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Biomedical subjects

W G Kerrick

Publications and source records attributed to W G Kerrick.

At least 19 recordsLinked to original sources
PubMed2000

Abnormal contractile function in transgenic mice expressing a familial hypertrophic cardiomyopathy-linked troponin T (I79N) mutation.

This study characterizes a transgenic animal model for the troponin T (TnT) mutation (I79N) associated with familial hypertrophic cardiomyopathy. To study the functional consequences of this mutation, we examined a wild type and two I79N-transgenic mouse lines of human cardiac TnT driven by a murine alpha-myosin heavy chain promoter. Extensive characterization of the transgenic I79N lines compared with wild type and/or nontransgenic mice demonstrated: 1) normal survival and no cardiac hypertrophy even with chronic exercise; 2) large increases in Ca(2+) sensitivity of ATPase activity and force in skinned fibers; 3) a substantial increase in the rate of force activation and an increase in the rate of force relaxation; 4) lower maximal force/cross-sectional area and ATPase activity; 5) loss of sensitivity to pH-induced shifts in the Ca(2+) dependence of force; and 6) computer simulations that reproduced experimental observations and suggested that the I79N mutation decreases the apparent off rate of Ca(2+) from troponin C and increases cross-bridge detachment rate g. Simulations for intact living fibers predict a higher basal contractility, a faster rate of force development, slower relaxation, and increased resting tension in transgenic I79N myocardium compared with transgenic wild type. These mechanisms may contribute to mortality in humans, especially in stimulated contractile states.

Animals↗
PubMed2000

Ca(2+) measurements in skinned cardiac fibers: effects of Mg(2+) on Ca(2+) activation of force and fiber ATPase.

In contrast to previous studies, a new fluorescent method was used to accurately determine the Ca(2+) concentration in test solutions used to activate skinned rat cardiac cells. This method used the calcium green-2 fluorescent indicator, which is shown to change its fluorescence over the Ca(2+) range responsible for Ca(2+) activation of force and ATPase. The dissociation constant (K(d)) of calcium green-2 for Ca(2+) was determined for three different Mg(2+) concentrations in solutions similar to those used in the experiment. Increasing Mg(2+) concentration from 1.0 to 8.0 mM had no significant effect on the Ca(2+) sensitivity of either force or actomyosin ATPase activity, in contrast to previous reported studies on force. The ATPase activity was activated at lower Ca(2+) concentration than the force. The ratio (ATPase/force) is proportional to the dissociation rate of force-generating myosin cross bridges and decreased during Ca(2+) activation. These findings are consistent with the hypothesis that cardiac muscle contraction is activated by a single Ca(2+)-specific binding site on troponin C.

Actomyosin↗
PubMed1999

Troponin C regulates the rate constant for the dissociation of force-generating myosin cross-bridges in cardiac muscle.

It is well known that cardiac troponin C (cTnC) regulates the association of force-generating myosin cross-bridges. We report here evidence for an additional role for cTnC. This hypothesis states that Ca2+ binds more strongly to cTnC when force-generating myosin cross-bridges are attached to actin and that removal of this bound Ca2+ accelerates the dissociation of force-generating myosin cross-bridges. Intact Fura-2-loaded rat papillary muscles and skinned (permeabilized) ventricular preparations were used. The preparations were mounted in the Guth Muscle Research System which is capable of measuring simultaneously fluorescence and force in response to length perturbations. All mechanical perturbations of muscle length (isotonic shortening, quick stretches and releases, and length vibrations) which cause dissociation of force-generating myosin cross-bridges during a twitch resulted in Ca2+ being released from troponin as judged from changes in the Ca2+ transients (Fura-2 (340/380) fluorescence ratio). Thus dissociation of force-generating myosin cross-bridges cause Ca2+ to be released from cTnC. Conversely, it would be expected that removal of strongly bound Ca2+ from cTnC would result in an increase in the rate of dissociation of force-generating myosin cross-bridges. To test this hypothesis actomyosin ATPase (NADH fluorescence change) and isometric force were measured in skinned cardiac preparations. The ratio of the ATPase/Force is proportional to the rate constant (gapp) for the dissociation of force-generating myosin cross-bridges. The data showed that decreasing the amount of Ca2+ bound to cTnC in skinned cardiac fibers caused an increase in the ratio of ATPase/Force, the rate of dissociation (gapp) of force-generating myosin cross-bridges.

Animals↗
PubMed1997

Fluorescent probes attached to Cys 35 or Cys 84 in cardiac troponin C are differentially sensitive to Ca(2+)-dependent events in vitro and in situ.

The goal of the current study was to generate recombinant cTnC proteins with single Cys residues as sites for attachment of fluorescent probes that can distinguish between the structural effects of myosin cross bridges and direct Ca2+ binding to cTnC (cardiac and slow skeletal troponin C) in skinned fibers. We anticipated that cTnC proteins which retain the endogenous Cys 35 (cTnC(C35)) or Cys 84 (cTnC(C84)) would provide fluorescent probes with distinct microenvironments, since these residues are on opposite sides of the globular regulatory domain. In vitro experiments that showed IAANS (2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) coupled to Cys 35 can induce unwanted structural perturbations as evidenced by a decreased affinity of site II for Ca2+ when IAANS-labeled cTnC(C35) is bound to cTnI. Important structural features involving Cys 35 in the inactive site I are suggested by a Ca(2+)-dependent increase in reactivity of Cys 35 with sulfhydryl specific reagents when cTnC(C35) is associated with cTnI. These characteristics are not seen for cTnC(C84). When incorporated in situ into skinned cardiac muscle fibers, native cTnC with IAANS bound to both Cys 35 and Cys 84 showed a pCa50 of fluorescence which preceded that of force, while the pCa50 values of both force and fluorescence were coincident for IAANS-labeled cTnC(C84). Disruption of force-producing myosin cross bridges had no effect on the pCa50 of fluorescence for IAANS-labeled cTnC(C84), but induced a rightward shift in the pCa50 of fluorescence for IAANS-labeled native cTnC. These data can be interpreted to indicate that cTnC with IAANS bound to both Cys 35 and C84 senses either myosin cross bridges or direct Ca2+ binding and myosin-induced cooperativity, while IAANS bound to Cys 84 alone senses conformations that are tightly coupled with force generation.

Amino Acid Sequence↗
PubMed1997

The effect of pH on the Ca2+ affinity of the Ca2+ regulatory sites of skeletal and cardiac troponin C in skinned muscle fibres.

It is known that intracellular pH drops rapidly after the onset of ischemia in cardiac muscle and may play some role in the rapid drop in force that ensues. It is also known that alpha 1-adrenoceptor agonists alkalinize intracellular pH by stimulating Na+/H+ exchange and may represent a mechanism which facilitates recovery of intracellular pH from acidosis. Lowering or raising pH shifts the Ca2+ dependence of force development in muscle fibres to higher or lower free Ca2+ concentrations, respectively, yet the precise mechanism is unknown. To investigate this phenomenon we have used skinned skeletal or cardiac muscle fibres whose endogenous troponin C (TnC) has been replaced with chicken skeletal TnC labelled with DANZ (STnCDANZ) or recombinant cardiac TnC labelled with IAANS (CTnC3(C84)[AANS), respectively. The fluorescence of the STnCDANZ or CTnC3(C84)IAANS was enhanced by Ca2+ binding to the Ca(2+)-specific (regulatory) site(s) of STnC or CTnC when incorporated into skinned fibres, and was measured simultaneously with force. When the pH was changed from 7.0 to 6.5 or 7.5 the shift in the Ca2+ dependence of force paralleled the shift in fluorescence. Since the force and fluorescence shift in parallel as the pH is lowered or raised, it can be concluded that these changes in Ca2+ sensitivity are caused by a decrease or increase, respectively, in the Ca2+ affinity of the Ca(2+)-specific site(s) of TnC. Since lowering or raising the pH also resulted in lower or higher, respectively, maximal Ca2+ activated force while maximal fluorescence remained unchanged, it is possible that H+ may act indirectly, as well, by reducing or increasing, respectively, the number or type of crossbridges attached to actin and thereby alter the crossbridge induced depression or elevation, respectively of the observed TnC Ca2+ affinity. Experiments with 2,3-butanedione monoxime, however, where force-generating crossbridges were greatly reduced, indicated that the pH effect may be primarily related to a direct change in the Ca2+ affinity to the regulatory sites of TnC.

Amino Acid Substitution↗
PubMed1994

Relationship between ATPase activity, Ca2+, and force in alpha-toxin- and beta-escin-treated smooth muscle.

Smooth muscle was made permeable with alpha-toxin and beta-escin. ATPase activity was measured using a phosphoenolpyruvate-pyruvate kinase regenerating system for ATP that was monitored by NADH fluorescence changes, and Ca2+ was measured using fura 2 fluorescence. alpha-Toxin-and beta-escin-treated bundles of cells had a high ATPase activity, which was reduced 80% when exposed to 1% Triton X-100. This Triton-sensitive ATPase activity was increased by approximately 20% when GTP or GTP gamma S was added to the solutions and was of much greater magnitude than the Ca(2+)-activated ATPase associated with contraction. This high membrane ATPase activity will cause a gradient of ATP into and ADP out of the bundle of cells. Thus modulation of this ATPase by G-protein-receptor mechanisms could alter the force at a constant Ca2+ concentration by changing the ADP/ATP ratio within the cells. Measurements of the fura 2 fluorescence ratio (340/380) in alpha-toxin-treated bundles of cells following sudden changes in extracellular Ca2+ showed that the cells were not freely permeable to Ca EGTA. Similar experiments in beta-escin-treated cells showed the cells to be much more permeable to Ca EGTA. These experiments indicate that great care must be taken in alpha-toxin- and beta-escin-treated fibers to make sure that the intracellular ATP, ADP, and Ca2+ are held constant.

Adenosine Triphosphatases↗
PubMed1993

Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP85/CD44)-cytoskeleton interaction.

The purposes of this study are to characterize the binding of hyaluronic acid (HA) to mouse T lymphoma cells, to measure changes in intracellular Ca2+ after HA binding, to elucidate the interaction between the HA receptor, GP85(CD44), and ankyrin in the membrane skeleton, and finally to correlate these events with HA receptor patching/capping and cell adhesion to HA. First, we established an in vivo assay using [3H]HA to measure the binding of HA to mouse T lymphoma cells, and found that the binding of [3H]HA to these cells is readily inhibited by the addition of anti-GP85(CD44) antibody suggesting that GP85(CD44) is the HA receptor. Next, we examined various signal transducing events that occur after HA binds to its receptor on mouse T lymphoma cells. The results of these studies indicate that the concentration of intracellular Ca2+ (as measured by Fura-2 fluorescence) begins to increase within seconds, and reaches a maximal level 5 min after the addition of HA to the cells. After this increase of intracellular Ca2+, HA induces both its receptors, GP85(CD44), to form patched/capped structures, and cell adhesion to HA-coated plates. Furthermore, we have determined that GP85(CD44) binds directly and specifically to ankyrin (Kd approximately 1.94 nM) in a saturable manner; and that ankyrin is preferentially accumulated underneath the HA-induced GP85(CD44) capped structures. The Ca2+ ionophore, ionomycin, was found to stimulate HA-induced receptor capping and adhesion while EGTA (a Ca2+ chelator), nefedipine/bepridil (Ca2+ channel blockers), W-7 (a calmodulin antagonist), and cytochalasin D (a microfilament inhibitor), but not colchicine (a microtubule disrupting agent), inhibit HA-induced receptor redistribution and adhesion to HA-coated plates. These findings strongly suggest that ankyrin plays an important role in linking the HA receptor, GP85(CD44), to the membrane-associated actomyosin contractile system during hyaluronic acid-mediated lymphocyte activation.

Animals↗
PubMed1993

Rapid stimulation of Ins (1,4,5)P3 production in rat aorta by NE: correlation with contractile state.

Rapid stimulation of Ins(1,4,5)P3 production in rat aorta by NE: correlation with contractile state. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H126-H132, 1993.--The isomeric composition of inositol phosphates generated in response to norepinephrine (NE) stimulation and the relationship of inositol phosphate production to release of intracellular Ca2+ as measured by contraction were characterized in rat aorta prelabeled with [3H]inositol. NE stimulated a rapid and transient increase in labeled D-myo-inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] levels. A maximal increase in labeled Ins(1,4,5)P3 occurred within 15 s of stimulation followed by a decline to control levels at 5 min. D-Myo-inositol 1,3,4-trisphosphate [Ins-(1,3,4)P3] and D-myo-inositol 1-monophosphate [Ins(1)P] levels also increased rapidly in response to NE. In contrast to the transient production of Ins(1,4,5)P3, Ins(1,3,4)P3 and Ins(1)P production was maintained in the presence of NE. Half-maximal stimulation of Ins(1,4,5)P3 production and Ca2+ release occurred at 0.3 microM NE, and maximal effects were obtained with 10 microM NE. The concentration-response curve and time course for production of Ins(1,4,5)P3 correlated with the neurotransmitter-induced Ca2+ release from intracellular stores, indicating that the level of Ins(1,4,5)P3 regulated the Ca(2+)-release mechanism. In the continued presence of NE, the intracellular pools did not completely refill with Ca2+ despite the return of Ins-(1,4,5)P3 levels to basal at 5 min. These results demonstrate that NE stimulates a rapid increase in Ins(1,4,5)P3 that correlates with contraction in Ca(2+)-free buffer. The reuptake of Ca2+ into intracellular stores is regulated by a mechanism that may not involve Ins(1,4,5)P3.

Animals↗
PubMed1992

Effects of the constitutively active proteolytic fragment of protein kinase C on the contractile properties of demembranated smooth muscle fibres.

The role of protein kinase C (PKC) in regulating the contractile state of smooth muscle was investigated using the constitutively active catalytic fragment of PKC (PKM) with skinned (demembranated) chicken gizzard fibres. PKM attenuated a submaximal contraction in gizzard smooth muscle skinned fibres, but not in rabbit cardiac skinned fibres. PKM-mediated relaxation of submaximal contractions of smooth muscle was accompanied by a reduction in the rate of ATP hydrolysis in the fibre and by phosphorylation of the 20 kDa light chain of gizzard myosin at the PKC sites (serine-1, serine-2 and threonine-9). In addition, several other endogenous proteins were phosphorylated by PKM. However, the inhibitory effects on tension and ATPase are consistent with the biochemical effects of PKC-catalysed phosphorylation of myosin, i.e. reduction of the actin-activated MgATPase activity of myosin prephosphorylated at serine-19 by myosin light chain kinase. Pretreatment of skinned fibres with PKM and ATP gamma S in the absence of Ca2+ had no inhibitory effect on the subsequent submaximal Ca(2+)-activation of force. Consistent with this observation, PKC was not able to utilize ATP gamma S as a substrate, confirming that the observed effects were the result of PKM-catalysed protein phosphorylation. We suggest that PKC may have two distinct effects on smooth muscle contraction: translocation of PKC to the sarcolemma on stimulation results in phosphorylation of a protein(s) other than myosin and a slow, sustained contraction; in some circumstances PKC may undergo proteolysis to PKM resulting in myosin phosphorylation at PKC-specific sites, a reduction in ATPase activity and relaxation of the muscle.

Adenosine Triphosphatases↗
PubMed1991

Functional characterization of the two isoforms of troponin C from the arthropod Balanus nubilus.

Two isoforms of troponin C (BTnC1 and BTnC2) from the striated muscle of the arthropod Balanus nubilus Darwin (giant barnacle) have been purified (Potter et al., 1987; Collins et al., 1991). Both isoforms were present in all of the white striated muscle fibres studied but not in the red fibres. The ratio of BTnC2 to BTnC1 in different fibre types varied between 3:1 and 1:1. Both forms of TnC could be readily extracted from myofibrillar bundles of barnacle muscle in low ionic strength EDTA solutions, reducing force activation to less than 10%. Both forms either separately or together reassociated with the TnC-depleted fibres in a relaxing (LR) solution (pCa greater than 8.0, [Mg2+] free = 1 mM, I = 0.15 M), and the reconstituted fibres could be subsequently activated in contraction (LA) solution (pCa = less than 3.8, [Mg2+] free = 1 mM, I = 0.15 M). The dissociation of BTnC 1 + 2 is blocked in low ionic strength solutions containing Mg2+ (greater than or equal to 10 mM). The two isoforms of crayfish TnC (CrTnC1 and CrTnC2) were also found to be equivalent to the barnacle TnCs in their ability to reactivate TnC-depleted barnacle myofibrillar bundles. Similar experiments using rabbit skeletal muscle TnC (STnC) (I = 0.15 M) in BTnC-depleted myofibrillar bundles of barnacle showed considerable variability. STnC could associate, although weakly, with the depleted bundles in either LR or LA, and force could be partially restored. In neither situation was it as effective as either BTnC or CrTnC. Interestingly, bovine cardiac TnC (CTnC), although it did not associate at pCa greater than 7.0, did associate and effectively activate force at pCa less than 3.8, but dissociated on return to pCa greater than 7.0 (LR). Neither barnacle TnC isoform associated with TnC-depleted skinned fibres from rabbit skeletal muscle at pCa greater than 7.0, but did associate and activate these fibres at pCa less than 3.8. Once these fibres were returned to LR and then placed in LA at pCa 3.8 all BTnC-restored force was lost, indicating a dissociation of BTnC once the Ca2+ is lowered, as observed with CTnC in barnacle myofibrillar bundles. Finally, the inhibitory effect of BTnI on force and the absence of an effect of calmodulin, trifluoperazine or ATP-gamma-S on force were all taken as evidence for a thin filament regulated Ca2+ control system.

Animals↗
PubMed1991

The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres.

The effect of Ca2+ activation on the apparent rate constant governing the dissociation of force generating myosin crossbridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21 +/- 1 degree C. Simultaneous measurements of Ca2(+)-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnCDANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnCDANZ. The Ca2+ activation of isometric force occurred at approximately two times higher Ca2+ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnCDANZ fluorescence and ATPase activity occurred over approximately the same Ca2+ concentration range at substantially lower concentrations of Ca2+ than did force, this data suggests that the TnCDANZ fluorescence is associated with the Ca2+ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constant gapp for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure of gapp shows approximately a fivefold decrease during Ca2+ activation of isometric force. This change in gapp is responsible for separation of the Ca2+ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change in gapp is reduced and consequently the difference in Ca2+ sensitivity between normalized steady state ATPase and force is also reduced.

Actins↗
PubMed1990

The relationship between ATPase activity, isometric force, and myosin light-chain phosphorylation and thiophosphorylation in skinned smooth muscle fiber bundles from chicken gizzard.

Isometric force developed by skinned gizzard muscle fiber bundles and levels of phosphorylation and thiophosphorylation of the 20,000-dalton myosin light chain were determined. These data showed a highly non-linear relationship between isometric force and myosin light-chain phosphorylation. Maximum force was developed at approximately 0.2 mol of phosphate/mol of light chain as reported previously (Hoar, P. E., Kerrick, W. G. L., and Cassidy, P. S. (1979) Science 204, 503-506). In contrast, the relationship between isometric force and myosin light-chain thiophosphorylation was linear, with maximum force occurring at 1.0 mol of thiophosphate/mol of myosin light chain. These observations are consistent with the latch-bridge hypothesis for conditions of varying myosin light-chain phosphatase/myosin light-chain kinase activity ratios as discussed by Hai and Murphy [1988) Am. J. Physiol. 254, C99-C106). To further test the latch-bridge hypothesis, ATPase activity was also measured during isometric force development in these fiber bundles. The relationship between isometric force and ATPase activity was linear whether the myosin light chains were phosphorylated or thiophosphorylated. Thus the number of cycling myosin cross-bridges, as measured by ATPase activity, was directly proportional to the force the muscle developed, not to the level of myosin light-chain phosphorylation. This finding that high levels of tension generated at low levels of light-chain phosphorylation are associated with high levels of ATPase activity is inconsistent with the latch-bridge model (Hai and Murphy, 1988).

Adenosine Triphosphatases↗
PubMed1989

The role of caldesmon in the regulation of receptor capping in mouse T-lymphoma cell.

Several complementary techniques, including immunocytochemical and immunobiochemical analyses, two-dimensional gel electrophoresis, and peptide mapping, were used in this study to examine the involvement of caldesmon in lymphocyte receptor capping. We have found a lymphoma 140-kDa polypeptide that is structurally similar to muscle caldesmon, suggesting that this polypeptide may be a lymphoma caldesmon. When lymphoma 140-kDa polypeptide is extracted from permeabilized cells using 25 mM MgCl2, capping is inhibited. Adding the 140-kDa protein or gizzard caldesmon back to the extracted cells restores their ability to cap. These findings suggest that actin-linked regulatory proteins such as caldesmon may be critically important to actomyosin-mediated contraction which, in turn, is responsible for collecting receptors into cap structures.

Animals↗
PubMed1988

Mn2+ activates skinned smooth muscle cells in the absence of myosin light chain phosphorylation.

Two effects of Mn2+ on skinned fibers from chicken gizzard smooth muscle were observed, dependent on the presence or absence of dithiothreitol (DTT) reducing agent. One involves protein oxidation (in the absence of DTT) with production of a "latch"-like state, and the other involves direct Mn2+ activation of contractile proteins. Cells activated by Mn2+ in the presence of ATP and the absence of Ca2+, Mg2+ and DTT did not relax when transferred to normal relaxing solutions. In contrast, when 5 mM DTT was included in the Mn2+ contracting solution to prevent protein oxidation by Mn2+, the cells still contracted when exposed to Mn2+, but relaxed rapidly when the Mn2+ was removed. In the presence of DTT both the Mn2+ activation and the relaxation following removal of Mn2+ were more rapid than normal Ca2+-activated contractions and relaxations. The skinned fibers activated by Mn2+ in the absence of DTT showed little active shortening unless DTT was added. This rigor-like state is probably due to oxidation of contractile proteins since the cells relaxed when exposed to a relaxing solution containing DTT (50 mM) and then contracted again in response to Ca2+ and relaxed normally. The Mn2+ activation was not associated with myosin light chain phosphorylation, in contrast to Ca2+-activated contractions.

Adenosine Triphosphate↗
PubMed1988

Skinned ventricular fibres: troponin C extraction is species-dependent and its replacement with skeletal troponin C changes Sr2+ activation properties.

Troponin C (TnC) was extracted from bundles of rat and mouse skinned cardiac ventricular cells by a method similar to that previously used to extract TnC from skinned rabbit skeletal muscle cells (Kerrick et al., J. biol. Chem. 260, 15687-93, 1985) and replaced with either bovine cardiac or rabbit fast-twitch skeletal TnC. In contrast, the same TnC extraction conditions will not extract TnC from either bovine or rabbit cardiac skinned fibres. When the extracted TnC was replaced by bovine cardiac TnC the Sr2+-activated tension relationship was not altered from control values. In contrast, replacement of the endogenous TnC with exogenous rabbit fast-twitch TnC caused the relationship between Sr2+ concentration and tension to shift towards higher concentrations of Sr2+. The Sr2+-activated tension of rat fibres with fast-twitch TnC was identical to that of rabbit fast-twitch skinned fibres. Partial skeletal TnC substitution in skinned cardiac cells gradually shifted the relationship between tension and [Sr2+] to higher Sr2+ concentrations and caused the cells to be activated over a wider range of Sr2+ concentrations. Thus it appears that the activation of rat cardiac skinned cells by Sr2+ is determined by characteristics of the TnC. In contrast, the Sr2+ activation of skinned rabbit fast-twitch skeletal fibres containing either cardiac or skeletal TnC is identical, strongly suggesting that protein-protein interactions determined the Sr2+ -activation properties in these fibres.

Animals↗