Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

An Attempt to Account for the Diversity of Crustacean Muscles

Crustacean muscles are known to contain muscle fibers of variableproperties and to be innervated by phasic and/or tonic motoneuronswhich may possess synapses of diverse physiological properties.Frequently, phasic motor axons innervate short-sarcomere phasicmuscle fibers and tonic motor axons innervate long-sarcomeretonic muscle fibers, but some muscles receiving a single (tonic)motor axon contain both phasic and tonic muscle fibers. Althoughit is not known whether neural trophic influences are involvedin muscle differentiation, some neural trophic effects havebeen found in crustaceans, and it is reasonable to assume thatsuch influences may be involved in establishing the definitiveproperties of the muscle. Several other postulates must be made:(1) Phasic and tonic motor axons differ in their trophic effectiveness:(2) muscle fibers innervated relatively early in developmentby a tonic motor axon acquire the properties of tonic musclefibers, while those innervated later become intermediate orphasic muscle fibers; (3) the developmental stage of a growingor regenerating axon terminal plays a role in determinationof synaptic properties. Studies on regenerating limb buds supportthe hypothesis, which can account for the genesis of all observedtypes of crustacean neuromuscular system. Further experimentalwork is necessary to test the hypothesis.     

Distribution of three types of muscle fiber in the skeletal muscles of Rana temporaria frogs (according to histochemical findings)]

On 15 skeletal muscles with different functional profile (flexors and extensors, trunk muscles and muscles of the head) and different embryonic origin (somatic and visceral), it has been shown that muscles of R. temporaria are mixed. Muscle fibers are divided into three groups which differ in the content of lipids, the activity of succinic dehydrogenase and localization of myofibrils on a cross section. On the basis of the data obtained and those in literature, these fiber groups are qualified as phasic, tonic and transitional (slow phasic) ones. Both phasic and tonic fiber groups are uniform, whereas transitional fibers form a broad specturm. Most of the muscles studied contain all the three types of fibers. In muscles of the extremities (especially in flexors), tonic fibers together with transitional ones usually form more or less compact bundles. The highest content of tonic and transitional fibers was found in muscles which do not directly participate in locomotion. If muscles contain only two types of fibers (phasic and transitional ones), these fibers form a mosaic structure.     

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H₂S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of K_ATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H₂S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of K_ATP-channels.     

Effects of agonists and antagonists of rhyanodine receptors on potassium contractures in twitch and tonic frog skeletal muscle fibers

A comparative analysis of the effects of the concentrations of Ca2+ in external medium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of rhyanodine-sensitive Ca2+ channels of carcoplasmic reticulum on the characteristics of potassium contracture in frog twitch and tonic skeletal muscles has been performed. It was shown that the duration of contracture in tonic muscles is not restricted by the presence of Ca2+, as distinct from twitch muscles. Dandrolene does not practically affect the contractile responses of tonic fibres, and the concentration of cresol eliciting the contracture for tonic fibres is substantially higher (1 mM) than for twitch fibers (0.25 mM). In twitch fibers, the potassium contracture activated in the presence of cresol is comparable in amplitude and dynamics with the contracture under control conditions, and in tonic fibers a summing of responses without relaxation after the washing of excessive potassium is observed. This suggests that, in twitch fibers, the influx of Ca2+ can directly create the concentration sufficient for the maintenance of contraction, and in tonic fibers its involvement is mediated through the Ca(2+)-dependent activation of the beta-isoform of rhyanodine-sensitive channels.     

Comparative characteristic of Mytilus muscle cells developed in vitro and in vivo

The mussel cells from premyogenic larval stages are capable of differentiation into smooth muscle cells in vitro. However, the behavior and protein composition of these cells are not completely identical to those of smooth muscle cells of adult mussels. In this study we compared some properties of mussel muscle cells forming from cells of trochophore (premyogenic larval stage) in vitro with those of muscle cells of veliger and adult mussel. We found a substantial difference between the contractile apparatus protein composition of veliger muscle and cultivated cells. Myorod, one of the molecular markers of the phenotype of mollusc smooth muscle cells (Shelud'ko et al., 1999, Comp Biochem Physiol 122:277-285), is not a constituent of the contractile apparatus of veliger muscle. At the same time the protein composition of contractile apparatus in cultivated cells was similar to that of adult Mytilus muscles. There were only few quantitative differences between them. The contractile activity of cultivated cells was changing in time. The kinetic parameters of first spontaneous contractions were similar to those of phasic contractions, while their period was close to that of tonic contractions. After 50-55 hrs cultivation the cells produced both phasic and tonic contractions, but the character of contractile activity of cultivated cells was regulated after six days of cultivation only. However, there were no muscle cells in vitro, whose contractile activity was similar to that of veliger muscle cells. So, we concluded that properties of muscle cells forming from premyogenic larval mussel cells in culture are similar to those of muscle cells of the adult mussel, but not of veliger.     

Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor

To investigate the relationship between the oxytocin (OT) receptor (OTR) quantity and the contractile features systematically, we measured the mRNA expression levels of OTR and L-type Ca(2+) channel alpha(1C)-subunit (alpha(1C)) and examined the regulatory mechanisms of OT-induced phasic or tonic contractions of the longitudinal smooth muscles in mouse uteri. The mRNA expression of OTR in 19.0 G (19.0 days of gestation) was greater than those in nonpregnant phases, and that of alpha(1C) in estrus and 19.0 G was higher than in diestrus. OT-induced contractions sparsely occurred in diestrus. The OT-induced all-or-none-type phasic contractions at low concentrations were abolished by verapamil in both estrus and 19.0 G. OT-induced tonic contractions had similar pD(2) values in both estrus and 19.0 G. However, the magnitude in 19.0 G was much greater than that in estrus. The large tonic contractions also occurred in PGF(2alpha) receptor (FP) knockout mice in 19.0 G despite a small amount of OTR. Verapamil and Y-27632 partially inhibited the tonic contractions in 19.0 G. Cyclopiazonic acid-induced tonic contractions were reciprocally decreased with the increase in the OT-induced ones in 19.0 G. These results indicate that the phasic contractions are dependent on alpha(1C). The tonic contractions in 19.0 G are dependent on both Ca(2+) influxes via L-type Ca(2+) channels and store-operated Ca(2+) channels, and the force is augmented by the Rho signal pathway, which increases the Ca(2+) sensitivity. Thus the uterine contractions are mainly controlled by the modification of contractile signal machinery rather than simply by the OTR quantity.     

The possible role of serotonin in the rhythmicity of the crop of Aplysia dactylomela

The isolated perfused crop of Aplysia dactylomela was used to assess the effects of applied serotonin on 1st order small phasic contractions, 2nd order large tonic contractions, and 3rd order slow changes in tonus. Very low concentrations of serotonin may potentiate spontaneous contractions. Concentrations of the order of 10(-7) M serotonin may regularize "beating" of the crop by suppressing large tonic contractions while potentiating phasic contractions. Concentrations higher than 10(-5) M serotonin induce prolonged tonic contracture.     

Signaling regulatory mechanisms of the contractile activity of smooth muscle

A comparative model been designed to study a contribution of proteinkinase C-(PKC)-activated intracellular signaling pathways in generation of different contractile responses of vascular (tonic) and visceral (phasic) smooth muscles. We have determined that, in tonic smooth muscle, PKC mediates activation of MAP-kinases that phosphorylate key regulatory proteins of the contractile system, myosin light chain kinase and caldesmon, leading to upregulation of actomyosine motor activity. In contrast, the MAP-kinase activation is uncoupled from the contractile machinery in phasic smooth muscles, which also reveal high levels of myosin light chain kinase-related protein KRP that contributes to relaxation. Phosphorylation of KRP following activation of PKC or cyclic nucleotide-dependent protein kinases enhances the KRP activity and further contributes to relaxion in phasic smooth muscle. A possibility is discussed for exploitation of the comparative model described herein for investigation of specific role of other regulatory intracellular pathways in generation of vascular tonic contraction.     

Function of Peripheral Inhibitory Axons in Insects

There are now many examples in insects of axons which elicithyperpolarizing junctional potentials in the muscle fibers theyinnervate. With the muscles bathed in haemolymph, electricalstimulation of these axons causes a decrease in the magnitudeof slow contractions. This property allows them to be definedas inhibitory. Although inhibitory axons have the ability toregulate the magnitude of maintained slow contractions, thereis little evidence that this is their normal function. The inhibitoryaxons supplying at least three insect muscles function to increasethe rate of relaxations following each contraction of a rhythmicsequence. Moreover, when the haemolymph potassium concentrationis high, some inhibitory axons probably ensure complete relaxationbetween rhythmic contractions by preventing potassium contractionsin tonic muscle fibers. There is no convincing evidence thatinhibitory axons can facilitate muscular contractions by becomingactive immediately before the excitatory input.     

Tonic contractions allow metabolic recuperation of the adductor muscle during escape responses of giant scallop Placopecten magellanicus

To escape from starfish predators, giant scallops, Placopecten magellanicus, swim using series of strong phasic contractions interrupted by tonic contractions. To investigate whether these tonic contractions allow metabolic recuperation of the adductor muscle, we sampled scallops at rest (Control), after an initial series of phasic contractions (Phasic) and after 1 min of tonic contraction following their initial phasic contractions (Phasic + Tonic) and compared muscle levels of phosphoarginine, adenylate nucleotides (ATP, ADP and AMP) and adenylate energy charge (AEC). Scallops in the two active groups did not differ in the numbers of phasic contractions or the mean phasic force production. Phosphoarginine concentrations in the adductor muscle decreased with phasic activity and remained low after 1 min of tonic contraction. ATP and ADP and total adenylate levels did not differ between the three groups, but AMP levels were higher in the scallops sampled after phasic contractions than in control scallops. The AEC was reduced by phasic contractions but returned to control levels after 1 min of tonic contraction. A significant negative correlation between AEC and the number of claps in the Phasic group disappeared in the Phasic + Tonic group. Thus, tonic contractions following phasic contractions allow partial metabolic recovery of the adductor muscle by returning AEC to control levels. However, phosphoarginine levels did not recover during tonic contractions, and a negative correlation between the number of claps and phosphoarginine levels remained in the Phasic + Tonic group. By interspersing tonic contractions between series of phasic contractions, scallops improved muscle energetic status, which should help maintain phasic force production during the remainder of the escape response.     

Acetylcholine contracture of the fast muscle fibers in the lamprey, Lampetra fluviatilis]

Fast fibers of m. longitudinalis linguae respond to Ach by a transient contracture with a half-decay period of 3-5 sec. The threshold concentration of Ach is approximately 10(-7) g/ml. Ach contracture is based on even depolarization of the whole muscle membrane. Threshold level of the MP for the onset of contracture lies between --50 and --40 mV. In the presence of Ach depolarization decreases twofold within 40-70 sec. Relaxation is not due to the decrease of depolarization. Contraction--Ach concentration curve has a small slope; it reaches maximum at a concentration 10(-4) g/ml, which corresponds to the MP ca. -10mV. Equilibrium Ach potential is significantly shifted to depolarization as compared to that in frog muscles, being equal to +1+/-1.8 mV.     

Crustacean Neuromuscular Mechanisms

Properties of crustacean muscle fibers and neuromuscular synapsesare discussed, with particular reference to the problems offast and slow contraction, synaptic diversity, and peripheralinhibition. Electrical and mechanical responses of crustacean muscle fibersare variable, and govern to a large extent the muscle's performance.Fast and slow contractions are often mediated by distinct "phasic"and "tonic" muscle fibers, as in abdominal muscles, in whichsuch fibers are segregated into two parallel sets of muscles.In leg muscles the fibers are often heterogeneous in propertiesand innervation. In doubly-motor-innervated muscles of crabsthe axons producing fast and slow contractions preferentiallyinnervate rapidly and slowly contracting fibers, respectively. Crustacean neuromuscular synapses vary greatly in electricalbehavior and in ultrastructural characteristics. Some motoraxons possess both facilitating and nonfacilitating synapses.The proportion of the different types of synapse associatedwith a motor axon probably determines in large measure the propertiesof the postsynaptic potentials evoked by that axon. Pre-synaptic and post-synaptic inhibition both occur, sometimesin the same muscle. The latter type is more common. Pre-synapticinhibition is thought to be mediated by the action of an inhibitorytransmitter-substance on receptors of the motor nerve terminals.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles

Laser flash photolysis of caged adenosine triphosphate (ATP), in the presence of Ca2+, was used to examine the time course of isometric force development from rigor states in glycerinated tonic (rabbit trachealis) and phasic (guinea-pig ileum and portal vein) smooth muscles. Photolytic liberation of ATP from caged ATP initiated force development, at 20 degrees C, with half-time (t1/2) of 5.4 s in trachealis and 1.2-2.2 s in the phasic muscles. Prior to photolysis, some muscles were phosphorylated with ATP plus okadaic acid (an inhibitor of myosin light-chain phosphatase) or thiophosphorylated with ATP gamma S to fully activate the regulatory system, before turning on the contractile apparatus. In these prephosphorylated muscles, force development, after caged ATP photolysis, was more rapid than in the unphosphorylated muscles, but the t1/2 values for trachealis (0.8-1.1 s) were still longer than for ileum and portal-vein muscles (0.20-0.25 s). The results suggest that both the contractile machinery and the regulatory system are slower in the tonic than in the phasic smooth muscles. The time course of force development for each muscle type was sigmoidal, with an initial delay (td) of approximately 10% of the t1/2 value. Some possible chemical and mechanical origins of the delay are discussed.     

The effects of changes of the tonicity of the bathing fluid upon the tension generated by atrial trabeculae isolated from the heart of the frog, Rana pipiens.

Raising the tonicity of the fluid bathing frog atrial trabeculae has three effects: an initial sustained relaxation, which depends on muscle length and probably originates from structures other than the contractile apparatus; an increase in contractility, which takes the form of a transient contracture if the muscle has previously undergone a high-potassium or a low-sodium contracture, and a further rise in contractility on return to isotonic fluid (off response). The hypertonic contractures, in high-potassium or sodium-free fluids, are antagonized by local anaesthetics and in Na-free media they are unaffected by removal of extracellular coat, whereas the 'off responses' are insensitive to both experimental manoeuvres. Hypotonic fluids applied in Na-free solutions evoke a phasic and a tonic contracture, neither of which are sensitive to local anaesthetics. The tonic response is reduced by lowering the [Ca]o, and occurs at tonicities where the permeability of the cell membrane is likely to have increased. The phasic part of the hypotonic contracture resembles the 'off response' which follows exposure to hypertonic solution. The effects of hypertonic fluids and of caffeine on frog heart are alike, and are also similar to the responses induced by the same experimental manoeuvres in skeletal muscle. The results can be interpreted by assuming that the intracellular relaxing system in frog heart is sensitive to changes in tonicity, and could be functionally divided.     

Histochemical mapping and fiber size analysis of mimic muscles.

Fourteen functionally relevant mimic muscles of nine human bodies were analyzed with respect to their muscle fiber sizes and their histochemical fiber type composition. In cryostat sections stained for actomyosin ATPase, type 1 and type 2 fibers were evaluated separately by means of computer-assisted image analysis. The fiber diameters varied between 20.24 and 41.45 microns. According to the proportions of the fiber types, the mimic muscles could be classified into three groups: (1) phasic muscles, with 14 to 15 percent type 1 fibers, (2) intermediate muscles, with 28 to 37 percent type 1 fibers, and (3) tonic muscles, containing 41 to 67 percent type 1 fibers. It is concluded that one has to consider this diversity of mimic muscles when planning the surgical reconstruction of facial paralysis.     

The effect of reduced activity on the enzymatic development of phasic and tonic muscles in the chicken

The effects of reduced activity (immobilisation) on the development of the contractile enzyme, Mg2+-activated myofibrillar ATPase was studied in a tonic muscle, the anterior latissimus dorsi and in a phasic muscle, the posterior latissimus dorsi of the chicken. Mg2+-activated myofibrillar ATPase activity showed a decreased and delayed activity peak in both the immobilised muscles. Large differences between the two muscles were observed using this marker enzyme. These data indicate that the activity of Mg2+-activated myofibrillar ATPase and the associated differential gene expression involved in fibre type differentiation are influenced by the early activity pattern of the muscles.     

Changes of nucleotide contents and of energy charge induced by contraction, catch and relaxation in smooth molluscan muscle fibres. An analysis using reversed-phase ion-pair high-performance liquid chromatography

1. The content of 14 different nucleotides, including cAMP, in isolated muscle fibres of the anterior byssus retractor muscle of Mytilus edulis was analysed. The nucleotide levels were determined after the muscle fibres performed phasic, tonic, or tetanic contractions and after serotonin-induced relaxation of tonic contraction. 2. Isolated resting muscle fibres revealed a lower energy charge than freshly-dissected ones. 3. During active force development adenosine energy charge decreased to stay at the same low level during catch, tetanus and serotonin-induced relaxation of catch respectively. 4. The energy charges of the guanosine, uridine and cytidine systems did not show changes parallel to the adenosine system. 5. The levels of cyclic AMP were only changed under the influence of serotonin.     

Expression and function of KCNH2 (HERG) in the human jejunum

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERG(USO)) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3-S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10(-6) M), atropine (10(-6) M). and l-nitroarginine (10(-4) M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10(-8) M) and MK-499 (3 x 10(-8) M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10(-6) M) produced a 10-20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10(-6) M) did not affect [(14)C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.