Ca2+ Release channels of pigs heterozygous for malignant hyperthermia

Porcine malignant hyperthermia (MH) is an autosomal recessive disorder resulting from a mutation in the skeletal muscle sarcoplasmic reticulum (SR) Ca²⁺ release channel. The Ca²⁺ release properties of SR vesicles isolated from pigs heterozygous for the MH gene have been demonstrated previously to be intermediate to those of vesicles isolated from MH-susceptible (MHS) and normal pigs. The Ca²⁺ release channel is tetrameric, so the intermediate Ca²⁺ release properties of heterozygous pig SR preparations could result either from populations of MHS and normal homotetramers, or populations of heterotetrameric Ca²⁺ release channels with properties unique from those of the two types of homozygous channels. To discriminate between these possibilities, the single channel percent open time (P_o) and channel dwell time distributions of SR Ca²⁺ release channels were analyzed. These data suggest that the heterozygous porcine Ca²⁺ release channel population must contain heterotetramers with properties distinct from those of either MHS or normal channels. The data also imply that the Ca²⁺ release channel population in MHS humans who are heterozygous for a dominant mutation in this protein also contains heterotetrameric channels.© 1995 John Wiley & Sons, Inc.     

Enhanced muscle shortening and impaired Ca2+ channel function in an acute septic myopathy model

Myopathies in critically ill patients are increasingly documented. Various animal models of chronic sepsis have been employed to investigate reduced membrane excitability or altered isometric contractility of skeletal muscle. In contrast, immediate changes occurring during acute sepsis are significantly under-characterised; L-type Ca²⁺ channel function or isotonic shortening are examples. We recorded slowly activating L-type Ca²⁺ currents (I _Ca) in voltage-clamped single intact mouse skeletal muscle fibres and tested the effects of acute challenge with serum fractions from critical illness myopathy patients (CIM). Using a high-speed camera system, we simultaneously recorded unloaded fibre shortening during isotonic contractions with unprecedented temporal resolution (~1,600 frames/s). Time courses of fibre lengths and shortening velocity were determined from automated imaging algorithms. CIM fractions acutely induced depression of I _Ca amplitudes with no shifts in I _Ca–V-relations. Voltage-dependent inactivation was unaltered and I _Ca activation and inactivation kinetics were prolonged compared to controls. Unexpectedly, maximum unloaded speed of shortening was slightly faster following CIM serum applications, suggesting a direct action of CIM serum on weak-binding-state cross-bridges. Our results are compatible with a model where CIM serum might acutely reduce a fraction of functional L-type Ca²⁺ channels and could account for reduced SR Ca²⁺ release and force production in CIM patients. Acute increase in isotonic shortening velocity might be an early diagnostic feature suitable for testing in clinical studies. The acute challenge model is also robust against atrophy or fibre type changes that ordinarily would have to be considered in chronic sepsis models.     

Aging impairs regulation of ryanodine receptors from extensor digitorum longus but not soleus muscles

ABSTRACT: Introduction: Because impaired excitation‐contraction coupling and reduced sarcoplasmic reticulum (SR) Ca²⁺ release may contribute to the age‐associated decline in skeletal muscle strength, we investigated the effect of aging on regulation of the skeletal muscle isoform of the ryanodine receptor (RyR1) by physiological channel ligands. Methods: [³H]Ryanodine binding to membranes from 8‐ and 26‐month‐old Fischer 344 extensor digitorum longus (EDL) and soleus muscles was used to investigate the effects of age on RyR1 modulation by Ca²⁺ and calmodulin (CaM). Results: Aging reduced maximal Ca²⁺‐stimulated binding to EDL membranes. In 0.3 μM Ca²⁺, age reduced binding and CaM increased binding to EDL membranes. In 300 μM Ca²⁺, CaM reduced binding, but the age effect was not significant. Aging did not affect Ca²⁺ or CaM regulation of soleus RyR1. Discussion: In aged fast‐twitch muscle, impaired RyR1 Ca²⁺ regulation may contribute to lower SR Ca²⁺ release and reduced muscle function. Muscle Nerve 57 : 1022–1025, 2018     

Malignant hyperthermia: Effects of sarcoplasmic reticulum Ca2+ pump inhibition

In porcine malignant hyperthermia-susceptible (MHS) skeletal muscles, calcium release is abnormal and resting calcium may be elevated. Thus MHS muscles may have prolonged twitch relaxation and lower fusion frequencies, which would be augmented by inhibition of sarcoplasmic reticulum (SR) Ca²⁺ adenosine triphosphatase (ATPase) activity; bundles of intact muscle cells from MHS and normal pigs were used to investigate this possibility. Cooling and low-frequency stimulation, in combination, enhanced twitch fusion and prolonged twitch relaxation significantly more in MHS than in normal muscles (e.g., 34 ± 4% versus 16 ± 4% fusion, and 82.4 ± 9.4 ms versus 43.2 ± 7.8 ms half-relaxation time, for MHS and normal muscles, respectively). Similarly, inhibition of the SR Ca²⁺ ATPase by cyclopiazonic acid resulted in significantly greater twitch fusion in MHS muscles. These results were consistent with predicted effects of enhanced SR Ca²⁺ release and/or elevated resting calcium in MHS muscles and indicate that cooling during a malignant hyperthermia crisis could actually increase the force of muscle contractures. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:361–366, 1998.     

Calendar of events

Intramyofiber accumulation of β‐amyloid fragments (Aβ) is a pathologic hallmark of inclusion‐body myositis (IBM), a progressive skeletal muscle disorder. We investigated the temporal pattern of alterations in the resting cytoplasmic [Ca²⁺] ([Ca²⁺]_i) as well as the depolarization‐evoked Ca²⁺ release from the sarcoplasmic reticulum in skeletal muscle from transgenic mice expressing human βAPP (MCK‐βAPP). MCK‐βAPP mice show an age‐dependent increase in [Ca²⁺]_i along with a reduction in depolarization‐evoked Ca²⁺ release, which appear well before the other reported aspects of IBM, such as inclusion formation, inflammation, centralized nuclei, atrophy, and skeletal muscle weakness. In the young MCK‐βAPP animals the increase in resting [Ca²⁺]_i can be attributed largely to Ca²⁺ influx through nifedipine‐sensitive Ca²⁺ channels. In the adult MCK‐βAPP mice, in addition to the nifedipine‐sensitive pathway, there is also a substantial contribution by the intracellular compartments to the increase in [Ca²⁺]_i. These results suggest that β‐amyloid‐induced disuption of Ca²⁺ handling may represent an early event in the pathogenesis of IBM. Muscle Nerve, 2010     

Pattern of summation with fatigue and inhibition of calcium release in rat muscle

Introduction:Fatigue disrupts muscle force summation and is associated with a decrease in cytoplasmic Ca²⁺ concentration. The purpose of this study was to compare summation during fatigue and recovery with summation during dantrolene‐induced inhibition of Ca²⁺ release.Methods:Rat medial gastrocnemius muscles were evaluated before and after fatigue, or during exposure to dantrolene. Summation was quantified by the ratio of the force transient associated with the final activation in a train of stimuli (Twf), obtained by subtraction of the force with one less stimulus, and the force of the twitch (Tw).Results:This ratio (Twf/Tw) decreased from 2.46 ± 0.11 (mean ± SEM) to 0.8 ± 0.1 during intermittent contractions, but was still significantly different from non‐fatigued muscle after 10 min of recovery. Dantrolene altered summation, as Twf/Tw was 1.7 ± 0.2 and 1.27 ± 0.15 at a low dose and a high dose, respectively.Conclusions:Inhibition of Ca²⁺ release alters summation, but repetitive stimulation leading to fatigue changes it more substantially. Muscle Nerve 44: 410–417, 2011     

From excitation to intracellular Ca2+ movements in skeletal muscle: Basic aspects and related clinical disorders

In skeletal muscle fiber, excitation-contraction coupling corresponds to the sequence of events occurring from action potential firing to initiation of contraction by an increase in cytosolic Ca²⁺. These events are elicited in response to excitation of the motor neuron which induces trains of action potentials in the muscle cell that spread along the sarcolemma and in depth along the T-tubule membrane. Depolarization of the T-tubule membrane induces a conformational change in a protein complex, called the dihydropyridine receptor, which opens a calcium channel anchored in the membrane of the sarcoplasmic reticulum, called the ryanodine receptor, in charge of release of Ca²⁺ ions that activate contractile proteins. Ryanodine receptors shut upon return of the T-tubule membrane potential to its resting value and muscle cell relaxation results from the removal of cytosolic Ca²⁺ that is pumped back into the SR lumen through the sarcoplasmic reticulum Ca²⁺ ATPase. Mutations in genes encoding either plasma membrane ion channels, the main subunit of the dihydropyridine receptor, ryanodine receptor, sarcoplasmic reticulum Ca²⁺ ATPase or proteins interfering with trans-sarcolemmal Ca²⁺ influx or sarcoplasmic reticulum Ca²⁺ efflux lead to clinical disorders that manifest as myotonia, muscle weakness, paralysis or muscle wasting.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

β-Adrenergic potentiation of E–C coupling increases force in rat skeletal muscle

We examined the mechanism(s) which allow terbutaline, a β₂-adrenergic agonist, to increase isometric force in bundles of normal and denervated rat soleus fibers. Terbutaline (10 μmol/L) potentiated tetanic contractions during exposure to 1 mmol/L ouabain, 10 μmol/L nifedipine, or 0.5 mmol/L iodoacetate. Terbutafine induced equivalent increases in submaximal potassium (K⁺) contracture and tetanic force: these effects were mimicked by 2 mmol/L dibutyrl-cyclic AMP. Therefore, terbutaline increased force by a cyclic AMP-dependent mechanism other than enhancement of sodium-pump activity, dihydropyridine sensitive Ca²⁺ currents, glycolysis, or action potentials. Pretreatment with 1 mmol/L caffeine induced submaximal potentiation of peak tetanic force but prevented further potentiation by terbutaline. This suggested that terbutaline did not influence the myofilaments, but acted on the sarcoplasmic reticulum (SR) to increase the myoplasmic Ca²⁺ concentration and hence force production. We speculate that force is potentiated following β-adrenoceptor activation by a cyclic AMP-dependent phosphorylation of Ca²⁺ release channels to facilitate SR calcium release during tetanic stimulation. © 1993 John Wiley & Sons, Inc.     

Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

Evidence for Modulation of DoDamine-neuronal Function by Tachykinin NK3 Receptor Stimulation in Gerbil Mesencephalic Cell Cultures

Primary cultures of gerbil mesencephalon were used for studying the modulation exerted by tachykinin NK₃ receptor activation on the activity of dopamine (DA) neurons. Dopamine neurons were identified by their ability to take up [³H]DA in a nomifensine-dependent manner. Moreover, tyrosine hydroxylase immunohistochemistry revealed that these neurons accounted for 5–7% of the total cell population. The NK₃ receptor agonists, senktide (EC₅₀= 0.58 nM) and [MePhe⁷]neurokinin B (EC₅₀= 3 nM), increased spontaneous [³H]DA release in a concentration-dependent manner. In contrast, tested at a supramaximal concentration (10-⁷ M), neither septide nor substance P were found to affect [³H]DA release. The senktide-evoked [³H]DA release was not observed when extracellular Ca²⁺ was chelated, but was unaffected by nomifensine. This indicates that this increase in [³H]DA outflow resulted more from an exocytotic process than from reversal of carrier-mediated DA uptake. Moreover, the senktide effect was unaffected by the Na⁺ channel blocker tetrodotoxin, a result suggesting a direct action of senktide on DA neurons. The non-peptide NK₃ receptor antagonist, SR 142801, shifted or blocked (ICs0 = 0.89 nM) the senktide-evoked [³H]DA release, while its (-)-antipode, SR 142806, was 80-fold less potent, in agreement with binding data. Selective antagonists for NK, (SR 140333) or NK₂ (SR 48968) receptors failed to reduce the senktide effect. Light scanning microscopic analysis of mesencephalic cells loaded with the Ca²⁺ sensitive dye, fluo-3, showed that senktide induced a rise in cytosolic Ca²⁺ in 8-10% of the cell population. The senktide-induced elevation in intracellular Ca²⁺ was rapid in onset and transient (at lo⁴ M) or more sustained with no further increase in fluorescence intensity (at 10^-7 M). The proportion of senktide-responsive cells was not significantly modified when extracellular Ca²⁺ was chelated, but was reduced by 87% in the presence of SR 142801 and by 75% in cultures that were pre-treated with the DA neurotoxin l-methyl-4-phenylpyridinium. The present study shows that enhancement of spontaneous [³H]DA release and intracellular Ca²⁺ mobilization may be observed after NK₃ receptor stimulation and that both biochemical events are likely to occur in DA neurons.     

Extracellular magnesium and calcium reduce myotonia in isolated ClC‐1 chloride channel‐inhibited human muscle

Introduction: Experimental myotonia induced in rat muscle by ClC‐1 chloride channel‐inhibited has been shown to be related inversely to extracellular concentrations of Mg²⁺ and Ca²⁺ ([Mg²⁺]_o and [Ca²⁺]_o) within physiological ranges. Because this implicates a role for [Mg²⁺]_o and [Ca²⁺]_o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg²⁺]_o and [Ca²⁺]_o on myotonia in human muscle. Methods: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC‐1 inhibition using 9‐anthracene carboxylic acid (9‐AC) and was assessed from integrals of force induced by 5‐Hz stimulation for 2 seconds. Results: Myotonia disappeared gradually when [Mg²⁺]_o or [Ca²⁺]_o were elevated throughout their physiological ranges. These effects of [Mg²⁺]_o and [Ca²⁺]_o were additive and interchangeable. Conclusions: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg²⁺ and Ca²⁺. Muscle Nerve 51 : 65–71, 2015     

Involvement of adenosine in ischemic and postischemic calcium regulation

In the CA1 region of the hippocampus, ischemia or high-frequency stimulation of the glutamatergic input induces neuronal calcium uptake that is reflected as a decrease of the extracellular concentration of calcium ([Ca²⁺]_ec. In this study, the effects of theophylline on these [Ca²⁺]_ec shifts were examined in doses (20 mg/kg iv) where theophylline is mainly acting by blocking adenosine receptors. By using calcium-sensitive microelectrodes, [Ca²⁺]_ec was concomitantly recorded in stratum pyramidale (SP) and stratum radiatum (SR) of the CA1 in adult Wistar rats, before, during, and for 6 h after transient forebrain ischemia. During ischemia (4-vessel occlusion, 20 min), the [Ca²⁺]_ec decrease in SR preceded (by 11± 4 s; mean ± SEM) the [Ca²⁺]_ec decrease in SP. Administration of theophylline prior to ischemia reduced the time from vessel-occlusion to the ischemic decrease in [Ca²⁺]_ec (from 3.0±0.3 to 0.9±0.1 min; mean±SEM;p<0.01). During electrically evoked burst firing, the [Ca²⁺]_ec shift was augmented by theophylline in nonischemic controls (by 29±4%; mean±SEM’p<0.05). After 6 h of reflow, i.e., at a time-point when the evoked calcium uptake is enhanced, theophylline had no effect on evoked [Ca²⁺]_ec shifts. In summary, during ischemia the uptake of calcium into CA1 pyramidal cells started in the dendrites and preceded that in the cell bodies. Removal of adenosine inhibition by theophylline accelerated ischemic calcium uptake and enhanced electrically evoked calcium uptake in control animals. In contrast, in the postischemic phase adenosine inhibition was lost with a secondary enhancement of the evoked calcium uptake that may be one critical factor in the development of delayed neuronal death.     

Roscovitine differentially facilitates cerebellar glutamatergic and GABAergic neurotransmission by enhancing Cav2.1 channel‐mediated multivesicular release

Synaptic vesicle exocytosis is triggered by Ca²⁺ influx through several subtypes of voltage‐gated calcium channels in the presynaptic terminal. We previously reported that paired‐pulse stimulation at brief intervals increases Ca_v2.1 (P/Q‐type) channel‐mediated multivesicular release (MVR) at glutamatergic synapses between granule cells (GCs) and molecular layer interneurons (MLIs) in rat cerebellar slices. However, it has yet to be determined how Ca_v2 channel subtypes take part in MVR in single axon terminal. This study therefore aimed at examining the effects of roscovitine on different types of cerebellar synapses that make contacts with Purkinje cells (PCs), because this compound has been shown to enhance Ca_v2.1 channel‐mediated MVR at GC‐MLI synapses. Bath application of roscovitine profoundly increased the amplitude of excitatory postsynaptic currents (EPSCs) at GC‐PC synapses by a presynaptic mechanism as previously observed at GC‐MLI synapses, whereas it caused a marginal effect on climbing fiber‐mediated EPSCs in PCs. At MLI‐PC synapses, roscovitine increased both the amplitude and decay time of inhibitory postsynaptic currents (IPSCs) by enhancing multivesicular GABA release. When extracellular Ca²⁺ concentration ([Ca²⁺]_e) decreased, roscovitine became less effective in increasing GC‐PC EPSCs. By contrast, roscovitine was able to augment MLI‐PC IPSCs in the low [Ca²⁺]_e. The Ca_v2.1 channel blocker ω‐agatoxin IVA suppressed the roscovitine‐induced facilitatory actions on both GC‐PC EPSCs and MLI‐PC IPSCs. These results demonstrate that roscovitine enhances MVR at the GC‐PC excitatory synapses in a manner dependent on the driving force of Ca_v2.1 channel‐mediated Ca²⁺ influx into the nerve terminal, while it also facilitates MLI‐PC inhibitory transmission via Ca²⁺‐insensitive mechanisms.     

Role of GABAB receptors in intracellular Ca2+ homeostasis and possible interaction between GABAA and GABAB receptors in regulation of transmitter release in cerebellar granule neurons

The expression of GABA_B receptors in cultured mouse cerebellar granule cells was investigated in binding experiments using [³H](S, R)-baclofen as well as in functional assessment of the ability of (R)-baclofen to interact with depolarization (15–40 mM KCI) coupled changes in intracellular Ca²⁺ homeostasis and neurotransmitter release. In the latter case a possible functional coupling between GABA_A and GABA_B receptors was investigated. The binding studies showed that the granule cells express specific binding sites for (R)-baclofen. The number of binding sites could be increased by exposure of the cells to the GABA_A receptor agonist THIP (4,5,6,7-tetrahy-droisoxazolo[5,4-c]pyridin-3-ol) during the culture period. Pretreatment of the neurons with pertussis toxin showed that the GABA_B receptors are coupled to G-proteins. This coupling was, however, less pronounced when the cells had been cultured in the presence of THIP. When ⁴⁵Ca²⁺ uptake was measured or the intracellular Ca²⁺ concentration ([Ca²⁺]_i) determined using the fluorescent Ca²⁺ chelator Fluo-3 it could be demonstrated that culturing the neurons in THIP influences intracellular Ca²⁺ homeostasis. Moreover, this homeostasis was found to be functionally coupled to the GABA_B receptors as (R)-baclofen inhibited depolarization-induced increases in ⁴⁵Ca²⁺ uptake and [Ca²⁺]_i. (R)-Baclofen also inhibited K⁺-induced transmitter release from the neurons as monitored by the use of [³H]D -aspartate which labels the neurotransmitter pool of glutamate. Using the selective GABA_A receptor agonist isoguvacine it could be demonstrated that the GABA_B receptors are functionally coupled to GABA_A receptors in the neurons leading to a disinhibitory action of GABA_B receptor agonists. © 1994 Wiley-Liss, Inc.     

Possible involvement of calmodulin-dependent kinases in Ca-dependent enhancement of [H]5-hydroxytryptamine uptake in rat cortex

Effects of Ca²⁺ on [³H]5-hydroxytryptamine (5-HT) uptake into rat cortical synaptosomes were studied. The uptake was enhanced in the presence of Ca²⁺ in Krebs-Ringer medium and the uptake at 0.3–5 mM Ca²⁺ was 2.4–2.7 times greater than that observed in the absence of Ca²⁺. The maximal increase at the concentration of 1 mM Ca²⁺ was achieved after 2 min preincubation. Ca²⁺-dependent enhancement of the [³H]5-HT uptake reflected an increase in V_max of the uptake process. However, K_d and B_max values for [³H]paroxetine were not significantly changed in the presence of 1 mM Ca²⁺ compared with Ca²⁺-free condition. On the other hand, uptake was still enhanced after synaptosomes were washed with Ca²⁺-free medium after preincubation with 1 mM Ca²⁺. Staurosporine (a protein kinase C inhibitor) and wortmannin (a myosin light chain kinase inhibitor) did not affect Ca²⁺-dependent enhancement of the uptake, whereas 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-

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-tyrosyl]-4-phenylpiperazine (KN-62, inhibitor of Ca²⁺/calmodulin-dependent kinase II) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a calmodulin antagonist) significantly reduced it. Moreover, L-type, but not P- or N-type, voltage-dependent Ca²⁺-channel blockers suppressed enhancement of the uptake. These results indicate that Ca²⁺-dependent enhancement of [³H]5-HT uptake is mediated by activation of calmodulin-dependent protein kinases, suggesting a possibility of calmodulin-dependent regulation of in vivo 5-HT uptake.     

Spatiotemporal Distribution of Ca2+ Following Axotomy and Throughout the Recovery Process of Cultured Aplysia Neurons

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca²⁺]_i with fura-2, and monitoring the neurons’resting potential and input resistance (R_i) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca²⁺]_i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca²⁺]_i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a [Ca²⁺]_i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating [Ca²⁺]_i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post-axotomy, [Ca²⁺]_i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca²⁺]_i recovers to the control level 7 – 10 min post-axotomy. In Ca²⁺-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca²⁺]_i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca²⁺]_i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca²⁺, Mg²⁺ and Na⁺ into intact axons do not induce the release of Ca²⁺ from intracellular stores, indicates that Ca²⁺ influx through voltage gated Ca²⁺ channels and through the cut end are the primary sources of [Ca²⁺]_i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca²⁺]_i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca²⁺]_i gradients. Other Ca²⁺ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.     

Pharmacological characterization of the specific binding of [H]ryanodine to rat brain microsomal membranes

High-affinity binding of [³H]ryanodine has been characterized in rat brain microsomal fractions. Membrane fractions from 4 brain regions (cerebral cortex, cerebellum, hippocampus and brainstem) have been isolated using sucrose density gradient purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the presence of a high-molecular weight protein (M_r 320kDa), similar to that of ryanodine receptor from muscle sarcoplasmic reticulum (SR). In the presence of high salt (1 M KCl), [³H]ryanodine binds to low density (0.8 M sucrose) cortical microsomal fraction with high affinity (K_d 1.5nM), and with the highest capacity (B_max 330fmol/mg protein). Kinetic analysis of the binding suggests multiple available binding sites for ryanodine. Binding of ryanodine is Ca²⁺ dependent (ED₅₀ 1 μM) and inhibited by Mg²⁺ and Ruthenium red. Adenine nucleotides have a biphasic effect on the binding of [³H]ryanodine. At low Ca²⁺ concentration caffeine and daunorubicin enhance the binding of [³H]ryanodine. The inositol 1,4,5-trisphosphate (IP₃) binding inhibitor, heparin, has no effect on ryanodine binding, and ryanodine and caffeine do not influence the binding of [³H]IP₃, which is enriched in the cerebellar fractions. These data demonstrate significant quantitative differences in the pharmacology of brain and muscle receptors and raise the question as to the physiological role of ryanodine binding proteins in the central nervous system and whether it is coupled to an endoplasmatic reticulum (ER) Ca²⁺ release channel.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

The bradykinin receptor — a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation

Bradykinin (BK) induced [³H]norepinephrine ([³H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [³H]NE release by BK is mediated by activation of BK-B₂-receptors, as determined using type specific BK receptor antagonists. BK induces [³H]NE release with a half maximal effective concentration of30 ± 0.5nM, and reaches maximal net fractional release of9.0 ± 1% with 200 nM BK. The BK-induced release is Ca²⁺ dependent, reaching maximal release at 1.0 mM Ca²⁺, is pertussis toxin insensitive (1 μg/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca²⁺ channel blockers, verapamil (10 μM), nifedipine (10 μM), and ω-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co²⁺ (ED₅₀ = 0.2mM) and Ni²⁺ (ED_50²⁺ = 1mM). These results indicate the involvement of a Ca²⁺ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca²⁺]_ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca²⁺]_ex is similar up to concentration of 1 μM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca²⁺ to BK-stimulated cells in Ca²⁺-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca²⁺ for induction of release. Protein kinase C inhibitors polymyxin B, K_252a, sangivamicin, and Ara-A, do not affect release induced by BK, indicating that also the diacylglycerol pathway activated by phospholipase C is not involved in the BK-mediated release. Since (a) the receptor-mediated release is absolutely calcium-dependent, with no release detected when Ca²⁺ is omitted from the extracellular medium, and (b) the receptor-triggered release of Ca²⁺ from intracellular stores is independent of [Ca²⁺]_ex⁷, it appears that calcium influx, and not Ca²⁺ released from intracellular stores, is the signal for stimulating release. Therefore, it is suggested that the primary signal stimulating release is Ca²⁺ influx via a specific calcium channel, and that the BK receptor may be coupled to this channel, which could be classified as a receptor-operated channel.