Effects of Removal of Muscle Receptor Organ Input on the Temporal Structure of Non-Giant Swimming Cycles in the Crayfish,Cherax Destructor

Most studies of the muscle receptor organs (MROs) of decapod crustaceans have focused on their role in local reflex loops. This may not be their only function. We examine their involvement in the regulation of non-giant swimming cycles by removing stretch receptor (SR) input from the MROs in abdominal segments 2-5 of the crayfish Cherax destructor. SR input was left intact in two control groups, one of which had sham surgery and the other no surgery at all. We recorded electromyograms (EMGs) from selected uropod muscles during tailflipping in sequences of non-giant swimming in tethered animals. The removal of SR input had a significant effect. The opener muscle period was shorter in the experimental group than in either of the control groups. This suggests that by using SR afference, crayfish sacrifice speed for increased control of the swimming movement.     

The activity of abdominal stretch receptors during non-giant swimming in the crayfish<Emphasis Type="Italic"> Cherax destructor</Emphasis> and their role in hydrodynamic efficiency

Recordings were made from the nerve innervating the stretch receptors of the abdominal muscle receptor organs and slow extensor muscles of tethered crayfish, Cherax destructor, during so-called non-giant swimming. The stretch receptors were active during the flexor phase of swimming but the duration and pattern of activity varied from cycle to cycle. Their pattern of firing was modified by the activity of the large accessory neurons which make direct inhibitory synapses upon them. Neither the stretch receptors nor the accessory neurons were active during the extensor phase of the cycle. The timing and extent of tailfan movements during the period of stretch receptor activity were measured from video records before and after the stretch receptor nerves were cut in the second to fifth segments. The promotion of the tailfan during flexion was significantly delayed and the minimum angle to which the uropods were remoted at the end of flexion significantly larger in denervated animals. We propose that afferent information from the stretch receptors coordinates the timing and extent of tailfan movements according to variations in the positioning and movement of the abdominal segments such that the hydrodynamic efficiency of the tailfan is enhanced on a cycle by cycle basis during non-giant swimming.Abbreviations A# abdominal segment number - Acc accessory neuron - LUU large unidentified unit - MRO muscle receptor organ - NGS non-giant swimming - SEMN slow extensor motor neuron - SR stretch receptor neuron     

Muscle receptor organs do not mediate load compensation during body roll and defense response extensions in the crayfish Cherax destructor.

It has been proposed that the abdominal muscle receptor organ (MRO) of decapod crustaceans acts in a sensory feedback loop to compensate for external load. There is not yet unequivocal evidence of MRO activity during slow abdominal extension in intact animals, however. This raises the possibility that MRO involvement in load compensation is context-dependent. We recorded from MRO tonic stretch receptors (SRs) in freely behaving crayfish (Cherax destructor) during abdominal extension occurring during two different behaviors: body roll and the defense response. Abdominal extensions are similar in many respects in both behaviors, although defense response extensions are more rapid. In both situations, SR activity typically ceased when the abdominal extension commenced, even if the joint of the SR being monitored was mechanically prevented from extending by a block. Since extensor motor neuron activity increased when the abdomen was prevented from extending, we concluded that the load compensation occurring in these behaviors was not mediated by the MROs.     

The abdominal motor system of the crayfish, Cherax destructor. II. Morphology and physiology of the deep extensor motor neurons

Two opposing muscle systems underlie abdominal contractions during escape swimming in crayfish. In this study we used extracellular and intracellular stimulation, recording and dye-filling to systematically identify each of the five deep extensor excitors and single inhibitor of the crayfish, Cherax destructor. Functional associations of each neuron were characterised by recording its responses to sensory and abdominal cord inputs, its extensor muscle innervation pattern, and its relationships with other neurons. Each excitor receives excitatory input from the tonic abdominal stretch receptors and the largest neuron also receives input from the phasic stretch receptor. The two largest excitors innervate the muscle bundle containing the fastest fibres and may be electronically coupled. The smaller neurons may also be electronically coupled and innervate the remaining deep extensor fibres which display dynamic characteristics from fast to medium-fast. The inhibitor does not receive input from the stretch receptors, but is strongly excited by tactile afferents. The implications of these findings for the current models of the control of abdominal tailflips and swimming are discussed. Accepted: 21 June 1998     

Morphological and physiological development of anterior thoracic stretch receptors in two isopods, <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> and <Emphasis Type="Italic">Ligia exotica</Emphasis>

Abdominal muscle receptor organs (MROs) monitor the position and movement of abdomen in crustaceans. Thoracic segments of decapods are fused and immovable. It is speculated that MROs had retrograded simple shape, N-cells that lost receptor muscles, a receptor cell and accessory nerves. We focused on the effect of segmental movement in respect to thoracic N-cells and MROs in isopods that have movable thoracic segments. Armadillidium vulgare rolled up its body segments. Ligia exotica swam by quick movement of the posterior thoracic segments. Both isopods possessed N-cells and MROs in the thorax. N-cells were a simple structure, but N-cells from the second and third thoracic segments of A. vulgare had a muscle strand. MROs^T3–T4 (from the third and fourth thoracic segments) of A. vulgare had two receptor muscles. MROs^T3–T4 of L. exotica had one long receptor muscle. N-cells of both species and MROs of A. vulgare showed slowly adapting stretch-activated discharges. MROs of L. exotica showed both slowly and rapidly adapting discharges. The stretch-activated responses of N-cells and MROs inhibited each other. N-cells or MROs in the thorax of isopods are not related to the segmental structure. The morphology and physiology of N-cells and MROs are specialized to species–specific behaviors.     

Neurotrophic regulation of GABA receptors at a crayfish inhibitory synapse

Experiments were done on the inhibitory synapse of crayfish tonic muscle receptor organs (MROs) to determine whether gamma-aminobutyric acid (GABA) receptors would be affected by denervation or axotomy. It was found that severing the dorsal nerve containing the MRO sensory and inhibitory neurons usually induced, in 30 days or less, a dramatic transformation in the responses of MROs to ionophoretically applied GABA. In contrast to normal MROs which show only inhibitory responses to GABA, transformed MROs were substantially depolarized and excited by GABA application to numerous points found on the axon, soma, dendrites. Interspersed among the points of excitation, normal inhibitory points could still be found on the transformed cells. The results suggested that chronic lesions can induced structural changes in GABA receptors, whereby the Cl- ionophore is replaced by a cationic channel. It was possible to compare the effects of postsynaptic axotomy alone with those of postsynaptic plus presynaptic axotomy by testing MROs from animals whose ventral nerve cord was sectioned. These experiments suggested that interruption of the presynaptic neuron is an important factor in the transformation. It was not determined whether complete degeneration of the inhibitory synapse is necessary for the transformation, but the rapidity of the effect, coupled with the probability of long-term survival of synaptic contacts, suggested that complete degeneration was not necessary. Similarities were found in the GABA responses of transformed MROs and those MROs which normally receive no innervation, which are located in the sixth abdominal segment. These results support the idea that trophic regulation from inhibitory neurons is a factor in stabilizing the association of the Cl- ionophore with GABA receptor.     

The relationship between body size and the field potentials generated by swimming crayfish

Aquatic animals generate electrical field potentials which may be monitored by predators or conspecifics. Many crustaceans use rapid, forceful contractions of the flexor and extensor muscles to curl and extend their abdomens during swimming in escape and locomotion. When crayfish swim they generate electrical field potentials that can be recorded by electrodes nearby in the water. In general, it is reasonable to assume that larger bodied crayfish will generate signals of greater amplitude because they have larger muscles. It is not known, however, how activity in particular muscles and nerves combines to produce the compound electrical waveform recorded during swimming. We therefore investigated the relationship between abdominal muscle, body size and the amplitude of nearby tailflip potentials in the freshwater crayfish (Cherax destructor). We found that amplitude was correlated positively with abdominal muscle mass. The mean amplitude recorded from the five smallest and five largest individuals differed by 440 microV, a difference sufficiently large to be of significance to predators and co-inhabitants in the wild.     

Release of 3H-gamma-aminobutyric acid (GABA) by inhibitory neurons of the crayfish

Inhibitory neurons innervating the muscle receptor organ (MRO) of crayfish were used to study the uptake and release of tritiated GABA. MROs that have been directly exposed to ³H GABA for 60–75 min release radioactivity during low-frequency electrical stimulation. When ganglia containing the inhibitory cell bodies are exposed to ³H GABA, the isotope travels along a pathway unique to the inhibitory axon, at rates that range between 160 and 240 mm per day. Electrical stimulation of inhibitory axons whose cell bodies have been exposed to ³H GABA for 4–5 hr produces release of isotope from isolated MROs. Low calcium, high magnesium exposure prevents the stimulus-dependent release of radioactivity. Thin layer chromatographic analyses indicate that GABA comprises at least a major fraction of the radioactivity collected from stimulated preparations. A number of unidentified radioactive compounds are usually present with GABA, and it is suggested that most of these are catabolites of GABA.     

A comparative electron microscope study of visceral muscle fibers in Cambarus,Drosophila and Lumbricus

Summary The arrangement of myofilaments in the striated visceral muscle fibers of two arthropods (crayfish and fruitfly) and in the unstriated visceral fibers of one annelid (earthworm) was studied comparatively. Transverse sections through the A bands of arthropod visceral fibers indicate that each thick myofilament is surrounded by approximately 12 thin filaments. The myofilaments are less organized in the visceral fibers of the earthworm than in muscle fibers of the crayfish and fruitfly. The thick myofilaments of the earthworm are composed of subunits, 20–30 Å in diameter. The presence of two distinct sets of myofilaments in these slowly contracting striated and unstriated visceral muscle fibers suggests that contraction is accomplished via a sliding filament mechanism.In crayfish visceral fibers the sarcolemma invaginates at irregular intervals to form a long and unbranched tubular system at any level in the sarcomere. Dyads formed by the apposition of T and SR membranes are observed frequently. The distribution of the T and SR systems in the visceral fibers of the fruitfly and the earthworm is markedly reduced and dyads are infrequently observed. The reduced T and SR systems may be related to the slow contraction of these fibers. Transport of specific substances across the sarcolemma could initiate contraction or relaxation in these fibers.This study was supported by a training grant GM-00582-06 from the U.S. Public Health Service.     

Extrinsic modulation of crayfish escape behaviour.

Extrinsic systems were shown to control the excitability of the neurones which mediate tail-flip escape in the crayfish. Restraint suppresses the escape mediated by giant fibres and some, but not all, categories of non-giant mediated escape; autotomy of claws increases the excitability of non-giant mediated escape without affecting the lateral giant reflex. The effects of restraint on the lateral giant reflex result from inhibition rather than reduced facilitation. The inhibition descends from thoracic and higher levels, and the lateral giant escape command neurone appears to be its primary target. Inhibition may serve to shift the control of escape behaviour from short latency 'reflex' systems to more flexible 'voluntary' ones which can produce responses at times most opportune for successful escape.     

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.     

The swimming performance of brown trout and whitefish: the effects of exercise on Ca2 + handling and oxidative capacity of swimming muscles

The swimming performance of two fish species, the brown trout and whitefish, having initially different swimming strategies, was measured after nine different training programs in order to relate the effects of exercise on Ca²⁺ handling and oxidative capacity of swimming muscles. The time to 50% fatigue was measured during the training period, and compared with the density of dihydropyridine (DHP) and ryanodine (Ry) receptors and succinate dehydrogenase (SDH) and phosphorylase activity determined by histochemical analysis of the swimming muscles. Overall, both trained brown trout and whitefish had superior swimming performance as compared to control ones. Interestingly, the training programs had different effect on the two species studied since brown trout achieved the highest swimming performance, swimming against the water flow velocity of 2 BL s⁻¹ while among whitefish the best efficiency was seen after training with lower swimming velocities. Training also induced a significant increase in DHP and Ry receptor density in both species. Generally, in brown trout the most notable increase in the receptor densities was observed in red muscle sections from the fish swimming for 6 weeks against water currents of 1 BL s⁻¹ (DHPR 176.5 ± 7.7% and RyR 231.4 ± 11.8%) and white muscle sections against 2 BL s⁻¹ (DHPR 129.6 ± 12.4% and RyR 161.9 ± 15.5%). In whitefish the most prominent alterations were noted in samples from both muscle types after 6 weeks of training against water current of 1.5 BL s⁻¹ (DHPR 167.1 ± 16.9% and RyR 190.4 ± 19.4%). Finally, after all the training regimens the activity of SDH increased but the phosphorylase activity decreased significantly in both the species. To conclude, our findings demonstrate an improved swimming performance and enhanced Ca²⁺ regulation and oxidative capacity after training. Moreover, there seems to be a connection between the swimming performance and receptor levels, especially in white swimming muscles of different fish species, regardless of their initially deviant swimming behaviours. However, depending on the training regimen the divergent swimming behaviours do cause a different response, resulting in the most prominent adaptational changes in the receptor levels of red muscle samples with lower swimming velocities in brown trout and with higher ones in whitefish.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

Repeated Administration of the Neurotensin Receptor Antagonist SR 48692 Differentially Regulates Mesocortical and Mesolimbic Dopaminergic Systems

Abstract: The purpose of the present study was to investigate the effects of repeated administration of the neurotensin receptor antagonist, SR 48692, on the activity of the mesocortical and mesolimbic dopaminergic (DA) systems. We showed that daily administration of SR 48692 for 15 days (1 mg/kg i.p.) to Wistar rats increased the expression of tyrosine hydroxylase mRNA and protein in the ventral mesencephalon. Simultaneous in vivo microdialysis in the shell part of the nucleus accumbens (AcbSh) and the medial prefrontal cortex (mPFC) revealed that blockade of neurotensin receptors for 15 days decreased basal extracellular levels of DA (∼50%) and its metabolites in the AcbSh, whereas no modification in DA levels was observed in the mPFC. In animals submitted to a forced swimming stress, which preferentially enhanced extracellular DA levels in the mPFC, treatment with SR 48692 failed to affect the stress-induced increase in DA. Moreover, given that glucocorticoids can modulate the activity of mesencephalic DA neurons, we examined the effect of the same SR 48692 treatment on corticosterone levels in dialysates from the AcbSh. We found that repeated SR 48692 did not affect the basal levels of free corticosterone, but significantly reduced the increase induced by forced swimming stress. The present results demonstrate that repeated treatment with SR 48692 modulates selectively the DA mesolimbic system when compared with the mesocortical pathway. These findings suggest that long-term treatment with selective neurotensin receptor antagonists could have potential clinical utility in the treatment of neuropsychiatric disorders associated with hyperactivity of the mesolimbic DA systems or the hypothalamic-pituitary-adrenal axis.     

Vanilloid receptor expressed in the sarcoplasmic reticulum of rat skeletal muscle

Vanilloid receptor subtype 1 (VR1) was cloned as a capsaicin receptor from neuronal cells of dorsal root ganglia. VR1 was subsequently found in a few non-neuronal tissues, including skeletal muscle [Onozawa et al., Tissue distribution of capsaicin receptor in the various organs of rats, Proc. Jpn. Acad. Ser. B 76 (2000) 68-72]. We confirmed the expression of VR1 in muscle cells using the RT-PCR method and Western blot analysis. Immunostaining studies with a confocal microscope and an electron microscope indicated that VR1 was present in the sarcoplasmic reticulum (SR), a store of Ca2+. The SR releases Ca2+ to cause a contraction when a muscle is excited. However, SR still releases a small amount of Ca2+ under relaxed conditions. We found that this leakage was enhanced by capsaicin and was antagonized by capsazepine, a capsaicin blocker, indicating that leakage of Ca2+ occurs through a channel composed of VR1.     

Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures

Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.     

Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses

The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.     

Loss of escape responses and giant neurons in the tailflipping circuits of slipper lobsters, Ibacus spp. (Decapoda, Palinura, Scyllaridae)

In many decapod crustaceans, escape tailflips are triggered by lateral giant (LG) and medial giant (MG) interneurons, which connect to motor giant (MoG) abdominal flexor neurons. Several decapods have lost some or all of these giant neurons, however. Because escape-related giant neurons have not been documented in palinurans, I examined tailflipping and abdominal nerve cords for giant neurons in two scyllarid lobster species, Ibacus peronii and Ibacus alticrenatus. Unlike decapods with giant neurons, Ibacus do not tailflip in response to sudden taps. Ibacus can perform non-giant tailflipping: the frequency of tailflips during swimming is adjusted by altering the gap between each individual tailflip. Abdominal nerve cord sections show no LG or MG interneurons. Backfilling nerve 3 of abdominal ganglia revealed no MoG neurons, and the fast flexor motor neuron population is otherwise identical to that described for crayfish. The loss of giant neurons in Ibacus represents an independent deletion of these cells compared to other reptantian decapods known to have lost these giant neurons. This loss is correlated with the normal posture in scyllarids, in which the last two abdominal segments are flexed, and an alternative defensive strategy, concealment by digging into sand.     

Locomotory capacity of Baltic Sea and freshwater populations of the threespine stickleback (Gasterosteus aculeatus)

Two freshwater populations and one marine population (Baltic Sea) of threespine stickeback (Gasterosteus aculeatus) from Northeastern Germany were studied with regard to locomotory capacity: sustained swimming performance, activities of key enzymes in axial muscle, pectoral fin muscle and heart, and morphology. We postulated that life history differences between migratory Baltic Sea and resident freshwater populations could have led to a divergence in their locomotory capacity. The activity of citrate synthase (CS) in pectoral muscle correlated with critical swimming speed. Critical swimming speed, aerobic and anaerobic capacity of the pectoral fin muscle were population-specific. The Baltic Sea sticklebacks had a higher locomotory capacity (activity of CS in pectoral muscle, critical swimming speed) than sticklebacks of one freshwater population. However, another freshwater population expressed a similar locomotory capacity as the Baltic Sea population. In addition, Baltic Sea sticklebacks had a greater mass and lower anaerobic capacity of the pectoral fin muscle than the freshwater sticklebacks. The results are interpreted as an indication of a proceeding divergence between marine and resident freshwater populations and between freshwater populations of G. aculeatus originating from marine ancestors. The migratory Baltic Sea sticklebacks had better morphological prerequisites for sustained swimming than both freshwater populations, but there was no general difference in the locomotory capacity between marine and freshwater sticklebacks. However, their morphology could favour a more effective locomotion in the Baltic Sea sticklebacks.     

A mechanism for both capacitative Ca(2+) entry and excitation-contraction coupled Ca(2+) release by the sarcoplasmic reticulum of skeletal muscle cells

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca(2+) uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca(2+) from cytosol to SR, and measurements of cytosolic Ca(2+). When Ca(2+) is added to Ca(2+)-free cells in the presence of Tg, the measured cytosolic Ca(2+) rises. This has been universally interpreted to mean that as SR Ca(2+) is depleted, exogenous Ca(2+) crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca(2+) in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca(2+) in the presence of Tg showed an increase in luminal Ca(2+) concentration in response to added exogenous Ca(2+). While we were able to reproduce the conventional demonstration of CCE-an increase of Ca(2+) in the cytosol in the presence of thapsigargin-we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca(2+) release channel, the ryanodine receptor (RyR). This was also unexpected if Ca(2+) enters the cytosol first. When Ca(2+) was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca(2+) and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca(2+) transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca(2+) inhibited its own uptake, a known feature of the RyR. We confirmed that Ca(2+) enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn(2+) in place of direct Ca(2+) measurements; we showed that Mn(2+) was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca(2+) flow in muscle cells. Our findings are entirely explained by a new model whereby Ca(2+) enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca(2+) subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca(2+) is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.