antagonist LY303870 has high affinity and specificity for human and guinea pig brain NK-1 receptors labeled with 125I-substance P. It has approximately 15- to 30-fold lower affinity for rat and mouse brain NK-1 receptors, consistent with previously reported species differences in the affinities of nonpeptide antagonists for NK-1 receptors. In vivo, LY303870 blocked the characteristic, caudally directed, biting and scratching response elicited by intrathecal administration of the selective NK-1 agonist Ac-[Arg6,Sar9,Met(O2)11]substance P6-11 in conscious mice. The potentiation of the tail-flick response elicited by intrathecal administration of the NK-1 agonist [Sar9,Met(O2)11]substance P in rats was also selectively blocked by LY303870. When tested in a model of persistent nociceptive activation induced by tissue injury (the formalin test), LY303870 blocked licking behavior in the late phase of the formalin test, in a dose-dependent manner. After oral administration of 10 mg/kg, the blockade of the late-phase licking behavior was evident for at least 24 hr. Ex vivo binding studies in guinea pigs showed that orally administered LY303870 potently inhibited binding to central and peripheral NK-1 receptors labeled with 125I-substance P. This inhibition was long-lasting, consistent with other in vivo activities. LY306155, the opposite enantiomer of LY303870, was less active in all of the functional assays. In rodents, LY303870 did not exhibit any neurological, motor, cardiovascular, gastrointestinal or autonomic side effects at doses of < or = 50 mg/kg p.o. Thus, LY303870 is a potent, centrally active, NK-1 antagonist in vivo, with long-lasting oral activity.

Animals↗
PubMed1996

3-Aryl-1,2-diacetamidopropane derivatives as novel and potent NK-1 receptor antagonists.

Early structure-activity studies on racemic tryptophan ester and amide NK-1 antagonists 5-7 led to the discovery that the potency of the series could be markedly increased by moving the carbonyl function in these molecules to an off-chain position as in the 3-aryl-1,2-diacetamidopropane 9. Further medicinal chemistry incorporating this change resulted in the discovery of a novel series of highly potent aryl amino acid derived NK-1 antagonists of the R stereoisomeric series (IC50's = 100 pM to > 5 microM). Compounds in this series were shown to be competitive antagonists using an in vitro NK-1 smooth muscle assay, and this data correlated well with observed human NK-1 binding affinities. Two of these agents, (R)-25 and (R)-32, blocked intrathecal NK-1 agonist-driven [Ac-[Arg6,Sar9,Met(O2)11]- substance P 6-11 (Ac-Sar9)] nociceptive behavior in mice. Both compounds potently blocked the neurogenic dural inflammation following trigeminal ganglion stimulation in the guinea pig after intravenous administration. Further, upon oral administration in this model, (R)-32 was observed to be very potent (ID50 = 91 ng/kg) and have a long duration of action (> 8 h at 1 micrograms/kg). Compound (R)-32, designated LY303870, is currently under clinical development as an NK-1 antagonist with a long duration of action.

Amides↗
PubMed1996

Quantitative measurements of force and displacement using an optical trap.

We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.

Biophysical Phenomena↗
PubMed1994

Single myosin molecule mechanics: piconewton forces and nanometre steps.

A new in vitro assay using a feedback enhanced laser trap system allows direct measurement of force and displacement that results from the interaction of a single myosin molecule with a single suspended actin filament. Discrete stepwise movements averaging 11 nm were seen under conditions of low load, and single force transients averaging 3-4 pN were measured under isometric conditions. The magnitudes of the single forces and displacements are consistent with predictions of the conventional swinging-crossbridge model of muscle contraction.

Actins↗
PubMed1993

Force on single actin filaments in a motility assay measured with an optical trap.

We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.

Actin Cytoskeleton↗
PubMed1988

Binding of [3H]prazosin and [3H]p-aminoclonidine to alpha-adrenoceptors in rat spinal cord.

alpha-Adrenoceptors in spinal cord appear to play a role in a number of physiologic processes including the control of blood pressure, pain and motor function. In order to evaluate more clearly these potential roles, the characteristics of binding of [3H]prazosin ([3H]PRZ) to spinal alpha 1 adrenoceptors and [3H]p-aminoclonidine ([3H]PAC) to spinal alpha 2 adrenoceptors were determined. Binding of each ligand to their respective adrenoceptors was saturable and Scatchard analysis revealed binding of each to a single class of adrenoceptors with characteristics of [3H]PRZ binding of Bmax = 78 fmol/mg protein and Kd = 0.75 nM and [3H]PAC binding Bmax = 70 fmol/mg protein and Kd = 1.39 nM. Whereas [3H]PRZ specific binding (Bmax) was unaltered by guanine nucleotides. [3H]PAC binding was increased with addition of 10 microM guanosine triphosphate (GTP) (P less than 0.05) and decreased with either 50 microM GTP or guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] (P less than 0.01). Competition for specific [3H]PRZ and [3H]PAC binding by various alpha 1 and alpha 2 adrenoceptor agonists and antagonists of known pharmacologic activity revealed that [3H]PRZ defines alpha 1 adrenoceptors (Ki = 2.1 nM for prazosin vs 4300 nM for yohimbine) and [3H]PAC defines alpha 2 adrenoceptors (Ki = 1.06 nM for yohimbine vs 15480 nM for prazosin). Regional spinal cord studies demonstrated that dorsal spinal cord in the lumbar region contains the highest density of both [3H]PRZ (Bmax = 93 +/- 14 fmol/mg protein) and [3H]PAC (Bmax = 101 +/- 6 fmol/mg protein) binding. In contrast, lowest binding was evident in thoracic cord with equal levels in both dorsal and ventral regions (Bmax = 44-48 fmol/mg protein). The regional distribution of both alpha 1 and alpha 2 adrenoceptors in spinal cord compares to the localization previously classified functionally utilizing various pharmacological agonists and antagonists at norepinephrine receptors.

Animals↗
PubMed1988

Synthesis and pharmacological characterization of 1-phenyl-, 4-phenyl-, and 1-benzyl-1,2,3,4-tetrahydroisoquinolines as dopamine receptor ligands.

A series of 1-phenyl-, 4-phenyl-, and 1-benzyl-1,2,3,4-tetrahydroisoquinolines have been prepared as ring-contracted analogues of the prototypical D1 dopamine receptor antagonist SCH23390 [(R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H- 3-benzazepine]. The affinity and selectivity of these isoquinolines for D1 receptors was determined by three biochemical endpoints in membrane homogenates prepared from rat corpus striatum: the potency to complete for [3H]SCH23390 binding sites; the potency to compete for [3H]spiperone (a D2 receptor ligand) binding sites; and effects on dopamine-stimulated adenylate cyclase. Competitive binding measurements at D1 sites showed SCH23390 to possess the highest affinity, followed by 1-phenyl greater than 1-benzyl greater than 4-phenyl for the isoquinolines. These results were highly correlated with the ability of the test compounds to antagonize dopamine-stimulated adenylate cyclase (r = 0.98). None of the compounds alone stimulated cAMP formation at concentrations of 10 nM to 100 microM. D2 competition binding showed the 1-benzyl derivative to possess the highest affinity, followed by 4-phenyl greater than SCH23390 greater than 1-phenyl. The tertiary 1-phenyl derivative was more potent than the secondary 1-phenyl analogue in all assays. Interestingly, resolution and single-crystal X-ray analysis of the tertiary N-methyl-1-phenyltetrahydroisoquinoline showed the most active enantiomer to possess the S absolute configuration, in contrast to the benzazepine (R)-SCH23390.

Adenylyl Cyclases↗
PubMed1988

X-ray diffraction studies on muscle during rapid shortening and their implications concerning crossbridge behaviour.

In isometric contraction, a high proportion of crossbridges are always in the attached state and crossbridge cycling is slow. During shortening, crossbridges must be entering the detached state at a higher rate, as they come to the end of their working strokes. The size of the population of detached crossbridges will then depend on the re-attachment rate and it is therefore of some interest to find out whether a significant detached population can be detected. Observations on the equatorial X-ray diffraction pattern indicate that this is the case at higher speeds of shortening, for example at a speed where the detachment rate must be of the order of 200 per second. In a muscle under these conditions, the 59 A and 51 A actin layer line reflections decrease in intensity compared to their values during isometric contraction. This decrease does not appear to be associated with a change in structure of the actin-troponin-tropomyosin complex, since the second actin layer-line reflection remains virtually unchanged in intensity. Thus the change is likely to arise from either a different total number of attached crossbridges, or a different number of attached crossbridges in the tension generating state. The result provides some further evidence for specific helical labelling of the actin structure by crossbridges during contraction, as do some recent electronmicroscope studies of rapidly frozen contracting muscle.

Actins↗
PubMed1986

Tension transients during the rise of tetanic tension in frog muscle fibres.

Tension transients were recorded from fibres isolated from the tibialis anterior muscle of the frog during the rise of tetanic tension at 0.8-2.5 degrees C. The length of a central segment of the fibre was controlled by feed-back from a spot-follower device. Length steps complete within 0.2 ms were applied at one end of the fibre, and tension changes were recorded at the other end with a transducer having a natural frequency of 10.8 kHz. The tension transients measured during the rise of force showed the four phases characteristic of transients recorded during the plateau of a tetanus and during shortening. The extreme tension change reached during a length change was smaller for a given size of step during the rise of tension than at the plateau, but by less than in proportion to the developed force, suggesting that stiffness increases earlier than tension. Stiffness changes were further assessed by matching the tension records from one fibre with the responses of an analogue circuit (delay line) representing the mechanical properties of the fibre and force transducer. Stiffness derived from these comparisons varied in approximately the same proportion as stiffness assessed from the extreme tension change. During the rise of tension, there was a roughly constant lag of tension behind stiffness, ranging from 11 to 16 ms in different fibres. Steps applied during the latent period showed a lag of about 10 ms from the first increase of stiffness to the first appearance of tension. The partial recovery of tension immediately following the step, phase 2, was faster at the low tension levels early in the tetanus. The intermediate level, T2, to which tension recovers during phase 2 scaled in approximate proportion to the tension level immediately preceding the step. This result is unlike the relative decrease in T2 levels we have recently described for steps applied during steady shortening, and suggests that the increased stiffness-tension ratio seen during the rise of tetanic force is not due to shortening within the sarcomeres. The results can be explained if the attachment of cross-bridges in the rising phase takes place in two steps, the initial state of attachment resulting in the production of little or no tension. Several such schemes are considered.

Animals↗
PubMed1986

The stiffness of frog skinned muscle fibres at altered lateral filament spacing.

When the surface membrane is removed from a frog muscle fibre the myofibrils swell, so that the spacing between the filaments increases by 10-30%. In this study, the stiffness of skinned fibres was measured when the lateral spacing of the filament lattice was reduced osmotically using polyvinyl pyrrolidone (PVP), a synthetic linear polymer. In the absence of PVP, the apparent stiffness of relaxed skinned fibres measured during ramp stretches was about 0.05-0.1 of the stiffness of intact fibres. The stiffness increased when the lattice spacing was decreased. The mechanical characteristics of resting stiffness in the presence of PVP (or bovine serum albumin) were similar to those of the short-range elastic component of resting intact fibres. However, when the spacing of skinned fibres was reduced osmotically to that of the intact lattice, the stiffness of skinned fibres was about 1.6 times higher. In the absence of PVP, Ca2+-activated skinned fibres were less stiff than are fully active intact fibres during isometric tetani. The skinned fibre force-extension relation was markedly curved. As the filament spacing was reduced, the stiffness of the Ca2+-activated skinned fibres increased, with little change in isometric tension, and the force-extension curve became more linear. Experiments at varied filament overlap and during feed-back control of sarcomere length, monitored by laser diffraction, showed that PVP increased the stiffness of the sarcomeres. The increase of active stiffness in the presence of PVP could be partially dissociated from the increase of resting stiffness by inclusion of Mg tripolyphosphate in the bathing solutions. It is concluded that the low stiffness and the non-linearity of the force-extension curve observed in fully activated skinned fibres are due primarily to the increase of filament separation that occurs when a fibre is skinned. The mechanism may be related to an increased angle between the subfragment-2 part of the cross-bridge and the backbone of the thick filament, perhaps leading to buckling of cross-bridges under compression.

Actin Cytoskeleton↗
PubMed1985

A mechanical study of regulation in the striated adductor muscle of the scallop.

Chemically skinned fibre bundles were prepared from the striated adductor muscle of the sea scallop, Placopecten magellanicus. The relation between tension and calcium concentration was determined in activating solutions containing 5 mM-MgATP, ionic strength 0.2, pH 7.1 at 20 degrees C. The isometric tension rose from zero to its maximum value between pCa 6.0 and 5.2. The steepness of the relation cannot be accounted for in terms of the binding of calcium to the two known sites on myosin and suggests that there must be an additional, co-operative mechanism. The regulatory light chain content of the fibre bundles was determined by urea gel electrophoresis and was found to be approximately 2 light chains per myosin molecule. The regulatory light chains were removed completely by treatment with EDTA at 25-30 degrees C. Fibre bundles then showed a total loss of control over contraction; a high tension was generated whether or not calcium was present in the bathing solution. Complete removal of the regulatory light chains did not greatly affect the tension generated or the stiffness in the rigor state. Control of contraction could be restored completely by the addition of regulatory light chains from scallop muscle. Treatment with EDTA at 0-12 degrees C resulted in the removal of 0.76-2.0 regulatory light chains per myosin molecule. Fibre bundles for which removal was less than complete were partially sensitive to calcium, i.e. tension was higher in the presence of calcium than in its absence. The results indicate that the normal mechanism of tension generation in scallop muscle is mediated primarily through myosin and not thin filament control. This finding is consistent with previous studies of the ATPase activity of myofibrils from scallop muscle.

Animals↗
PubMed1985

Tension transients during steady shortening of frog muscle fibres.

Single intact fibres from frog muscle at 0-1 degrees C were stimulated to produce isometric tetani at a sarcomere length of about 2.25 micron, using a spot-follower apparatus to control the length of the central part of a fibre. When the plateau of the tetanus was reached the fibre was forced to shorten by applying a step and ramp length change in an approximation to an isotonic release. When tension had reached a steady level, Ti, during shortening, tension transients were elicited by applying step changes of length, complete within 0.2 ms, ranging from a stretch of 1.5 nm per half-sarcomere to a release of 6 nm per half-sarcomere. The tension transients recorded during shortening were qualitatively similar to those previously recorded in isometric tetani. There were four phases: phase 1, the change of tension during the step; phase 2, a rapid partial recovery of tension; phase 3, a delay or reversal of recovery; phase 4, a slower recovery of tension to the level before the step was applied. Measurements were made of the extreme tension, T1, attained during a step, and the level, T2, to which tension recovers in phase 2. The excursion of tension, [T1-Ti], during a small step of given size, fell with increase of shortening velocity, reaching about 40% of the isometric value near the maximum velocity of shortening. T2 fell as shortening velocity was increased and the fraction of steady tension recovered, T2/Ti, also decreased, so that the proportion of tension recovery in phase 4 increased. All the recovery phases became progressively more rapid with increase of shortening velocity. The early tension response was matched with a delay-line simulator so as to estimate the value of the instantaneous stiffness. Stiffness during shortening was found to decrease approximately linearly with tension, reaching about 35% of the isometric value as tension approached zero. It was impossible to match the early tension response in a rapidly shortening fibre without assuming decreased stiffness. The decline of stiffness is interpreted as due largely to reduced number of attached cross-bridges, but quantitative estimates would be affected by possible filament compliance and non-linearity of cross-bridge stiffness. The decrease in T2 also suggests fewer cross-bridges are attached as shortening velocity increases, but uncertainties about the processes determining phase 2 during shortening do not permit a precise estimate of stiffness to be made.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Changes in the lateral filament spacing of skinned muscle fibres when cross-bridges attach.

When a skinned fibre prepared from frog skeletal muscle goes from the relaxed to the rigor state at a sarcomere length of about 2.2 micron, the 1, 0 transverse spacing of the filament lattice, measured by X-ray diffraction, decreases by about 11%. In measurements at various sarcomere lengths, the decrease in the spacing was approximately proportional to the degree of overlap between the thick and thin filaments. This suggests that the shrinkage of the lattice is caused by a lateral force produced by cross-bridges. In order to estimate the magnitude of the lateral force, the decrease of spacing between relaxed and rigor states was compared with the shrinkage caused osmotically by adding a high molecular weight polymer, polyvinylpyrrolidone, to the bathing solution. The results indicate that the lateral force produced per unit length of thick filament in the overlap zone is of the same order of magnitude as the axially directed force produced during maximum isometric contraction (10(-10) to 10(-9) N/micron). Experiments in the presence of a high concentration of polyvinylpyrrolidone (100 g/l) show that when the lattice spacing is decreased osmotically beyond a certain value, the lateral force produced when the fibre goes into rigor changes its direction, causing the lattice to swell. This result can be explained by assuming that there is an optimum interfilament spacing at which the cross-bridges produce no lateral force. At other spacings, the lateral force tends to displace the filament lattice toward that optimum value.

Animals↗