ABSTRACT
Vascular endothelial growth factors(165) (VEGF(165)) is the most potent and widely used pro-angiogenic factor. Here we determined optimal culture condition of recombinant human VEGF(165) (rhVEGF(165)) in Escherichia coli (E. coli). rhVEGF(165) expression was the highest in 0.25% of L-arabinose induction concentration, at 20 °C induction temperature, and for 5 h induction time under the control of araBAD promoter using pBADHisA vector. In biological activity test, rhVEGF(165) significantly increased the proliferative activity of CPAE cells (p<0.001) and upregulated the expressions of endothelial cell growth-related genes, such as platelet endothelial cell adhesion molecule (PECAM-1), endothelial-specific receptor tyrosine kinase (TEK), kinase insert domain protein receptor (KDR), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1) in calf pulmonary artery endothelial (CPAE) cells.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Vascular Endothelial Growth Factor A/biosynthesis , Animals , Arabinose , Cattle , Cell Proliferation/drug effects , Cells, Cultured , Culture Media , Escherichia coli/cytology , Gene Expression Regulation/drug effects , Humans , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Cerebellar Purkinje neurons have intracellular regulatory systems including Ca2+-binding proteins, intracellular Ca2+ stores, Ca2+-ATPase and Na+-Ca2+ exchanger (NCX) that keep intracellular Ca2+ concentration ([Ca2+]i) in physiological range. Among these, NCX interacts with AMPA receptors, activation of which induces cerebellar synaptic plasticity. And the activation of metabotropic glutamate receptor 1 (mGluR1) is also involved in the induction of cerebellar long-term depression. The interaction of NCX with mGluR1 is not known yet. Thus, in this study, the functional relationship between NCX and mGluR1 in modulating the [Ca2+]i in rat Purkinje neurons was investigated. The interaction between NCX and mGluR1 in Purkinje neurons was studied by measuring intracellular Ca2+ transients induced by an agonist of group I mGluRs, 3,5-dihydroxyphenylglycine (DHPG). The DHPG-induced Ca2+ transient was significantly reduced by treatments of NCX inhibitors, bepridil and KB-R7943. When cells were pretreated with antisense oligodeoxynucleotides of NCX, the DHPG-induced Ca2+ transient was also inhibited. These results suggest that NCX modulates the activity of mGluR1 in cerebellar Purkinje neurons. Therefore, NCX appears to play an important role in the physiological function of cerebellar Purkinje neurons such as synaptic plasticity.
Subject(s)
Calcium/metabolism , Cerebellum/cytology , Purkinje Cells/metabolism , Receptors, Metabotropic Glutamate/physiology , Sodium-Calcium Exchanger/metabolism , Animals , Animals, Newborn , Bepridil/pharmacology , Calcium Channel Blockers/pharmacology , Cells, Cultured , Drug Interactions , Enzyme Activation/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Oligodeoxyribonucleotides, Antisense/pharmacology , Purkinje Cells/drug effects , Quinoxalines/pharmacology , Rats , Sodium-Calcium Exchanger/antagonists & inhibitors , Sodium-Calcium Exchanger/genetics , Thiourea/analogs & derivatives , Thiourea/pharmacologyABSTRACT
Variations in intracellular calcium activity ([Ca2+]i) play crucial roles in information processing in Purkinje neurons such as synaptic plasticity. Although Na+-Ca2+ exchanger (NCX) has been shown to participate in the regulation of homeostasis and secretion in neuronal cells, the physiological role of NCX in Purkinje neurons, such as a role in cerebellar synaptic plasticity, is not well understood. NCX in acutely dissociated rat Purkinje neurons was identified by double staining with anti-calbindin D-28k antibody and anti-NCX antibody. The physiological activity of NCX was examined by measuring transient intracellular Ca2+ changes resulting from the Ca2+ influx via reverse mode of NCX (with 0 mM Na+/2.5 mM Ca2+ solutions) and the efflux via the forward mode of NCX (with 140 mM Na+/0 mM Ca2+ solutions). This transient increase in Ca2+ concentration was not elicited in the cells pretreated with NCX antisense oligodeoxynucleotides. And the Ca2+ influx resulting from the reverse mode of NCX was significantly reduced by 2-[2-[4-(4-nitrobenyloxy) phenyl] ethyl] isothiourea methanesulfonate, while the Ca2+ efflux via forward mode was inhibited by bepridil. The physiological role of NCX in synaptic function was studied by measuring Ca2+ transients induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolone-propionate (AMPA) receptor activation. This AMPA-evoked response was decreased with the inhibition of NCX forward mode and also, to less degree, with the inhibition of reverse mode. In antisense oligodeoxynucleotides pretreated cells, the AMPA-evoked response was also reduced, as was the case in NCX-inhibitor treated cells. The inhibition of NCX activity had depressant effects on Ca2+ transients induced by AMPA receptor activation. These results suggest that NCX plays a physiological role in modulating the activity of cerebellar Purkinje neurons, such as synaptic plasticity, via interaction with AMPA receptors in Purkinje neurons.
Subject(s)
Calcium/metabolism , Cerebellum/cytology , Excitatory Amino Acid Agonists/pharmacology , Isoxazoles/pharmacology , Propionates/pharmacology , Purkinje Cells/drug effects , Sodium-Calcium Exchanger/physiology , Thiourea/analogs & derivatives , Agatoxins , Animals , Animals, Newborn , Bepridil/pharmacology , Calbindins , Calcium Channel Blockers/pharmacology , Cells, Cultured , Diagnostic Imaging/methods , Drug Interactions , Excitatory Amino Acid Antagonists/pharmacology , Fluorescent Antibody Technique/methods , Glutamic Acid/pharmacology , Microscopy, Confocal/methods , Oligonucleotides, Antisense/pharmacology , Polyamines/pharmacology , Purkinje Cells/metabolism , Quinoxalines/pharmacology , Rats , S100 Calcium Binding Protein G/metabolism , Sodium/metabolism , Sodium Channel Blockers/pharmacology , Sodium-Calcium Exchanger/antagonists & inhibitors , Sodium-Calcium Exchanger/drug effects , Thiourea/pharmacologyABSTRACT
This study examined the acute effects of ethanol (EtOH) on the firing patterns of Purkinje cells (PCs) using an intracellular recording in slice preparation of rat cerebellum. The experiments were performed in sagittal cerebellar slices (400 micrometer) of adult Sprague-Dawley rats (80-100g). Ethanol was applied by a bath superfusion with a known concentration expressed as the percentage of solution by volume (v/v) at 0.1, 0.5, 1, 2, and 4%. The result of the Chi-square test illustrated that the firing patterns were altered significantly after EtOH (p=0.007). However, the firing patterns that were altered by EtOH application were not affected by EtOH concentration (p= 0.1296). Among the 54 PCs tested, 30 PCs did not display any spontaneous firing activity and 24 PCs displayed spontaneous spike activity, either spiking in the simple manner (n=14) or cyclicly oscillating (n=10). In the presence of EtOH, 31 PCs were quiet, 22 PCs exhibited simple spiking activity and 1 PC continued to oscillate. Most PCs that displayed spontaneous activity before EtOH application progressively slowed their spike activity after EtOH superfusion. Especially, it was evident that 9 out of 10 oscillating PCs stopped their regular cyclic activity. In addition, 9 out of 14 PCs that displayed simple spike activity ceased to fire after EtOH application. Eleven out of 30 quiet PCs began to fire irregularly after EtOH application and this phenomenon usually occurred with membrane depolarization. EtOH induced spontaneous activity in 36.7% (11/30) of the quiescent PCs. In conclusion, there was differential EtOH sensitivity in the vitro slice preparation. EtOH depressed the endogenously generated spontaneous activity, especially the oscillatory firing activity. In contrast, the silent PCs were excited after EtOH application. Since this differential sensitivity persists in the presence of tetrodotoxin (TTX), it is suggested that this differential sensitivity is peculiar to the PCs.
Subject(s)
Ethanol/toxicity , Purkinje Cells/drug effects , Animals , In Vitro Techniques , Purkinje Cells/physiology , Rats , Rats, Sprague-Dawley , Tetrodotoxin/pharmacologyABSTRACT
Transient myocardial ischemia during cardiac surgery causes a loss of energy sources, contractile depression, and accumulation of metabolites and H+ ion resulting in intracellular acidosis. The reperfusion following ischemic cardioplegia recovers intracellular pH, activates Na+-H+ exchange and Na+-Ca2+ exchange transports and consequently produces Ca2+ overload, which yields cell death. Among the various Ca2+ entry pathways, the Na+-Ca2+ exchanger is known to play one of the major roles during the ischemia/reperfusion of cardioplegia. Consequently, information on the changes in intracellular Ca2+ activities of human cardiac myocytes via the Na+-Ca2+ exchanger is imperative despite previous measurements of Ca2+ current of human single myocytes. In this study, human single myocytes were isolated from the cardiac tissues obtained during open-heart surgery and intracellular Ca2+ activity was measured with cellular imaging techniques employing fluorescent dyes. We report that the Na+-Ca2+ exchanger of adult cardiac myocytes is more susceptible to hypoxic insult than that of young patients.
Subject(s)
Calcium/metabolism , Hypoxia/metabolism , Sodium-Calcium Exchanger/physiology , Adult , Child , Child, Preschool , Female , Humans , Hydrogen-Ion Concentration , Infant , Male , Middle AgedABSTRACT
In this study, we investigated the effects of tertiary-butyl hydrogen peroxide (tBHP) on the large-conductance Ca2+-activated K+ (Maxi-K) channel of rat brain using lipid bilayer. When tBHP was applied to the cytosolic side, the open probability (Po) of both fast- and slow-gating Maxi-K channels increased within 1 min in dose-dependent manner. tBHP effects did not reverse immediately, suggesting tBHP induces some chemical modification on the channel protein. From kinetic analysis of single channel data, the increase in the Po appears to be mainly due to shortening of closed dwell time in both types of the Maxi-K channels. 50 microM diamide, a sulfhydryl-specific oxidant, irreversibly decreased the Po. However, further addition of 7.3 mM tBHP still increased the Po, suggesting that tBHP does not share the target for oxidation with diamide.
Subject(s)
Brain/drug effects , Potassium Channels, Calcium-Activated , Potassium Channels/metabolism , Potassium/metabolism , tert-Butylhydroperoxide/pharmacology , Animals , Brain/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Large-Conductance Calcium-Activated Potassium Channels , Lipid Bilayers , Liposomes/metabolism , Rats , tert-Butylhydroperoxide/administration & dosageABSTRACT
BACKGROUND AND PURPOSE: MRI has superior capabilities for the detection of cerebral infarcts compared with CT. CT was used to locate infarcts in most previous studies of atherothrombotic middle cerebral artery (MCA) territory infarcts. Thus, there was a possibility of missing concomitant small infarcts. More accurate identification of topographic lesions in MCA territory with MRI may help to establish the pathogenesis of stroke. The present study determines topographic patterns, distribution of vascular lesions, and probable mechanisms. METHODS: Forty-two patients with MCA territory infarcts on routine MRI and no major cause of cardioembolism were studied with conventional angiography or MR angiography. RESULTS: The topographic patterns seen on MRI were subdivided into 4 groups: cortical border-zone infarcts (n=6), pial territory infarcts without insular infarct (n=3), pial territory infarcts with insular infarct (n=14), and large subcortical infarcts (n=19). Of 6 patients with cortical border-zone infarcts, 4 had concomitant small cortical or subcortical multiple lesions. Angiography showed intrinsic MCA disease in 4 patients. Of 3 patients with pial territory infarcts without insular infarct, 2 also had small multiple centrum ovale lesions. All had intrinsic MCA disease. Pial territory infarcts with partial or whole insular lesions were present in 10 and 4 patients, respectively. Five patients had additional multiple cortical or subcortical lesions. Ten patients had intrinsic MCA disease. Of the 19 patients with large subcortical infarcts, 12 had centrum ovale infarcts, and 4 had both basal ganglia and centrum ovale lesions. Ten had concomitant small cortical or subcortical lesions. Six patients had intrinsic MCA disease. CONCLUSIONS: Similar vascular lesions induce different topographic patterns in MCA territory infarction, which are related to individual vascular variability, degree of primary and secondary collateralization, and pathogenesis of infarcts. Our study indicates that concomitant small cortical or subcortical lesions are also commonly associated findings in diverse patterns of MCA territory infarction, which can mostly be explained by probable embolic mechanism.
Subject(s)
Brain/pathology , Infarction, Middle Cerebral Artery/diagnosis , Magnetic Resonance Imaging , Adult , Aged , Aged, 80 and over , Cerebral Angiography , Diabetes Complications , Female , Humans , Hypercholesterolemia/complications , Hypertension/complications , Infarction, Middle Cerebral Artery/etiology , Infarction, Middle Cerebral Artery/pathology , Magnetic Resonance Angiography , Middle AgedABSTRACT
Traveling across time zones causes disruption to the normal circadian rhythms and social schedules because of travelers' shift in time. As the endogenous circadian timing system adapts slowly to new time cues, the phase relationship between biological rhythms and external time cues are out of synchronization for a period of time. This disturbance of circadian rhythms has been shown to impair physical and psychological health (Winget et al., 1984). To test the effects of repeated jet lag on mental abilities, airline cabin crew were compared with ground crew. Salivary cortisol was used as a physiological marker for circadian disruption. The cabin crew group, who had a history of repeated jet lag, had significantly higher salivary cortisol levels in an average working day. In addition, this elevated level of cortisol was only seen in the same subjects when the cabin crew were on transmeridian flights but not domestic flights. Cabin crew also exhibited cognitive deficits, possibly in working memory, that became apparent after several years of chronic disruption of circadian rhythms.
Subject(s)
Aerospace Medicine , Jet Lag Syndrome/psychology , Memory, Short-Term , Adult , Circadian Rhythm , Female , Humans , Hydrocortisone/metabolism , Jet Lag Syndrome/metabolism , Reaction Time , Saliva/metabolismABSTRACT
BACKGROUND AND PURPOSE: Correlation of MRI findings with atherosclerotic vascular lesions has rarely been attempted in patients with cerebellar infarction. The aim of this study was to correlate the MRI lesions with the vascular lesions seen on conventional cerebral angiography in cerebellar infarction. METHODS: The subjects included 31 patients with cerebellar infarcts who underwent both MRI and conventional cerebral angiography. We analyzed the risk factors, clinical findings, imaging study, and angiography results. We attempted to correlate MRI lesions with the vascular lesions shown in the angiograms. RESULTS: The vascular lesions seen on angiograms were subdivided into 3 groups: large-artery disease (n=22), in situ branch artery disease (n=6), and no angiographic disease with hypertension (n=3). The proximal segment (V1) lesions of vertebral artery were the most common angiographic features in patients with large-artery disease in which stroke most commonly involved the posterior inferior cerebellar artery (PICA) cerebellum. The V1 lesions with coexistent occlusive lesions of the intracranial vertebral and basilar arteries were correlated with cerebellar infarcts, which had no predilection for certain cerebellar territory. The intracranial occlusive disease without V1 lesion was usually correlated with small cerebellar lesions in PICA and superior cerebellar artery (SCA) cerebellum. The subclavian artery or brachiocephalic trunk lesion was associated with small cerebellar infarcts. The in situ branch artery disease was correlated with the PICA cerebellum lesions, which were territorial or nonterritorial infarct. No angiographic disease with hypertension was associated with small-sized cerebellar infarcts within the SCA, anterior inferior cerebellar artery, or SCA cerebellum. CONCLUSIONS: Our study indicates that the topographic heterogeneity of cerebellar infarcts are correlated with diverse angiographic findings. The result that large-artery disease, in which nonterritorial infarcts are more common than territorial infarcts, is more prevalent than in situ branch artery disease or small-artery disease, suggest that even a small cerebellar infarct can be a clue to the presence of large-artery disease.
Subject(s)
Cerebellum/blood supply , Cerebral Infarction/etiology , Intracranial Arteriosclerosis/complications , Intracranial Thrombosis/complications , Magnetic Resonance Imaging , Adult , Aged , Aged, 80 and over , Arteries/pathology , Basilar Artery/diagnostic imaging , Basilar Artery/pathology , Brachiocephalic Trunk/diagnostic imaging , Brachiocephalic Trunk/pathology , Cerebellum/diagnostic imaging , Cerebral Angiography , Cerebral Infarction/classification , Cerebral Infarction/diagnosis , Cerebral Infarction/diagnostic imaging , Female , Humans , Hypertension/complications , Intracranial Arteriosclerosis/diagnosis , Intracranial Arteriosclerosis/diagnostic imaging , Intracranial Thrombosis/diagnosis , Intracranial Thrombosis/diagnostic imaging , Male , Middle Aged , Prevalence , Risk Factors , Stroke/diagnosis , Stroke/diagnostic imaging , Stroke/etiology , Subclavian Artery/diagnostic imaging , Subclavian Artery/pathology , Vertebral Artery/diagnostic imaging , Vertebral Artery/pathologyABSTRACT
BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a rare transmissible disease that typically causes a rapidly progressive dementia and leads to death in less than 1 year. Although a few anecdotal reports suggest that diffusion-weighted magnetic resonance imaging may help substantiate premortem diagnosis of CJD, detailed correlation between radiographic data and clinical, electrophysiologic, and metabolic parameters is not available. METHODS: Signal abnormalities on diffusion-weighted images in 3 consecutive patients with probable CJD were correlated with psychometric features, electroencephalographic findings, and functional images with either positron emission tomography or single photon emission computed tomography. RESULTS: Focality of abnormalities on diffusion-weighted image, not apparent on routine magnetic resonance images, correlated closely with clinical manifestations of CJD. The topographic distribution of signal abnormality on diffusion-weighted image corresponded with abnormal metabolism or perfusion on positron emission and single photon emission computed tomographic scans. In 2 cases, the laterality of diffusion abnormalities correlated with periodic sharp wave activity on electroencephalograms. CONCLUSION: These findings extend previous observations that suggested a diagnostic and localizing utility of diffusion-weighted imaging in CJD.
Subject(s)
Creutzfeldt-Jakob Syndrome/diagnosis , Aged , Brain/diagnostic imaging , Brain/pathology , Cognition Disorders/diagnosis , Disease Progression , Electroencephalography , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Neuropsychological Tests , Probability , Severity of Illness Index , Tomography, Emission-Computed , Tomography, Emission-Computed, Single-PhotonABSTRACT
Intracellular recordings in cerebellar slice preparation showed that applications of 4-AP altered the pattern of oscillatory firing activity in Purkinje cells (PCs), especially yielding pronounced changes in action potential shape. 4-AP increased the amplitude and duration of action potential significantly and decreased the spike frequency. After 4-AP application, the duration of bursting was prolonged and the duration of after-burst hyperpolarization was progressively shortened. In all PCs tested, the rhythmicity of oscillatory firing activity was abolished completely at the steady state. These results suggest that 4-AP-sensitive currents determine the shape and frequency of individual Ca(2+)-dependent action potentials as well as maintaining oscillatory firing activity in PCs.
Subject(s)
4-Aminopyridine/pharmacology , Action Potentials/drug effects , Calcium/physiology , Purkinje Cells/drug effects , Purkinje Cells/physiology , Animals , Electrophysiology , In Vitro Techniques , Oscillometry , Rats , Rats, Sprague-DawleyABSTRACT
The effects of various concentrations (20, 50, and 100 mumol litre-1) of mepivacaine were studied in isolated guinea pig and rat right ventricular papillary muscles by measuring the effects on myocardial contractility and electrophysiological parameters. Mepivacaine produced dose-dependent depression of peak force during 0.5 to 3 Hz stimulation rates in guinea pig papillary muscles. Conduction block was frequently noted, especially at higher stimulation rates (2 and 3 Hz) with mepivacaine 50 and 100 mumol litre-1. In rat papillary muscle experiments, about 20% depression of peak force was shown at rested state contraction. Shortening of action potential (AP) duration (APD50: about 10%, APD90: about 10%) and rate-dependent depression of dV/dt max was observed with mepivacaine 100 mumol litre-1. In 26 mmol litre-1 K+ Tyrode's solution, mepivacaine 50 and 100 mumol litre-1 produced a dose-dependent depression of early (50 mumol litre-1: about 20%, 100 mumol litre-1: about 30%) and late (50 mumol litre-1: about 30%, 100 mumol litre-1: about 50%) force development. In slow APs, neither shortening of AP duration nor changes of dV/dt max were shown by mepivacaine 100 mumol litre-1. An approximate 30% depression of contracture induced by rapid cooling after 2 Hz stimulation rates was observed with mepivacaine 100 mumol litre-1. It may be concluded that the direct myocardial depressant effect of mepivacaine is likely to be caused by inhibition of Ca2+ release from the sarcoplasmic reticulum. The Na+ channel blocking action may contribute indirectly to the depression of contractility.
Subject(s)
Anesthetics, Local/pharmacology , Mepivacaine/pharmacology , Myocardial Contraction/drug effects , Animals , Culture Media , Culture Techniques , Depression, Chemical , Dose-Response Relationship, Drug , Electrophysiology , Female , Guinea Pigs , Hypothermia, Induced , Isotonic Solutions , Papillary Muscles/drug effects , Rats , Rats, Sprague-DawleyABSTRACT
Using lipid bilayer reconstitution technique, we investigated the oxidation effect of t-butyl hydrogen peroxide (tBHP) on the single channel activity of the sarcoplasmic reticulum (SR) calcium release channels isolated from canine latissimus dorsi muscles. When 0.7% tBHP was added in the cytosolic side, the channel activity became suppressed (n = 7), and it was recovered by changing the solution to the control solution. The suppression was due to the change in the gating mode of the channel: before tBHP the channel opened to four sub-conductance levels, but it opened to only one level after tBHP. These effects by tBHP were different from the previous finding using hydrogen peroxide (H2O2), which may be explained by different oxidation patterns between the two oxidants.
Subject(s)
Calcium Channels/drug effects , Peroxides/pharmacology , Sarcoplasmic Reticulum/drug effects , Animals , Dogs , Hydrogen Peroxide/pharmacology , Sarcoplasmic Reticulum/metabolism , tert-ButylhydroperoxideABSTRACT
Using the planar lipid bilayer technique, we tested whether NO directly activates calcium-activated potassium (Maxi-K) channels isolated from rat brain. We used streptozotocin (STZ) as NO donor, and the NO release was controlled with light. In the presence of 100-800 microM STZ, the Maxi-K channel activity increased up to 3-fold within several tens of seconds after the light was on, and reversed to the control level several minutes after shutting off the light. Similar activation was observed with other NO donors such as S-nitroso-N-acetylpenicillamine and sodium nitroprusside. The degree of activity increase was dependent upon the initial open probability (P[init]). When the P(init) was lower, the activity increase was greater. These results demonstrate that NO can directly affect the Maxi-K channel activity, and suggest that the Maxi-K channel might be one of the physiological targets of NO in brain.
Subject(s)
Calcium/pharmacology , Cerebral Cortex/chemistry , Liposomes/metabolism , Nitric Oxide/pharmacology , Potassium Channels, Calcium-Activated , Potassium Channels/metabolism , Action Potentials , Animals , Large-Conductance Calcium-Activated Potassium Channels , Light , Lipid Bilayers , Nitric Oxide/physiology , Nitroprusside/metabolism , Nitroprusside/pharmacology , Penicillamine/analogs & derivatives , Penicillamine/metabolism , Penicillamine/pharmacology , Rats , S-Nitroso-N-Acetylpenicillamine , Streptozocin/metabolism , Streptozocin/pharmacologyABSTRACT
BACKGROUND: The effects of anesthetic concentrations of sevoflurane were studied in isolated myocardial tissue to delineate the mechanisms by which cardiac function is altered. METHODS: Isometric force of isolated guinea pig ventricular papillary muscle was studied at 37 degrees C in normal and 26 mM K+ Tyrode's solution at various stimulation rates. Normal and slow action potentials were evaluated using conventional microelectrodes. Effects of sevoflurane on sarcoplasmic reticulum function in situ were also evaluated by its effect on rapid cooling contractures, which are known to activate Ca2+ release from the sarcoplasmic reticulum, and on concentrations of rat papillary muscle. Finally, Ca2+ and K+ currents of isolated guinea pig ventricular myocytes were examined using the whole-cell patch clamp technique. RESULTS: Sevoflurane equivalent to 1.4% and 2.8% depressed guinea pig myocardial contractions to approximately 85 and approximately 65% of control, respectively, although the maximum rate of force development at 2 or 3 Hz and force in rat myocardium after rest showed less depression. In the partially depolarized, beta-adrenergically stimulated myocardium, sevoflurane selectively depressed late peak force without changing early peak force, whereas it virtually abolished rapid cooling contractures. Sevoflurane did not alter the peak amplitude or maximum depolarization rate of normal and slow action potentials, but action potential duration was significantly prolonged. In isolated guinea pig myocytes at room temperature, 0.7 mM sevoflurane (equivalent to 3.4%) depressed peak Ca2+ current by approximately 25% and increased the apparent rate of inactivation. The delayed outward K+ current was markedly depressed, but the inwardly rectifying K+ current was only slightly affected by 0.35 mM sevoflurane. CONCLUSIONS: These results suggest that the direct myocardial depressant effects of sevoflurane are similar to those previously described for isoflurane. The rapid initial release of Ca2+ from the sarcoplasmic reticulum is not markedly decreased, although certain release pathway, specifically those induced by rapid cooling, appear to be depressed. Contractile depression may be partly related to the depression of Ca2+ influx through the cardiac membrane. The major electrophysiologic effect of sevoflurane seems to be a depression of the delayed outward K+ current, which appears to underlie the increased action potential duration.
Subject(s)
Anesthetics, Inhalation/pharmacology , Ethers/pharmacology , Methyl Ethers , Myocardial Contraction/drug effects , Animals , Calcium/metabolism , Depression, Chemical , Female , Guinea Pigs , In Vitro Techniques , Papillary Muscles/drug effects , Papillary Muscles/physiology , Potassium Channels/drug effects , Rats , Rats, Sprague-Dawley , SevofluraneABSTRACT
The ryanodine receptor/channel (RyR) mediates the release of calcium from the sarcoplasmic reticulum (SR) in both skeletal and cardiac muscle cells. There are three isoforms of the RyR: RyR1, RyR2, and RyR3. RyR1 is specifically expressed in skeletal muscles and RyR2 in cardiac muscles. RyR3 is yet another isoform found in non-muscle cells such as neuronal cells. Single channel recordings of RyR1 and RyR2 reconstituted in artificial lipid bilayer show that the characteristics of two isoforms are very distinct. RyR1 has a shorter mean open time and is activated at a higher concentration of Ca2+ than RyR2. In this study, we isolated the heavy SR membranes from canine latissimus dorsi muscles and investigated the single channel activities from the heavy SR membrane fraction using Cs+ as a charge carrier. Two different types of activities were observed. The fast-gating type (FG) with the mean open time of 0.9 ms was more frequently recorded (n = 12) than the slow-gating type (SG) with the mean open time of 269.2 ms. From the I-V relation, the slope conductance of the FG was calculated to be 514.7 pS and the SG, to 625.6 pS. The activity of the fast gating type increased by raising the concentration of Ca2+ in the cis-solution up to 100 microM. The appearance of the SG in the canine heavy SR membrane fraction suggests a possibility that two types of RyR isoform are co-expressed in mammalian skeletal muscle as well as in avian, amphibian and piscine fast twitch muscles.
Subject(s)
Calcium Channels/metabolism , Ion Channel Gating , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Dogs , Lipid Bilayers , Microsomes/metabolism , Ryanodine Receptor Calcium Release Channel , Thorax , Time FactorsABSTRACT
Cardiac dysfunctions such as myocardial functional failure and ventricular arrhythmia have been largely attributed to intracellular Ca2+ overload. One of the mechanisms of intracellular Ca2+ overload involves a rapid influx of Ca2+ via Na(+)-Ca2+ exchange during the reperfusion which utilizes the accumulation of Na+ in myocytes during ischemic cardiac arrest. Possible sources of the intracellular Na+ accumulation include Na+ channel, Na(+)-H+ exchange, Na(+)-Ca2+ exchange, and Na+ background current. In this study, we studied the role of the Na+ background current in intracellular Na+ accumulation during the cardiac arrest by measuring the Na+ background current in guinea pig ventricular myocytes with whole cell clamp method and evaluating the effects of cardioprotective drugs on the Na+ background current. The results were as follows: (1) The Na+ background inward current at -40 mV membrane potential was larger at Ca2+ free solution than 1.8 mM Ca2+ solution. (2) The Na+ background current was not affected by verapamil. (3) 2 microM O-(N, N-hexamethylene)-amiloride (HMA) decreased the Na+ background current at negative membrane potential. (4) The new cardioprotective drug, R 56865, decreased the Na+ background current. These results suggest that the Na+ background current plays a role in increasing the intracellular Na+ activity during high K+ cardioplegia and the blocking effect of myoprotective drugs, such as R 56865, on the Na+ background current may contribute to myocardial protection after cardioplegia.
Subject(s)
Heart/drug effects , Sodium/metabolism , Amiloride/pharmacology , Animals , Benzothiazoles , Guinea Pigs , Heart Arrest, Induced , Myocardium/metabolism , Piperidines/pharmacology , Potassium/pharmacology , Thiazoles/pharmacology , Verapamil/pharmacologyABSTRACT
The Na(+)-Ca2+ exchange transport operating in outward mode has been suggested to cause Ca2+ entry during reperfusion or reoxygenation, exchanging extracellular Ca2+ for intracellular Na+ that has accumulated during ischemia or cardioplegia. During cardioplegia, however, an increase in Ca2+ entry via this mechanism can be decreased due to increased intracellular H+ activity and a decrease in cellular ATP content. In this study giant excised cardiac sarcolemmal membrane patch clamp technique was employed to investigate the effect of cytosolic pH change on the Na(+)-Ca2+ exchanger, excluding the effect of ATP, in guinea pig cardiac myocytes. The outward Na(+)-dependent current, which has a characteristics of Hill equation, was decreased as pH was decreased in the range of 7.5-6.5. The current density generated by the Na(+)-Ca2+ exchange transport was 56.6 +/- 4.4 pA/pF (Mean +/- S.E.M.) at pH 7.2 and decreased to 42.9 +/- 3.0 pA/pF at pH 6.9. These results imply that Na(+)-Ca2+ exchange transport, operating in a reverse mode during cardioplegia, decreases due to increased intracellular H+, and further suggest that consequent intracellular Na+ accumulation is one of aggravating factors for Ca2+ influx during reoxygenation or reperfusion.
Subject(s)
Acidosis/metabolism , Calcium/metabolism , Myocardium/metabolism , Sodium/metabolism , Animals , Electric Conductivity , Guinea Pigs , Heart Ventricles/metabolism , Hydrogen-Ion Concentration , Ion Transport , Sodium-Hydrogen Exchangers/physiologyABSTRACT
Ryanodine has different effects on the contractility of rat and guinea pig ventricular muscle. Thus we investigated the effect of ryanodine on the intracellular Ca2+ and Na+ activities of the rat and guinea pig ventricular myocytes with two specific aims; whether there are any differences in intracellular Na+ activities between rat and guinea pig ventricular muscle cells, and if any, how the differences in intracellular Na+ activities are related to the effect of Na(+)-Ca2+ exchange on the action potential configuration and excitation-contraction coupling of the rat and guinea pig ventricular myocytes. Ryanodine (10(-7) M) diminished the slow repolarization phase of the rat ventricular action potential while the duration of the rapid repolarization phase increased. Ryanodine (10(-7) M) significantly increased the plateau of the action potential. At the steady state of 0.2 cps, intracellular Na+ activities (aiNa) of the rat and guinea pig ventricular myocytes were 8.7 +/- 5.2 mM (n = 16, 4 rats) and 10.0 +/- 4.1 mM (n = 25, 7 guinea pigs) respectively, but there were no statistically significant differences. The contractility of the rat ventricular muscle nearly disappeared due to ryanodine (10(-7) M) with little changes in aiNa. Monensin (10 mM) not only increased the resting tension but also remarkably increased aiNa from 2.0 mM to 20 mM. Ryanodine (10(-7) M) continuously decreased aiNa of the guinea pig ventricular muscle after the contraction ceased to decrease. Monensin increased the contractility as well as aiNa. These results suggest that the contractility of rat and guinea pig ventricular myocytes is determined by the change in the action of the Na(+)-Ca2+ exchange mechanism depending upon the plateau of action potential and the intracellular Na+ and Ca2+ activities. So ryanodine could decreases the contractility via its effect on Na(+)-Ca2+ exchange transport which could be one of possible mechanisms of negative inotropism by ryanodine.
Subject(s)
Heart/drug effects , Myocardial Contraction/drug effects , Myocardium/metabolism , Ryanodine/pharmacology , Sodium/metabolism , Action Potentials/drug effects , Animals , Female , Guinea Pigs , Heart Ventricles , Intracellular Membranes/metabolism , Male , Myocardium/cytology , RatsABSTRACT
The removal of Ca2+ from the cardioplegic solutions could cause the danger of inducing a "calcium paradox" during reperfusion. Since intracellular Ca2+ activities are coupled to Na+ activities via Na(+)-Ca2+ exchange, an increase in intracellular Na+ activities during the cardioplegia could cause an abrupt Ca2+ influx when reperfused. To study the effects of Na+ and Ca2+ concentrations in cardioplegic solutions on intracellular Ca2+ activities during the cardioplegia and subsequent recovery period, the membrane potential and intracellular Na+ and Ca2+ activities of guinea pig ventricular papillary were measured. 1) A cardioplegia with low Ca2+ cardioplegic solution significantly decreased the overshoot and duration of the first action potential after cardioplegia, but the changes in action potential configuration were minimized after a cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 2) Intracellular Na+ activity was continuously decreased during the cardioplegia, and the intracellular Na+ activity 20 minutes after cardioplegia was the highest with low Ca2+ cardioplegic solution. 3) Intracellular Na+ and Ca2+ activities were continuously decreased during the cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 4) During a reperfusion of Tyrode solution after cardioplegia intracellular Na+ and Ca2+ activities were increased. Intracellular Ca2+ activity was increased more rapidly than intracellular Na+ activity. 5) The rate of increase in intracellular Ca2+ activity with reperfusion of Tyrode solution was dependent upon intracellular Na+ activity during cardioplegia, in such a way that the higher the intracellular Na+ activity was, the faster the intracellular Ca2+ activity increased. These data suggest that Na(+)-Ca2+ exchange mechanism may play an important role in the regulation of intracellular Ca2+ activity during recovery after cardioplegia.