Butanedione Monoxime Research Articles

Left ventricular diastolic dysfunction during demand ischemia: rigor underlies increased stiffness without calcium-mediated tension. Amelioration by glycolytic substrate

OBJECTIVESThe goal of this study was to determine the subcellular mechanism(s) underlying increased left ventricular (LV) diastolic chamber stiffness (DCS) during angina (demand ischemia).BACKGROUNDIncreased DCS may result from increased diastolic myocyte calcium concentration and/or rigor. Therefore, we assessed the effects of direct alterations of both calcium-activated tension and high-energy phosphates on increased DCS.METHODSDemand ischemia was reproduced in isolated, isovolumic, red-cell perfused rabbit hearts by imposing low-flow ischemia and pacing tachycardia. This resulted in increased DCS. Interventions were performed after LV end-diastolic pressure had increased approximately 7 mm Hg. Initially, to determine the effects of altered calcium concentration or myofilament calcium responsiveness, hearts received either: 1) 5 or 14 mmol/L calcium chloride; 2) 8 mmol/L egtazic acid; 3) 5 mmol/L butane-dione-monoxime (BDM); or 4) 50 mmol/L ammonium chloride (NH4Cl). Then, to assess the contribution of decreased high-energy phosphate supply, hearts received 5) glucose (25 mmol/L) and insulin (400 μU/ml).RESULTS1) Calcium chloride, 5 and 14 mmol/L, increased LV systolic pressure by 42% and 70%, respectively (p < 0.001), indicating increased calcium-activated tension, but did not further increase DCS, implying intact diastolic calcium resequestration. 2) Egtazic acid reduced LV systolic pressure by 30% (p < 0.001), indicating reduced intracellular calcium, but failed to reduce increased DCS. 3) Butane-dione-monoxime and NH4Cl chloride affected contractile function (i.e., a calcium-driven force) but did not alter increased DCS. 4) Glucose and insulin, which increase high-energy phosphates during ischemia, reduced increased DCS by 50% (p < 0.001).CONCLUSIONSIncreased DCS during demand ischemia was insensitive to maneuvers altering intracellular calcium concentration or myofilament calcium-responsiveness, that is, evidence against an etiology of calcium-activated tension. In contrast, increased glycolytic substrate ameliorated increased DCS, supporting a primary mechanism of rigor-bond formation.

Specific changes to the mechanism of cell locomotion induced by overexpression of beta-actin.

Overexpression of beta-actin is known to alter cell morphology, though its effect on cell motility has not been documented previously. Here we show that overexpressing beta-actin in myoblasts has striking effects on motility, increasing cell speed to almost double that of control cells. This occurs by increasing the areas of protrusion and retraction and is accompanied by raised levels of beta-actin in the newly protruded regions. These regions of the cell margin, however, show decreased levels of polymerised actin, indicating that protrusion can outpace the rate of actin polymerisation in these cells. Moreover, the expression of beta*-actin (a G244D mutant, which shows defective polymerisation in vitro) is equally effective at increasing speed and protrusion. Concomitant changes in actin binding proteins show no evidence of a consistent mechanism for increasing the rate of actin polymerisation in these actin overexpressing cells. The increase in motility is confined to poorly spread cells in both cases and the excess motility can be abolished by blocking myosin function with butanedione monoxime (BDM). Our observations on normal myoblasts are consistent with the view that they protrude by the assembly and cross linking of actin filaments. In contrast, the additional motility shown by cells overexpressing beta-actin appears not to result from an increase in the rate of actin polymerisation but to depend on myosin function. This suggests that the additional protrusion arises from a different mechanism. We discuss the possibility that it is related to retraction-induced protrusion in fibroblasts. In this phenomenon, a wave of increased protrusion follows a sudden collapse in cell spreading. This view could explain why it is only the additional motility that depends on spreading, and has implications for understanding the differences in locomotion that distinguish tissue cells from highly invasive cell types such as leucocytes and malignant cells.

Calyculin‐A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggs

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex “actin cup” since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a 32P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. Cell Motil. Cytoskeleton 48:245–261, 2001. © 2001 Wiley-Liss, Inc.

Calyculin-A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggs.

When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a (32)P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis.

ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres.

The contractile properties and ATPase activity of skinned human skeletal muscle fibres from vastus lateralis were examined. Fibre types were resolved from single fibre segments by SDS-polyacrylamide gel electrophoresis. ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. The partitioning of ATPase activity into (a) calcium-activated activity due to actomyosin (AM) interaction, (b) calcium-activated activity of the sarcoplasmic reticular (SR) calcium pump, and (c) basal (calcium independent) activity was investigated by comparing ATP utilization before and after exposure of the preparations for 30 min to a solution containing 0.5 % Triton X-100, which effectively abolished the SR ATPase activity. Partitioning of ATPase activity was also determined by measuring ATP utilization and force at different concentrations of butanedione monoxime (BDM), which inhibits AM interaction. The results obtained with Triton X-100 and BDM were similar. At saturating Ca2+ concentrations and 20 degrees C, the AM, SR and basal ATPase activities per litre cell volume (+/- S.E.M.) amounted to 46 +/- 4, 51 +/- 4 and 19 +/- 2 muM s-1 in type I fibres (n = 21), 139 +/- 14, 69 +/- 8 and 30 +/- 3 muM s-1 in type IIA fibres (n = 25), 137 +/- 22, 175 +/- 28 and 26 +/- 8 muM s-1 in type IIA/B fibres (n = 4) and 108 +/- 13, 169 +/- 42 and 32 +/- 8 muM s-1 in type IIB fibres (n = 2). These results indicate that ATP utilization for SR Ca2+ pumping in fast fibres is considerably larger than in slow fibres. The SR ATPase activity in human muscle represents a considerable fraction of the total (AM + SR + basal) ATPase activity.

Reprogramming of gene expression in cultured cardiomyocytes and in explanted hearts by the myosin ATPase inhibitor butanedione monoxime.

Butanedione monoxime (BDM) is a reversible myosin ATPase inhibitor. Its use in transplantation medicine may be of benefit in the preservation of hearts. As little is known about its ability to prevent stress and metabolic deregulation, we wanted to investigate the genomic response in cultured cardiomyocytes and explanted, preserved hearts at the transcriptional level. We thus investigated the gene expression of the transcription factors GATA-4, Nkx2.5, MEF-2c, and Oct-1 and of the downstream target genes atrial and brain natriuretic peptide, alpha- and beta-myosin heavy chain, alpha-cardiac actin, and alpha-skeletal actin. Additionally, lactate dehydrogenase and creatine kinase enzyme activities were measured as markers for membrane integrity and metabolic deregulation of cardiomyocytes. In untreated cardiomyocyte cultures, expression of GATA-4 and Nkx2.5 was increased 7- and 4-fold, 72 hr after isolation, but the gene expression of MEF-2c and Oct-1 was reduced to 10% and 70%, at day 3 in culture. We show atrial natriuretic peptide and brain natriuretic peptide gene expression to be maximal 24 and 72 hr after isolation, the level being 3- and 2-fold, when compared with freshly isolated cells. The gene expression of alpha- and beta-myosin heavy chain was reduced to approximately 30% at day 3 in culture and similar observations were made for alpha-cardiac and alpha-skeletal actin, which declined to approximately 20% and 10% of control values, 72 hr after isolation. BDM prevented at the transcriptional level enhanced expression of markers for stress and metabolic deregulation, and the activities of lactate dehydrogenase and creatine kinase were highly significantly reduced. Similar results were obtained when explanted hearts were stored in BDM-containing organ preservation solution. Preservation of metabolic function in donor organs is of critical importance in transplantation medicine, and we show gene markers for stress and metabolic deregulation in cultures of cardiomyocytes and explanted hearts to be significantly reduced by BDM. Reprogramming of gene expression of nuclear transcription factors and downstream target genes may prolong the acceptable storage time between explantation and transplantation.

Butanedione monoxime increases the viability and yield of adult cardiomyocytes in primary cultures.

Various protocols for the isolation and cultivation of adult rat cardiomyocytes were compared, and the cytoprotective potential of the reversible myosin ATPase inhibitor butanedione monoxime (BDM) was evaluated based on cell yield, cell vitality, lactate dehydrogenase (LDH) and creatine kinase (CK) release, and the mRNA expression of atrial natriuretic peptide (ANP). Overall, a yield of 11.9 x 10(6)cells with >92% cell vitality was obtained when BDM was added to the isolation and cultivation buffers. In contrast, cell vitality ranged from 30% to 70% and cell yield was (4-10) x 10(6) when standard methods for the isolation of cardiomyocytes were used. Butanedione monoxime, at a 15 mM concentration, was cytoprotective during the isolation and cultivation of heart muscle cells, as judged by the morphological appearance (rod shape, lack of bleb formation, and other cytoskeleton defects) and the mRNA expression of the ANP gene. The activities of LDH and CK were also significantly reduced (p < 0.05%) when BDM was added to the isolation and cultivation buffer. The results obtained with BDM warrant further investigation into its cytoprotective potential during ischemia and damage to the cytoskeleton.

EFFECTS OF THE MYOSIN INHIBITOR 2,3-BUTANEDIONE MONOXIME (BDM) ON CELL SHAPE, LOCOMOTION AND FLUID PINOCYTOSIS IN HUMAN POLYMORPHONUCLEAR LEUCOCYTES

We investigated the role of myosin in polymorphonuclear leucocyte (PMN) shape changes, locomotion, and fluid pinocytosis using the myosin inhibitor 2,3 butanedione monoxime (BDM). Treatment of resting spherical PMNs with BDM produced spheroid cells showing small continuous shape changes (IC50=15.5m m BDM) and occasionally small blebs. Cell polarity, as induced by the chemotactic peptide fNLPNTL or by colchicine, and locomotion were completely suppressed (IC50=8.4 to 10m m). Suppression of fNLPNTL- or colchicine-induced cell polarity produced spheroid cells, suppression of PMA-induced shape changes and fluid pinocytosis produced non-motile spherical cells (IC50=25 to 30m m BDM). BDM suppressed formation of lamellipodia but not formation of blebs. Suppression of microvilli by BDM as observed in resting spherical cells was partially antagonized by PMA. The results suggest that myosin is involved in stabilizing the shape of resting spherical cells, including microvilli, and that myosin is required for cell polarity, locomotion, fluid pinocytosis and for formation of lamellipodia, but not for formation of blebs.

Contractility affects stress fiber remodeling and reorientation of endothelial cells subjected to cyclic mechanical stretching.

We studied the effect of contractility on stress fiber remodeling and orientation response of human aortic endothelial cells subjected to cyclic mechanical stretching. The cells were grown on silicone membranes subjected to 10% cyclic pure uniaxial stretching in the presence or absence of 2,3 butanedione monoxime (BDM), a proven inhibitor of cellular contractility. It was found that treatment of the cells with BDM (40 mM) abolished stress fibers and blocked cell reorientation in response to cyclic stretching, indicating that contractility is required for these two cellular responses. When cells were stretched in the presence of N-acetylcysteine (NAG, 20 mM), a hydrogen peroxide (H2O2) scavenger, stress fibers were still formed and the cells reoriented--but more slowly. Specifically, compared with untreated cells, NAG treated cells after 0.5, 1, and 3 h of 10% stretching had significantly (p<0.005) less skewed orientation distributions than those of untreated cells. After the cells were treated with both NAG (20 mM) and nordihydroguaiaretic acid (NDGA, 50 microM), another antioxidant, however, stress fibers were abolished and cell reorientation was completely blocked. These results indicate that reactive oxygen species (ROS), including H2O2, affect stress fiber remodeling and reorientation of endothelial cells in response to cyclic stretching. We suggest that the effect of ROS on stress fiber remodeling and cell reorientation is due to the ability of ROS to regulate cellular contractility, which is crucial for these cellular responses.

A myosin I is involved in membrane recycling from early endosomes.

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA(-)/B(-), and myoB(-) cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA(-)/B(-) cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.

Nerve growth factor stimulates coupling of beta1 integrin to distinct transport mechanisms in the filopodia of growth cones.

The cycling of membrane receptors for substrate-bound proteins via their interaction with the actin cytoskeleton at the leading edge of growth cones and other motile cells is important for neurite outgrowth and cell migration. Receptor delivered to the leading edge binds to its ligand, which induces coupling of the receptor to a rearward flowing network of actin filaments. This coupling is thought to facilitate advance. We show here that a soluble growth factor stimulates this cycling. We have used single particle tracking to monitor the effects of nerve growth factor (NGF) on the movements of beta1 integrin in the plane of the plasma membrane of the filopodia of growth cones. Beta1 integrin was visualized by its binding of 0.2 microm beads coated with a monoclonal Ab directed against an extracellular epitope distant from the binding site for extracellular matrix ligands. The beads were observed by video microscopy. Beads coated with a low concentration of antibody, and therefore bound to unliganded receptor with little cross-linking, showed an increase in both diffusion and directed forward transport in response to NGF. Transport had a net velocity of 37 microm/minute and was characterized by brief periods of sustained forward excursions with a velocity of 75-150 microm/minute. There was a 2-fold increase in the number of beads accumulated at the tips of filopodia after 10 minutes, indicating that NGF enhanced the delivery of beta1 integrin to the periphery. Forward transport was dependent on an intact actin cytoskeleton and myosin ATPase, since treatment with cytochalasin D or the myosin ATPase inhibitor butanedione monoxime inhibited the transport but not the diffusion of receptors. NGF also greatly increased the steady rearward migration of beads coated with a high density of (&bgr;)1 integrin antibody, indicating that coupling of cross-linked receptor to the retrograde flow of actin is also enhanced. The rate of the retrograde flow of actin was unaffected by NGF. These studies show that a soluble factor can stimulate the coupling of a receptor for substrate-bound factor to two actomyosin-based transport mechanisms and thus facilitate the response of the growth cone to the substrate-bound factor by increasing cycling of the receptor at the periphery.

Role of Actin Cortex in the Subplasmalemmal Transport of Secretory Granules in PC-12 Cells

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 × 10 −4 μm 2/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg 2+ and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.

Endothelial cell retraction is induced by PAK2 monophosphorylation of myosin II.

The p21-activated kinase (PAK) family includes several enzyme isoforms regulated by the GTPases Rac1 and Cdc42. PAK1, found in brain, muscle and spleen, has been implicated in triggering cytoskeletal rearrangements such as the dissolution of stress fibers and reorganization of focal complexes. The role of the more widely distributed PAK2 in controlling the cytoskeleton has been less well studied. Previous work has demonstrated that PAK2 can monophosphorylate the myosin II regulatory light chain and induce retraction of permeabilized endothelial cells. In this report we characterize PAK2's morphological and biochemical effect on intact endothelial cells utilizing microinjection of constitutively active PAK2. Under these conditions we observed a modification of the actin cytoskeleton with retraction of endothelial cell margins accompanied by an increase in monophosphorylation of myosin II. Selective inhibitors were used to analyze the mechanism of action of PAK2. Staurosporine, a direct inhibitor of PAK2, largely prevented the action of microinjected PAK2 in endothelial cells. Butanedione monoxime, a non-specific myosin ATPase inhibitor, also inhibited the effects of PAK2 implicating myosin in the changes in cytoskeletal reorganization. In contrast, KT5926, a specific inhibitor of myosin light chain kinase was ineffective in preventing the changes in morphology and the actin cytoskeleton. The additional finding that endogenous PAK2 associates with myosin II is consistent with the proposal that cell retraction and cytoskeletal rearrangements induced by microinjected PAK2 depend on the direct activation of myosin II by PAK2 monophosphorylation of the regulatory light chain.

Role of cell contractions in cAMP-induced cardiomyocyte atrial natriuretic peptide release

Incubation of spontaneously beating ventricular cardiomyocytes from neonatal rats with prostaglandin E(2) (0.1 microM) or forskolin (0.1 microM) simultaneously increased the rate of cellular contraction and atrial natriuretic peptide (ANP) secretion. Both responses were maximal within 10-20 min of application and were accompanied by three- to fourfold increases in cAMP formation. By contrast, a higher regimen of forskolin (10 microM) promoted a 20- to 30-fold increase in basal cAMP production, which was accompanied by the abolition of contractile activity and ANP release. Low regimens of forskolin (0.1 microM) doubled the occurrence of cytosolic Ca(2+) transients associated with monolayer contraction, whereas higher regimens of forskolin (10 microM) completely suppressed Ca(2+) transients. Moreover, in quiescent cultures that were pretreated with ryanodine, tetrodotoxin, nifedipine, or butanedione monoxime, prostaglandin E(2) (0.1 microM) and forskolin (0.1 microM) failed to elicit significant ANP secretion, suggesting that cAMP-elevating agents promote ANP secretion to a great extent via an increase in cellular contraction frequency in ventricular cardiomyocytes.

Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system.

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.

Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Two components of transducer adaptation in auditory hair cells.

Mechanoelectrical transducer currents in turtle auditory hair cells adapted to maintained stimuli via a Ca(2+)-dependent mechanism characterized by two time constants of approximately 1 and 15 ms. The time course of adaptation slowed as the stimulus intensity was raised because of an increased prominence of the second component. The fast component of adaptation had a similar time constant for both positive and negative displacements and was unaffected by the myosin ATPase inhibitors, vanadate and butanedione monoxime. Adaptation was modeled by a scheme in which Ca(2+) ions, entering through open transducer channels, bind at two intracellular sites to trigger independent processes leading to channel closure. It was assumed that the second site activates a modulator with 10-fold slower kinetics than the first site. The model was implemented by computing Ca(2+) diffusion within a single stereocilium, incorporating intracellular calcium buffers and extrusion via a plasma membrane CaATPase. The theoretical results reproduced several features of the experimental responses, including sensitivity to the concentration of external Ca(2+) and intracellular calcium buffer and a dependence on the onset speed of the stimulus. The model also generated damped oscillatory transducer responses at a frequency dependent on the rate constant for the fast adaptive process. The properties of fast adaptation make it unlikely to be mediated by a myosin motor, and we suggest that it may result from Ca(2+) binding to the transducer channel or a nearby cytoskeletal element.

Pharmacologic evaluation of isometric contraction-relaxation coupling indexes in rabbit ventricular muscle

Investigations of the coupling between contraction and relaxation (contraction-relaxation [CRC] process) in isometric conditions are essential in determining whether pharmacologic interventions or cardiac diseases specifically modify isometric relaxation (intrinsic lusitropic effect) or change it in proportion with the accompanying changes in contractility (or inotropy). For this purpose, the CRC process is quantified by various indexes, derived from differentiation and/or curve fitting the whole or relaxation phase of the isometric twitch, one of the most used being τ, the time constant of the final iso(volu)metric phase of relaxation. Nevertheless, the possible redundancy and validity of such indexes have not been thoroughly investigated. Accordingly, we performed a pharmacologic evaluation of such indexes in isolated rabbit ventricular muscles isometrically contracting in vitro, using modifiers of either intracellular Ca 2+ handling (nifedipine, ryanodine, 2,5-di- tert-butyl-benzohydroquinone, all negative inotropic compounds, and BAY K 8644, a positive inotropic drug), or myofibrillar Ca 2+ sensitivity (CGP 48506, a Ca 2+ sensitizer, and butanedione monoxime, a Ca 2+ desensitizer, respectively positive and negative inotropic compounds). The isometric twitch in control conditions and in the presence of increasing concentration of each compound was analyzed to determine the classically used CRC and/or lusitropic indexes, derived either from single parameters such as the maximal rate or contraction and relaxation (+dT max and −dT max, respectively), or from curve fitting of the whole, or part, of the twitch. As the rate of isometric relaxation is dependent on myofilament properties, we expected that compounds modifying myofibrillar Ca 2+ sensitivity in an opposite direction (CGP 48506 vs butanedione monoxime) would be the only drugs exerting an intrinsic lusitropic and opposite effect on a validated CRC index. Results showed that (1) none of the tested compounds affected the slope of the linear relationship between peak twitch tension and dT max, a previously assumed CRC index, sensitive only to myofibrillar Ca 2+ sensitivity modifiers; (2) the lusitropic parameter B, derived from mathematical curve fitting of the whole isometric twitch, and the ratio +dT max/dT max, exhibited similar drug- and dose-dependency, but no opposite sensitivity to CGP 48506 and BDM for either index; and (3) negative inotropic compounds dose-dependently slowed relaxation (and conversely for positive inotropes), whether the latter was quantified by the rate constant β, derived from double exponential curve fitting of the whole relaxation phase, or by the time constants τ L and τ E, derived from the curve fitting (logistic and monoexponential, respectively) of the final phase of relaxation. Nevertheless, the pharmacologicly induced changes in β were statistically significant at lower concentrations and exhibited less individual variability, compared with the time constants. We demonstrate that intrinsic lusitropic changes can be quantified by the value of the slope of the relationship relating β to peak isometric tension: the slope value was unchanged by Ca 2+ handling modifiers, decreased by CGP 48506, and reversed by BDM (indicating number, negative, and positive intrinsic lusitropic effects respectively). Based on these data, we propose that the linear relationship between β and peak isometric tension could be used a new method to assess whether pharmacologic interventions or cardiac diseases exert intrinsic effects on isometric relaxation.

Myosin regulation of NKCC1: effects on cAMP-mediated Cl− secretion in intestinal epithelia

The basally located actin cytoskeleton has been demonstrated previously to regulate Cl- secretion from intestinal epithelia via its effects on the Na+-K+-2Cl- cotransporter (NKCC1). In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the role of myosin in the regulation of secretagogue-stimulated Cl- secretion in intestinal epithelia. The human intestinal epithelial cell line T84 was used for these studies. Prevention of myosin light chain phosphorylation with the MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase with butanedione monoxime (BDM) attenuated cAMP but not Ca2+-mediated Cl- secretion. Both ML-9 and BDM diminished cAMP activation of NKCC1. Neither apical Cl- channel activity, basolateral K+ channel activity, nor Na+-K+-ATPase were affected by these agents. Cytochalasin D prevented such attenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin light chain and subsequent contraction of basal actin-myosin bundles are crucial to the cAMP-driven activation of NKCC1 and subsequent apical Cl- efflux.

Diatom gliding is the result of an actin-myosin motility system.

Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility. No effect on diatom gliding was observed using the cytochalasins, known actin inhibitors, or the microtubule-inhibitors oryzalin and nocodazole. The latrunculins are a new group of anti-actin drugs, and we show here that they are potent inhibitors of diatom gliding, resulting in the complete disassociation of the raphe-associated actin cables. The recovery of actin staining and motility following latrunculin treatment was extremely fast. Cells exposed to latrunculin for 12 h recovered full function and actin staining within 5 sec of the drug being removed, demonstrating that the molecular components required for this motility system are immediately available. Butanedione monoxime (BDM), a known myosin inhibitor, also reversibly inhibited diatom gliding in a manner similar to the latrunculins. This work provides evidence that diatom gliding is based on an actin/myosin motility system.