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

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

Three-dimensional location of the imperatoxin A binding site on the ryanodine receptor.

Cryo-electron microscopy and three-dimensional, single-particle image analysis have been used to reveal the specific binding site of imperatoxin A (IpTx(a)) on the architecture of the calcium release channel/ryanodine receptor from skeletal muscle (RyR1). IpTx(a) is a peptide toxin that binds with high affinity to RyR1 and affects its functioning. The toxin was derivatized with biotin to enhance its detection with streptavidin. IpTx(a) binds to the cytoplasmic moiety of RyR1 between the clamp and handle domains, 11 nm away from the transmembrane pore. The proposed mimicry by IpTx(a) of the dihydropyridine receptor (DHPR) II-III loop, thought to be a main physiological excitation-contraction trigger, suggests that the IpTx(a) binding location is a potential excitation-contraction signal transduction site.     

Location of divergent region 2 on the three-dimensional structure of cardiac muscle ryanodine receptor/calcium release channel

Ryanodine receptors (RyRs) are a family of calcium release channels found on intracellular calcium-handing organelles. Molecular cloning studies have identified three different RyR isoforms, which are 66-70% identical in amino acid sequence. In mammals, the three isoforms are encoded by three separate genes located on different chromosomes. The major variations among the isoforms occur in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and DR3). In the present study, a modified RyR2 (cardiac isoform) cDNA was constructed, into which was inserted a green fluorescent protein (GFP)-encoding cDNA within DR2, specifically after amino acid residue Thr1366 (RyR2(T1366-GFP)). HEK293 cells expressing RyR2(T1366-GFP) cDNAs showed caffeine-sensitive and ryanodine-sensitive calcium release, demonstrating that RyR2(T1366-GFP) forms functional calcium release channels. Cells expressing RyR2(T1366-GFP) were identified readily by the characteristic fluorescence of GFP, indicating that the overall structure of the inserted GFP was retained. Cryo-electron microscopy (cryo-EM) of purified RyR2(T1366-GFP) showed structurally intact receptors, and a three-dimensional reconstruction was obtained by single-particle image processing. The location of the inserted GFP was obtained by comparing this three-dimensional reconstruction to one obtained for wild-type RyR2. The inserted GFP and, consequently Thr1366 within DR2, was mapped on the three-dimensional structure of RyR2 to domain 6, one of the characteristic cytoplasmic domains that form part of the multi-domain "clamp" regions of RyR2. The three-dimensional location of DR2 suggests that it plays roles in the RyR conformational changes that occur during channel gating, and possibly in RyR's interaction with the dihydropyridine receptor in excitation-contraction coupling. This study further demonstrates the feasibility and reliability of the GFP insertion/cryo-EM approach for correlating RyR's amino acid sequence with its three-dimensional structure, thereby enhancing our understanding of the structural basis of RyR function.     

Effect of temperature on the electrical and mechanical activity of lizard ventricular myocardium

Intracellular action potentials and isometric twitches were recorded from lizard ventricles electrically driven at 20 and 4 beats/min and submitted to temperatures changes between 10.5 and 21 degrees C. It was found that cooling induced a depolarization of the diastolic membrane potential ER, which below 15 degrees C exceeded that predictable for a diffusion potential; on the contrary, during the recovery from hypothermia ER underwent a transitory hyperpolarization. Other effects of the low temperature were a decrease of the maximum rate of depolarization, a lengthening of both the action potential duration and the time to peak contraction, an increase of the strength of contraction, in the hearts driven at 20/min it became apparent also an increase of the action potential overshoot. The hypothesis is discussed that the positive inotropic effect of low temperatures may be due not only to a slowing down of the repolarization of the action potential, but also to an increase of the slow inward current intensity.     

Effect of induced hypothyroid state on the mechanical and electrical properties of the rat ventricular myocardium

The effect of hypothyroid state on the transmembrane potential was studied in isolated cardiac ventricular trabeculae of rats. Hypothyroid state was induced by methimazole treatment or thyroidectomy and checked by determining serum thyroxine level. Hypothyroidism decreased the maximum rate of depolarization (Vmax) and the resting potential, increased the overshoot and the duration of action potential at 20, 50 and 90% repolarization. These changes were more pronounced after methimazole treatment than after thyroidectomy. The results strongly suggest that in hypothyroidism the significant alterations in the voltage-time course of the transmembrane action potential influencing Ca2+-movement across the sarcolemma may have an indirect role in the decreased myocardial contractility. On the other hand, methimazole has an aspecific cardiac effect which may modify the cardiac effect of hypothyroidism induced by the drug.     

2 Hz与100 Hz刺激在诱导大鼠视皮层长持续长时程增强中的不同作用

在哺乳动物的视皮层,多种不同参数的刺激可诱导出长时程增强(long-term potentiation,LTP)现象。但关于刺激参数与持续时间长于3h的长持续LTP(long lasting LTP,L-LTP)之间关系的研究较少。本研究用3周龄的大鼠视皮层脑片标本,在Ⅳ层刺激而在Ⅱ／Ⅲ层记录场电位,待场电位稳定后施加强直刺激诱导LTP,探讨2Hz与100Hz的强直刺激在诱发持续时间长于3h的L-LTP中的作用。结果表明,多于300个脉冲不同频率的刺激可稳定地诱导出L-LTP;2Hz与100Hz的刺激诱发的L-LTP有明显不同的表达形式,100Hz刺激可诱导出较大的L-LTP;频率相同而脉冲数不同的强直刺激诱发的L-LTP有相同的表达形式。以上结果提示,不同频率的强直刺激诱发的L-LTP机制可能不同;相同频率的刺激(脉冲数不同)诱发的L-LTP可能有相同的机制。     

Effect of temperature and temperature acclimation on the ryanodine sensitivity of the trout myocardium

The influence of acute temperature change and temperature acclimation on the sensitivity of contracture development to ryanodine were examined in the rainbow trout myocardium using two preparations: in vitro isolated ventricular strips and in situ working perfused hearts. Ryanodine effects in vitro were dependent on test temperature (8 and 18 °C), pacing frequency (0.2–1.5 Hz) and acclimation temperature (8 and 18 °C). At a pacing frequency of 0.2 Hz and a test temperature of 18 °C, ryanodine depressed isometric tension development in ventricular strips both from trout acclimated to 8 and 18 °C but the decrease was significantly greater in strips from 8 °C-acclimated trout. No ryanodine effect was observed in either acclimation group at a test temperature of 8°C. The effect of ryanodine in vitro was reduced or lost at pacing frequencies greater than 0.2 Hz and at 0.6 Hz ryanodine depressed tension development at 18 °C only in strips from 8 °C-acclimated trout. Ryanodine did not affect tension development at stimulation rates above 0.6 Hz in any test group. Likewise, ryanodine did not significantly impair cardiac performance of in situ working perfused heart preparations which operated at intrinsic beat frequencies in excess of 0.6 Hz. These results suggest that the sarcoplamic reticulum calcium release channel of the trout myocardium is expressed but is not functionally involved in beat-to-beat regulation of contractility at either (1) low temperature (8 °C), or (2) at routine physiological heart rate (>0.6 Hz). However, under conditions in which involvement of the sarcoplasmic reticulum is observed (18 °C and a heart rate < 0.6 Hz), prior acclimation to low temperature results in either a greater capacity of the sarcoplasmic reticulum to store releasable calcium or an increase in the amount of calcium that is in releasable form.Abbreviations bm body mass - E-C coupling, excitation-contraction coupling - IVS isometric ventricular strip - SR sarcoplasmic reticulum - TES N-tris[hydroxy-methyl]methyl-2-aminoethane sulfonic acid - WPH in situ working perfused heart     

Effects of adrenocortical steroids on long-term potentiation in the limbic system: Basic mechanisms and behavioral consequences

Summary Hippocampal structures are a major target for adrenal steroid hormones, and hence these neural regions are some of the most likely mediators of the effects of adrenocortical steroids on behavior. Memory disturbance, in particular biasing toward negative contents, are part of the symptomatology presented by depressive patients. In turn, a sizeable subset of depression also presents with hypercortisolemia. Adrenocortical hormones are also known to affect memory processes. Hippocampal formatio is essential for declarative memory. We thought it appropriate then to study the effects of adrenal steroids on long-term potentiation, a putative memory mechanism in the hippocampus. Two clearly distinguished components of the evoked response to perforant path stimulation can be studied in the hippocampus: the excitatory postsynaptic potential (EPSP) which denotes the graded depolarization of the somadendritic region of the neuron and the population spike (PS), a manifestation of the all-or-none-discharge of the cell action potential. Corticosterone had a significant depressant effect on the EPSP component of the evoked response immediately and 15 min after injection. Thereafter EPSP amplitudes were within normal values. Corticosterone significantly decreased the PS immediately after the train, the component remaining low 30 min after the train. 5-Dihydrocorticosterone (a ring A-reduced metabolite of corticosterone) significantly reduced the PS component of the response at all times after injection. 18-Hydroxydeoxycorticosterone and deoxycorticosterone significantly decreased both EPSP and PS components of the evoked response from the time of infusion. Contrary to expectation, tetrahydrodeoxycorticosterone was ineffective in decreasing and if anything, enhanced the development of long-term potentiation. 18-Hydroxydeoxycorticosterone 21-acetate behaved like vehicle, except for the first 30 min after injection when the EPSP was decreased. Allotetrahydroprogesterone decreased all EPSP's values and had no effect in the PS development in comparison with vehicle. The suggestion is made that the study of steroidal effects on hippocampal LTP can serve as a preclinical model of some aspects of depression in a specific subset of the disease.     

Effects of Chronic Stress and Phenytoin on the Long-term Potentiation （LTP） in Rat Hippocampal CA1 Region

Stress is the response to stimulation from inside andoutside with complicated effects on organisms. Appropri-ate stressful reactions are helpful in resisting diseases byactivating unspecific modulation system, while severe orprolonged stresses are harmful and even induce mentaland physical disorders such as recurrent depression, post-traumatic stress disorder (PTSD), Alzheimer’s disease andepilepsy [1]. Hippocampus, a main brain region of keyimportance for learning, memory and emotion, is t…     

Toward decrypting the allosteric mechanism of the ryanodine receptor based on coarse‐grained structural and dynamic modeling

The ryanodine receptors (RyRs) are a family of calcium (Ca) channels that regulate Ca release by undergoing a closed‐to‐open gating transition in response to action potential or Ca binding. The allosteric mechanism of RyRs gating, which is activated/regulated by ligand/protein binding >200 Å away from the channel gate, remains elusive for the lack of high‐resolution structures. Recent solution of the closed‐form structures of the RyR1 isoform by cryo‐electron microscopy has paved the way for detailed structure‐driven studies of RyRs functions. Toward elucidating the allosteric mechanism of RyRs gating, we performed coarse‐grained modeling based on the newly solved closed‐form structures of RyR1. Our normal mode analysis captured a key mode of collective motions dominating the observed structural variations in RyR1, which features large outward and downward movements of the peripheral domains with the channel remaining closed, and involves hotspot residues that overlap well with key functional sites and disease mutations. In particular, we found a key interaction between a peripheral domain and the Ca‐binding EF hand domain, which may allow for direct coupling of Ca binding to the collective motions as captured by the above mode. This key mode was robustly reproduced by the normal mode analysis of the other two closed‐form structures of RyR1 solved independently. To elucidate the closed‐to‐open conformational changes in RyR1 with amino‐acid level of details, we flexibly fitted the closed‐form structures of RyR1 into a 10‐Å cryo‐electron microscopy map of the open state. We observed extensive structural changes involving the peripheral domains and the central domains, resulting in the channel pore opening. In sum, our findings have offered unprecedented structural and dynamic insights to the allosteric mechanism of RyR1 via modulation of the key collective motions involved in RyR1 gating. The predicted hotspot residues and open‐form conformation of RyR1 will guide future mutational and functional studies. Proteins 2015; 83:2307–2318. © 2015 Wiley Periodicals, Inc.     

Age-related effects of the neuromodulator D-serine on neurotransmission and synaptic potentiation in the CA1 hippocampal area of the rat

The effects of the co-agonist of the N-methyl-D-aspartate receptor (NMDAr) D-serine on glutamatergic neurotransmission and synaptic potentiation were studied in the CA1 hippocampal field of young (3-5 months old) and aged (25-27 months old) Sprague-Dawley rats using ex vivo extracellular electrophysiological recording techniques. Exogenous d-serine depressed fast neurotransmission mediated by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate subtype of glutamate receptors in young but not in aged rats by acting on inhibitory glycinergic interneurons. In contrast, D-serine dose-dependently enhanced NMDAr-mediated synaptic responses in both groups of animals, but with a larger magnitude in aged rats, thus preventing the age-related decrease in NMDAr activation. D-serine also increased the magnitude of long-term potentiation in aged but not in young rats. Finally, D-serine levels were dramatically reduced in hippocampal tissues of aged rats. Taken together, these results indicate a weaker activation of the NMDAr glycine modulatory site by endogenous D-serine in aged animals, which accounts for a reduced NMDAr contribution to synaptic plasticity in ageing.     

Influence of dilated cardiomyopathy and a left ventricular assist device on vortex dynamics in the left ventricle

Together with new developments in mechanical cardiac support, the analysis of vortex dynamics in the left ventricle has become an increasingly important topic in literature. The aim of this study was to develop a method to investigate the influence of a left ventricular assist device (LVAD) on vortex dynamics in a failing ventricle. An axisymmetric fluid dynamics model of the left ventricle was developed and coupled to a lumped parameter model of the complete circulation. Simulations were performed for healthy conditions and dilated cardiomyopathy (DCM). Vortex structures in these simulations were analysed by means of automated detection. Results show that the strength of the leading vortex ring is lower in a DCM ventricle than in a healthy ventricle. The LVAD further influences the maximum strength of the vortex and also causes the vortex to disappear earlier in time with increasing LVAD flows. Understanding these phenomena by means of the method proposed in this study will contribute to enhanced diagnostics and monitoring during cardiac support.     

Influence of temperature on calcium sensitivity in the ventricular myocardium of the South American lungfish: Effects of extracellular calcium and adrenaline

The mechanical properties of ventricular myocardium of the South American lungfish, Lepidosiren paradoxa, acclimated to 25 °C, were evaluated in vitro at 15, 25 and 35 °C. The inotropism (Fc—% of initial values) of ventricle strips was examined in response to adrenaline (from 10⁻⁸ to 10⁻⁵ M) and extracellular calcium (from 2.5 to 14.5 mM) in all experimental temperatures. At 15 and 25 °C, Fc rose when extracellular Ca²⁺ or adrenaline were increased, while Fc remained unchanged at 35 °C. These results suggest that at lower temperatures Ca²⁺ availability is a limiting step to cardiac performance and can be ameliorated by adrenergic stimulation. In contrast, since inotropic agents failed to increase cardiac inotropism at 35 °C, lungfish myocytes seem to show a high temperature sensitivity, which increases Ca²⁺-buffering capacity and/or Ca²⁺ transportation from and to cytosol as well as myofilaments Ca²⁺ sensitivity.     

Effect of 120 days of bed-rest with and without countermeasures on the mechanical properties of the triceps surae muscle in young women

The effect of a 120-day 6° head-down tilt (HDT) bed rest with and without countermeasures on the mechanical properties of the human triceps surae muscle was studied in eight healthy young women subjects. One group [n = 4, mean age 31.5 (SEM 1.7) years] underwent a 120-day HDT only and a second group [n = 4; mean age 28.0 (SEM 1.1) years] underwent HDT with countermeasures (physical training). The results showed that the contractile properties of the skeletal muscle studied changed considerably. After HDT without countermeasures the maximal voluntary contraction (MVC) had decreased by 36% (P < 0.05), and the electrically evoked tetanic tension at 150 Hz (P _o) and isometric twitch contraction (P _t) had decreased by 24% (P < 0.02) and 12% (P < 0.05), respectively. Time- to-peak tension (TPT) of the twitch had significantly increased by 14% (P<0.05), but half-relaxation time (1/2RT), and total contraction time (TCT) had decreased by 19% (P<0.05) and 18% (P<0.05), respectively. The difference between P _o and MVC expressed as a percentage of P _o and referred to as force deficiency (FD), was also calculated. The FD had increased by 40% (P<0.001). The rate of increase of voluntary contractions calculated according to a relative scale had significantly reduced, but for the electrically evoked contraction no substantial changes were observed. After HDT with countermeasures TPT, 1/2RT and TCT of the twitch had decreased by 4%, 7%, 19%, respectively in relation to the control condition. Training had caused a decrease of 3% (P>0.05) in MVC, and P _t, and in P _o of 14%, and of 9% (P>0.05), respectively. The FD had decreased significantly by 10% (P<0.02). The rate of increase of electrically evoked tetanic tension did not change significantly during HDT with countermeasures but the rate of increase in isometric voluntary tension development was increased. Physical training provided a reserve of neuromuscular function, which attenuated the effect of bed rest. The experimental findings indicated that neural as well as muscle adaptation occurred in response to HDT with countermeasures. Accepted: 7 November 1997     

Interaction between interferon gamma and insulin-like growth factor-1 in hippocampus impacts on the ability of rats to sustain long-term potentiation

There is compelling evidence to suggest that inflammation significantly contributes to neurodegenerative changes. Consistent with this is the observation that several neurodegenerative disorders are accompanied by an increase in the concentration of interleukin (IL)-1beta. IL-1beta has a negative impact on synaptic plasticity and therefore an increased concentration of IL-1beta, such as that in the hippocampus of the aged rat, is associated with a deficit in long-term potentiation (LTP). IL-1beta is derived mainly from activated microglia but the trigger leading to this activation, specifically in the aged brain, remains to be identified. Here we examined the possibility that interferon (IFN)gamma may stimulate microglial activation and increase IL-1beta concentration, thereby inhibiting LTP. The IFNgamma concentration was increased in hippocampus prepared from aged, compared with young, rats and inversely correlated with the ability of rats to sustain LTP. Intracerebroventricular injection of IFNgamma inhibited LTP, and increased microglial activation was observed in both IFNgamma-injected and aged rats. The age-related increase in IFNgamma was accompanied by a decrease in the hippocampal concentration of insulin-like growth factor (IGF)-1. The evidence presented suggests that IGF-1 acts to antagonize the IFNgamma-induced microglial activation, the accompanying increase in IL-1beta concentration and the consequent deficit in LTP.