Mg++-sensitivity of neuromuscular transmission in two crustaceans: Correlation with blood Mg++ levels

Neuromuscular transmission was measured in muscles of spider crabs (Hyasareneus) and lobsters (Homarus americanus). Solutions containing 40 and 10 mM/1 Mg⁺⁺, which were approximately the same as those measured in the blood of Hyas and Homarus, respectively, were used to soak the preparations prior to testing. In Homarus, neuromuscular transmission was severely depressed by 40 mM Mg⁺⁺. In spider crabs, neuromuscular transmission was not severely depressed. Although the amount of transmitter released by nerve impulses was reduced, total membrane depolarization during trains of impulses was not reduced because a compensating increase in muscle fiber membrane resistance occurred in Hyas preparations exposed to 40 mM Mg⁺⁺. Hyas, but not Homarus, is physiologically adapted to function at relatively high blood Mg⁺⁺ concentrations.     

Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

Is Embryonic Limulus Heart Really Myogenic?

Although the neurogenic nature of the heartbeat in adult Limulushas been well studied and is undisputed, we contest the reportsthat the embryonic heartbeat is myogenic. This notion, basedon histological, calorimetric, and drug studies, is challengedby evidence from transmission electron microscopy and intracellularrecording. The first, infrequent heartbeats occur at the timeof the third embryonic molt when only the anterior portion ofthe heart tube is formed and functional. Contractions extendfurther caudad concomitant with lumen formation in the rearheart segments. All lumen-containing heart sections that wehave examined, from the earliest on, have revealed neural elementsin a bundle at the dorsal midline of the heart. Axons 1/m orless in diameter are prevalent: vesicle-filled terminal-likeareas adjacent to muscle cells are often present as well, evenin the youngest beating hearts. Myocardial cells show excitatorypostsynaptic potentials as soon as heartbeat has begun, butthey often fail to summate in the earlier stages so that contractionsare few. Resting potentials remain at –65 to –70mV from the onset of heartbeat until well after the larva hashatched, but heartbeat frequency, regularity, depolarizationheight (never overshooting) and duration all increase as embryosget older, probably as innervation of muscle fibers increasesand coordination between pacemaker and follower neurons improves.We have found no evidence that embryonic Limulus heart passesthrough a myogenic phase and believe that it is neurally drivenfrom the beginning.     

Transient Responses to Sudden Illumination in Cells of the Eye of Limulus

Responses recorded from visual cells of Limulus (presumably eccentric cells) following abrupt and maintained illumination consist of depolarization with superimposed spikes. Both the depolarization and the frequency of firing are greater at the beginning of the response than later on. Frequency of firing decreases with time also during stimulation with constant currents, but the decay is then less than it is during constant illumination. Early and steady-state responses do not increase in the same proportion following illumination at different intensities. Membrane conductance is higher during the early peak of the response than in steady state. Early and late potential changes appear to tend to the same equilibrium value. The results support the assumptions that: (a) discharge of impulses is the consequence of depolarization of a specialized "pacemaker region" in the axon; (b) depolarization induced by light is the consequence of increase of membrane conductance. The major conductance changes occurring during constant illumination may be due to corresponding changes of the "stimulus" supplied by the photoreceptor or to changes of sensitivity of the eccentric cell's membrane to this stimulus. Some accessory phenomena may be the consequence of regenerative properties of the nerve cell itself.     

Neurotrophic control of the transmembrane Cl− pump in mammalian muscle fibers

Resting membrane potential of both innervated and denervated rat diaphragm muscle fibers was investigated when the concentration of potential-producing ions was changed in the external fluid and following treatment with furosemide. It was found that equilibrium potential ( ) equalled resting potential level in innervated muscle for Cl^–, but shifts to more positive values compared with resting membrane potential following section of the nerve. Furosemide retards development of post-denervation depolarization of the muscle membrane. It is deduced that trophic influences originating from the motor nerve activates the furosemide-sensitive Cl^– influx system, leading to raised intracellular concentration of Cl^–, a shift in (E_Cl) and depolarization of the muscle membrane.S. V. Kurashov Medical Institute, Minsitry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 766–771, November–December, 1987.     

Differentiation of Nerve Terminals in the Crayfish Opener Muscle and Its Functional Significance

Junctional potentials (jp's) recorded from superficial distal fibers of the crayfish opener muscle are up to 50 times larger than jp' in superficial central fibers when the single motor axon that innervates the muscle is stimulated at a frequency of 1/sec or less. At 80/sec, in contrast, central jp's are up to four times larger than those observed in distal fibers. The tension produced by single muscle fibers of either type is directly proportional to the integral of the time-voltage curve minus an excitation-contraction coupling threshold of 3 mv. Distal fibers therefore produce almost all the total muscle tension at low frequencies of stimulation and central fibers add an increasingly greater contribution as their nerve endings begin to facilitate in response to increased rate of motor discharge. Differentiation of muscle membrane characteristics (input resistance, space constant, time constant) cannot account for these differences in facilitation ratios. The mechanism of neuronal differentiation is not based upon the size or effectiveness of transmitter quanta, since equal sized jp's have equal variances;: mjp sizes and variances are also equal. No differences were found between fiber types in rates of transmitter mobilization, density of innervation, or the relationship between transmitter release and terminal depolarization. Single terminals on distal fibers were found to release transmitter with a greater probability than central terminals. More effective invasion of distal terminals by the nerve impulse at low frequencies can account for the difference.     

Neuromuscular Organization for "Wing" Control in a Mollusc (Clione limacina) and a Bird (Columba livia): Parallels in Design

Flapping as a means of locomotion is shared by divergent groupsranging from pteropod molluscs to birds. The pteropod, Clionelimacina, exhibits two modes of locomotion, slow and fast swimming.The motor units which control swimming consist of small motoneuronsand relatively nonfatigable muscle fibers, while those involvedin fast swimming consistof large motoneurons and relativelyfatigable fibers (Satterlie et al., 1990).The pectoralis muscleof the pigeon, Columba livia, consists of two populations ofmuscle fibers distinguished by histochemistry and size. Cinematographicand electromyographic experiments suggest that the large fibersare used for takeoff and landing and the small fibers for levelflight (Dial et al., 1988). We have employed a suite of experimentaltechniques similar to those used for studies of the neuromuscularsystem of Clione to analyze a limited sample of motor unitsfrom the pectoralis of Columba. The peak tetanic tension andcontractile fatigue resistance during electrical stimulationof single alpha axons, functionally isolated from nerve filaments,was studied in 30 motor units. All units but one generated peaktetanic tensions which were less than 0.22% of whole muscletension. A high proportion (75%) of units demonstrated fatigueresistance, reflective of the demands of sustained flappingflight. These preliminary data suggest that the peripheral neuromuscularsystems of Clione and Columba share some common components forthe execution of at least two distinct modes of flapping locomotion.     

Cardioacceleratory reflexes triggered by mechanoproprioceptors of the swimmerets in the stomatopod crustacean Squilla oratoria

The heart of Squilla oratoria is innervated by processes arising from the cardiac ganglion, which lies on the outer surface of the heart wall. The ganglion is regulated by one pair of cardioinhibitory nerves and two pairs of cardioacceleratory nerves. Cardiac acceleration accompanied activation of the five pairs of swimmerets in the first to the fifth abdominal segments. The cardioacceleratory nerves were activated when swimmerets beat strongly. Activation of the cardioacceleratory nerves was caused by electrical stimuli to a nerve branch extending to the swimmerets from the first nerve root of the abdominal ganglion. Bursts of afferent impulses were recorded from the nerve branch of the first nerve root corresponding to periods of protractive and retractive swimmeret movements. Afferent impulses were recorded from the nerve branch when the articular membrane was artificially boosted up. Cardiac acceleration during active swimmeret movements in Squilla is attributable to a reflexive response triggered by the movements. Putative mechanoproprioceptors on the articular membrane between the sterna and basipodite in the swimmerets may be responsible for the cardioacceleratory reflex.     

Role of calcium ions and spike activity in the neurotrophic control of membrane potential in rat muscle fibers

Blockade of calcium permeability produced an increase in postdenervation depolarization of rat diaphragm muscle fibers during in vitro experiments, while increased Ca²⁺ concentration in the sarcoplasm induced by caffeine led to hyperpolarization of the muscle membrane. Direct stimulation of the muscles or carbamylcholine application retarded the reduction of membrane potential in the muscle fibers. Verapamil and d-tubocurarine eliminated the hyperpolarizing effect of stimulation. The hyperpolarizing effects of carbamylcholine applied in conjunction with stimulation did not produce an accumulated action on the membrane. It is deduced that the factors governing membrane potential in the muscle fibers are acetylcholine and Ca²⁺ reaching the sarcoplasm mainly through the acetylcholine-sensitive ionic channels during the process of nerve impulse fluxes.S. V. Kurashov Medical Institute, Kazan', Ministry of Public Health of the RSFSR. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 449–456, July–August, 1987.     

The Sites of Action of Pericardial Organ Extract and 5-Hydroxytryptamine in the Decapod Crustacean Heart

Responses of isolated, perfused hearts of Homarm americanusto brief, internal application of extracts of pericardial organs(PO's) ofCancer borealis, or 5-hydroxytryptamine (5HT) are verysimilar over a thousand-told range of concentration: an increasein rate and amplitude of beating. These reach their maxima afterwashing out has begun, and recover within ten minutes. Externalapplication is ineffective and the substances do not interactwith effects of stretch stimulation. Intracellular recordingfrom heart muscle fibers reveals facilitation of depolarizationto bursts at heart beat frequencies. There may be some effectof 5HT directly on neuromuscular facilitation. Responses recordedfrom isolated cardiac ganglia show increased burst rate, burstduration, or both. Thresholds and the range of concentrationsfor which coordinated responses are recorded correspond to thosefor perfused hearts. It is concluded that the major sites ofaction of PO extract and 5HT are in the cardiac ganglion. 5HTtachyphlaxis and LSD block effects of 5HT, but not of PO extractor accelerator nerve stimulation. Intracellular recordings fromthe large ganglion cells show no effects on resting, synaptic,or spike potentials. Changes in membrane potential to currentpulses revealed no changes in membrane resistance or in theresistance of the electrotonic pathway between cells. Resultsof selective application to large or small cells suggested thatPO extract may contain a rate-increasing substance and one prolongingthe duration of bursts. The former, and 5HT, may influence pacemakerpotentials; the latter may increase the number of spikes a unitcan produce before becoming refractory.     

Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat

Using a single, mechanically skinned fiber approach, we tested the hypothesis that denervation (0 to 50 days) of skeletal muscles that do not overlap in fiber type composition [extensor digitorum longus (EDL) and soleus (SOL) muscles of Long-Evans hooded rats] leads to development of different fiber phenotypes. Denervation (50 day) was accompanied by 1) a marked increase in the proportion of hybrid IIB/D fibers (EDL) and I/IIA fibers (SOL) from 30% to >75% in both muscles, and a corresponding decrease in the proportion of pure fibers expressing only one myosin heavy chain (MHC) isoform; 2) complex muscle- and fiber-type specific changes in sarcoplasmic reticulum Ca²⁺-loading level at physiological pCa 7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca²⁺, particularly for SOL fibers (by 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by 35%); and 6) increased occurrence of biphasic behavior with respect to Sr²⁺ activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels. mechanically skinned fibers; myosin heavy chain isoforms; lineage; sarcoplasmic reticulum; Ca²⁺; Sr²⁺ sensitivity; Long-Evans hooded rat     

A NOTE ON THE ACTION OF CURARE,ATROPINE, AND NICOTINE ON THE INVERTEBRATE HEART

1. The alkaloids (curare, atropine, and nicotine) in molluscs and arthropods stimulate and paralyze the central nervous system and peripheral (visceral) ganglia, but do not paralyze the motor nerve endings to skeletal or visceral muscle. 2. They stimulate and paralyze the denervated heart. 3. They paralyze or block the cardioinhibitory nerves, but not the cardioaccelerator nerves. 4. In the Limulus heart these drugs act primarily on the heart ganglion, not on the heart muscle or the intrinsic motor nerve fibers.     

Inhibition in the eye of Limulus

In the compound lateral eye of Limulus each ommatidium functions as a single receptor unit in the discharge of impulses in the optic nerve. Impulses originate in the eccentric cell of each ommatidium and are conducted in its axon, which runs without interruption through an extensive plexus of nerve fibers to become a fiber of the optic nerve. The plexus makes interconnections among the ommatidia, but its exact organization is not understood. The ability of an ommatidium to discharge impulses in the axon of its eccentric cell is reduced by illumination of other ommatidia in its neighborhood: the threshold to light is raised, the number of impulses discharged in response to a suprathreshold flash of light is diminished, and the frequency with which impulses are discharged during steady illumination is decreased. Also, the activity that can be elicited under certain conditions when an ommatidium is in darkness can be inhibited similarly. There is no evidence for the spread of excitatory influences in the eye of Limulus. The inhibitory influence exerted upon an ommatidium that is discharging impulses at a steady rate begins, shortly after the onset of the illumination on neighboring ommatidia, with a sudden deep minimum in the frequency of discharge. After partial recovery, the frequency is maintained at a depressed level until the illumination on the neighboring receptors is turned off, following which there is prompt, though not instantaneous recovery to the original frequency. The inhibition is exerted directly upon the sensitive structure within the ommatidium: it has been observed when the impulses were recorded by a microelectrode thrust into an ommatidium, as well as when they were recorded more proximally in single fibers dissected from the optic nerve. Receptor units of the eye often inhibit one another mutually. This has been observed by recording the activity of two optic nerve fibers simultaneously. The mediation of the inhibitory influence appears to depend upon the integrity of nervous interconnections in the plexus: cutting the lateral connections to an ommatidium abolishes the inhibition exerted upon it. The nature of the influence that is mediated by the plexus and the mechanism whereby it exerts its inhibitory action on the receptor units are not known. The depression of the frequency of the discharge of nerve impulses from an ommatidium increases approximately linearly with the logarithm of the intensity of illumination on receptors in its vicinity. Inhibition of the discharge from an ommatidium is greater the larger the area of the eye illuminated in its vicinity. However, equal increments of area become less effective as the total area is increased. The response of an ommatidium is most effectively inhibited by the illumination of ommatidia that are close to it; the effectiveness diminishes with increasing distance, but may extend for several millimeters. Illumination of a fixed region of the eye at constant intensity produces a depression of the frequency of discharge of impulses from a nearby ommatidium that is approximately constant, irrespective of the level of excitation of the ommatidium. The inhibitory interaction in the eye of Limulus is an integrative process that is important in determining the patterns of nervous activity in the visual system. It is analogous to the inhibitory component of the interaction that takes place in the vertebrate retina. Inhibitory interaction results in the exaggeration of differences in sensory activity from different regions of the eye illuminated at different intensities, thus enhancing visual contrast.     

Fever induced in marine arthropods by prostaglandin E1

Injections of prostaglandin E₁ into the haemocoel of the marine arthropods $\text{Limulus}$$\text{polyphemus}$, $\text{Homarus}$$\text{americanus}$ and $\text{Penaeus}$$\text{duorarum}$ induced a behaviorally mediated fever (increase in preferred temperature, resulting in increased body temperature). The finding that PGE₁ is pyorgenic to arthropods as well as to mammals suggests that arthropods can be used as simple experimental models for further investigations of the neuropharmacological role of prostaglandins in fever.     

Inotropism and Contracture of Aplysiid Ventricles as Related to the Action of Neurohumors on Resting and Action Potentials of Molluscan Hearts

The postsynaptic actions of neurohumors on molluscan musclemay be exerted through control of force as well as by meansof excitation and inhibition. The control of force may appearas potentiation of the response to excitation, as increasedinotropism in spontaneous contractions, or as an increase intonus. We have directed our attention to the alterations offorce induced in aplysiid ventricles by applied postulated neurohumors.The results are interpreted in terms of the known effects ofneurohumors on resting potential and action potentials of molluscanhearts. We recorded compound membrane potentials of aplysiidventricles extracellularly, using a single sucrose gap apparatustogether with a force-displacement transducer to measureforcein contractions or contracture. Aplysia californica ventriclehas a membrane potential of –52.5 ± 9.4 mV. Ventriclesof Aplysia dactylomela or Aplysia californica are depolarizedby increased concentrations of external potassium ion, withan accompanying contracture. After incubation in calcium-freemedium, KCl contracture-force is directly dependent on calciumion concentration. A depolarization of 8.3 ±2.14 mV inpotassium-free medium is blocked by substitution of lithiumfor sodium in the medium, suggesting an electrogenic sodiumpump. There is a sustained depolarization in low chloride medium,which suggests a significant chloride contribution to the restingpotential. Ventricles of Dolabella auriculana or Aplysia dactylomelaare depolarized by acetylcholine (ACh). Threshold for depolarizationis lower than threshold for contracture-force. The ventricleof A. dactylomela is depolarized by 5- hydroxytryptamine (5HT)with threshold at 10^–9 M and a maximum depolarizationof 30 mV at 10^–4M. Depolarization by 5HT may induce beatingbut does not induce contracture. Ventricles of Aplysia californicaare not depolarized by ACh although beating ventricles are inhibited,and a depolarized ventricle in a tonic contracture may be hyperpolarizedand relaxed by low concentrations. The force of contractionof the ventricle of Dolabella auricularia is dependent on theduration of the plateau phase of the cardiac action potential.The plateau is lengthened by 5HT with an accompanying increasein force of beat, and shortened by ACh, with an accompanyingdecrease in force of beat. The action potential in the ventricleof Aplysia californica is not differentiated into spike andplateau phases, and neither ACh nor 5HT has any marked effecton the form of the action potential. Nevertheless, isolatedventricles of Dolabella auricularia, Aplysia dactylomela, andAplysia californica are all excited by 5-hydroxytryptamine,with a threshold at about 10^–M. Both spontaneous beatingand excitation induced in A. californica ventricles by 5HT areblocked by lack of the sodium ion, which may be responsiblefor pacemaker potentials in molluscan hearts.     

Muscle and Muscle Fiber Type Transformation In Clawed Crustaceans

SYNOPSIS. The first pair of thoracic limbs in many crustaceansis elaborated into claws in which the principal muscle is thecloser. Changes in the fiber composition of the closer muscleduring claw development, regeneration and reversal are reviewedhere and the hypothesis is advanced that such changes are nerve-dependent.In adult lobsters, Homarus amencanus, the paired claws and closermuscles are bilaterally asymmetric, consisting of a minor orcutter claw with predominantly fast fibers and a small ventralband of slow and a major or crusher claw with 100% slow fibers.Yet in the larval and early juvenile stages the paired clawsand closer muscles are symmetric consisting of a central bandof fast fibers sandwiched by slow. Differentiation into a cutteror crusher muscle during subsequent juvenile development isby appropriate fiber type transformation. Experimental manipulationof the claws or the environment in early juvenile stages whenthe claws are equipotent revealed that the determination ofclaw and closer muscle asymmetry is dependent on the convergenceof neural input from the paired claws: the point of convergencemost likely being the CNS. Bilaterally symmetrical input resultsin the development of paired cutter claws while bilaterallyasymmetric input gives rise to dimorphic, cutter and crusherclaws. In the northern crayfish, Orconectes rusticus, wherethe paired claws are bilaterally similar, the closer muscletransforms its central band of fast fibers to slow, both duringprimary development and regeneration. Whether these fiber typetransformations are nerve-dependent is unknown. In adult snappingshrimps, Alpheus sp., the paired claws and closer muscles areasymmetric: the minor or pincer claw has a central band of fastfibers flanked by slow while the major or snapper claw has 100%slow fibers. Claw reversal occurs with removal of the snapperresulting in the transformation of the existing pincer to asnapper and the regeneration of a new pincer at the old snappersite. Transformation of the closer muscle from pincer to snappertype is by degeneration of the fast fiber band and hypertrophyof the slow fibers. Claw transformation can be either preventedif the pincer nerve is sectioned at the time of snapper removalor promoted if the snapper nerve is sectioned: both resultsimplicating a neural basis for muscle transformation.     

Structure and Function of the Transverse Tubular System in Crustacean Muscle Fibers

The structure and function of the transverse tubular system(TTS) in two types of crustacean muscle fibers are examined.Giant fibers from the barnacle,Balanus nubilus, which are gradedlycontracting, are compared with allor-none twitch fibers fromthe crab, Carcinus maenas. Both fiber types were found to havedeep sarcolemmal invaginations which serve both to increasethe fiber surface area and to kfeep the length of the tubulesshort enough for electrotonic propagation.The ultrastructureof the tubular system in both types of fiber is compared.Thesystem is better developed in Carcinus than in Balanus, butthe slow Balanus fibers do have a relatively well developedTTS and sarcoplasmic reticulum in contrast to slow vertebratefibers. The apparent high, membrane-capacitance values of crustaceanfibers are the result of investigators not taking into considerationthe large increase in surface area due to the sarcolemmal infoldings.Thetubular membranes in Carcinus fibers were found to be permselectiveto chloride ions, and could be made to swell (as confirmed byelectron microscopy) by establishing an outward gradient forchloride across them. The capacitance of the tubular membranerelative to the plasma membrane was found to increase when thetubuleswere swollen. The implication of a fiber having two spatiallyseparated, differentially permeable membranes on excitation-contractioncoupling is discussed.     

Trauma-induced changes of skeletal muscle membrane: decreased K+ and increased Na+ permeability

Hong, S. J., and C. C. Chang.Trauma-induced changes of skeletal muscle membrane: decreasedK⁺ and increasedNa⁺ permeability.J. Appl. Physiol. 83(4):1096-1103, 1997.Trauma of skeletal muscle causes membranedepolarization and reduces membrane resistance. The underlyingmechanisms were studied in isolated mouse phrenic nerve diaphragmssubject to sharp transections of muscle. Depolarization was most markedat the vicinity (~1 mm) of trauma, where the membrane potentialdropped rapidly from about 80 mV to zero and repolarized toabout 25 mV. At the end-plate region (located ~3 mm away fromthe cut end), the membrane gradually attained a plateau potentialaround 45 mV. The magnitude of depolarization was not reduced byinhibition of Na⁺,Ca²⁺, orCl channel, whereas theprogress of depolarization was delayed in low-Na⁺ medium. Activation of theK⁺ channel with lemakalim inducedsome hyperpolarization at damaged site but produced aglybenclamide-sensitive outward current and hyperpolarization ofend-plate region to the levels before trauma, as if there was nodiminution of transmembrane K⁺gradient in this area. Appropriate elevation of extracellular K⁺ to stimulateK⁺ conductance also hyperpolarizedthe end-plate region. The results suggest that depolarization atregions remote from trauma is related to decreasedK⁺ and increasedNa⁺ permeability. The cytoplasmacompartmentalization and permeability changes may protect muscle fiberfrom trauma.

     

The Role of Cell Calcium in the Contraction of Single Cannulated Muscle Fibers

Intracellular injections of the calcium-binding agent, EGTA,into single cannulated fibers of Balanus and Maia were ableto suppress, almost completely, the contractions induced byvarious contractile agents. The amount oE EGTA required in Maia fibers for the suppressionof the contractile response produced by caffeine and high-Ksaline, as has already been reported, was similar to the meanfiber calcium level. In Balanus fibers, however, although theamount of EGTA needed for the suppression of the caffeine-salineresponse was similar to the estimated level of fiber calcium,the amount required in the raised-K-saline experiments was considerablygreater. It has been suggested that membrane depolarizationunder these conditions allows calcium to enter the fiber fromthe external saline, the amount entering being related, at leastin part, to a large effective sarcolemmal surface area and thepresence of binding agent internally. The results of intracellularand plate-electrode stimulation of Belanus fibers also suggestedthat the fiber under certain conditions could utilize externalcalcium ions, while the results of plate-electrode stimulationof Maia fibers could be explained most easily in terms of mobilizationof mainly intracellular calcium for the process of contraction. Efflux of Sr⁸⁹ ions from Balanus fibers under various conditionssuggested that this ion is bound and mobilized internally ina manner similar to calcium. The results are seen not to contradict a chanelled-current theoryfor e-c coupling of the type proposed for crayfish fibers.     

Obidoxime antagonizes the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate via different mechanisms

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC₅₀: 300 µM) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC₅₀: 0.6 µM) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC₅₀ of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC₅₀ against DFP, at a concentration (30 µM) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation.