Polymorphisms of SLC22A9 (hOAT7) in Korean Females with Osteoporosis

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SNP within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SNPs in the osteoporosis group (A645G and T1277C), three SNPs in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and 12.74 microM, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

Distinct Cellular Calcium Metabolism in Radiation-sensitive RKO Human Colorectal Cancer Cells

Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.

Association of rOAT1 with Caveolin-1 in Rat Kidney / 대한신장학회지

PURPOSE: A family of organic anion transporters (OAT) has been identified, and several isoforms have been reported. The regulatory mechanisms of OATs functions, however, still remain to be elucidated. The rat OAT1 contributes to move a number of negatively-charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. To address this issue, we investigated the protein-protein interaction between rOAT1 and Cav-1. METHODS: The expressions of rOAT1 and Cav-1 (mRNA and protein) were evaluated using RT-PCR and Western blot analysis. The localization of rOAT1 and Cav-1 was determined in the caveolae-rich membrane fraction isolated by sucrose density gradient ultra-centrifugation. For the direct binding between the rOAT1 and Cav-1 proteins, the immuno-precipitation method and confocal microscopy were employed. To perform functional analysis, a Xenopus oocytes expression system with the antisense oligonucleotides (ODN) technique was used. RESULTS: The expressions of rOAT1 and Cav-1 were detected in the kidney. The caveolae-rich membranous fractions from the kidney contained both rOAT1 and Cav-1 in the same fractions. The immuno-precipitation experiments showed the formation of a complex between the rOAT1 and Cav-1. The confocal microscopy using primary cultured renal proximal epithelial cells also supported the co-localization of rOAT1 and Cav-1 at the plasma membrane. The uptake function of rOAT1, as assessed by using a Xenopus oocytes expression system, was inhibited by the Xenopus Cav-1 antisense ODN. CONCLUSION: rOAT1 co-localizes with caveolin-1 in the caveolae, and caveolin-1 plays an important role in regulating the function of rOAT1.

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+ -Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+](ER)) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+](i)). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+](i). In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.

Effects of Reactive Oxygen Species on ATP-induced intracellular Ca2+ Activity in Osteoblasts / 대한마취과학회지

BACKGORUND: The physiological activity of osteoblsts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+ ]i) in osteoblasts. The cellular regulation of ([Ca2+ ]i) in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or as toxins in cells. METHODS: Osteoblasts were isolated from the femurs and tibias of neonatal Sprague-Dawley rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+ -sensitive fluorescent dye, Fura-2 AM ester, and fluorescence images were monitored using a cooled CCD camera. Ca-spike changes upon ATP application were checked for (1) osteoblasts in Ca2+ -free and 2.5 mM CaCl2 normal Tyrode solution, (2) osteoblasts in which the Ca2+ of the endoplastic reticulumin had been depleted with ryanodine, thapsigargin ord caffein, and (3) osteoblasts pretreated with H2O2, in which the expression of iP3 receptor was checked by Western blotting. RESULTS: ATP increased intracellular free Ca2+ regardless of extracellular Ca2+ concentration. When the intracellular Ca2+ store was depleted, the level of increased Ca2+ activity by ATP was suppressed. H2O2 sustained the Ca2+ increase induced by ATP. The expression of iP3 receptor was enhanced by H2O2. CONCLUSiONS:H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.

H2O2 Enhances Ca2+ Release from Osteoblast Internal Stores

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.

Hypoxia Delays the Intracellular Ca2+ Clearance by Na+ - Ca2+ Exchanger in Human Adult Cardiac Myocytes

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.

A Comparison of the Efficacy and Safety of Tropisetron and Tropisetron Plus Dexamethasone as Antiemetics for Elective Thyroidectomy / 대한마취과학회지

BACKGROUND: Thyroidectomy is associated with a relatively high incidence of postoperative nausea and vomiting (PONV) ranging from 63% to 84%. In this study, we evaluated the safety and the antiemetic effects of tropisetron 30 microgram/kg or tropisetron 30 microgram/kg plus dexamethasone 5 mg in patients undergoing thyroidectomy under a standard anesthetic technique without narcotics. METHODS: Sixty-eight patients undergoing thyroidectomy were randomized to receive a placebo (Group C, n = 28), tropisetron 30 microgram/kg (Group T, n = 23) or tropisetron 30 microgram/kg plus dexamethasone 5 mg (Group T + D, n = 17) IV over 2 5 minutes immediately before the induction of anesthesia. The effects of these regimens on the development of PONV, adverse events and need for rescue antiemetics were analyzed for the 0 to 1 hour and 1 to 24 hours postoperative periods. RESULTS: In the 0 to 1 hour postoperative periods, the incidence of PONV in group C, T and T + D was 35.7%, 17.4% and 17.6% respectively, which showed no significant difference among the three groups (P > 0.05). In the same period, the incidence of retching or vomiting in Group C, T and T + D was 14.3%, 0% and 0% respectively, which showed a significantly lower incidence in Group T and T + D than Group C (P 0.05). During the first 24 hours postoperatively, the overall incidences of PONV was 67.9% for group C, 60.9% for group T and 58.8% for group T + D, which showed no siginificant difference among the three groups (P > 0.05). Group T + D patients had more headache compared to other groups, but there was no significant difference in theincidences of overall adverse events. CONCLUSIONS: Neither tropisetron or tropisetron plus dexamethasone was significantly different from the placebo for the prevention of PONV after thyroidectomy during the first 24 hour postoperative period. Only vomiting during the first 1 hour postoperatively was prevented in the tropisetron and combination of tropisetron plus dexamethasone groups compared to the control group.

Acute Effect of Ethanol on Firing Patterns of Purkinje Cells in the Rat Cerebellar Slice Preparation

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 microm) 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.

Anesthetic Management for Nutcracker Syndrome Patient / 대한마취과학회지

Nutcracker syndrome consists in the compression of the left renal vein by an aortomesenteric clamp. This results in left renal venous hypertension leading to the development of collateral veins with intrarenal and perirenal varicosities which can cause hematuria. The main presenting symptom is hematuria with or without left flank pain. It responds successfully to surgical treatment. We report a case of anesthesia for a nutcracker syndrome patient.

Retrial of Anesthetic Management for a Patient with Malignant Hyperthermia during Previous General Anesthesia / 대한마취과학회지

Malignant hyperthermia is a subclinical myopathy, usually triggered by anesthetics and associated with a mortality rate of up to 70%, when left untreated. But with early diagnosis using capnography and with the advent of dantrolene, the mortality rate could be reduced to less than 5%, which implies the significance of early diagnosis and proper treatment. Owing to the reduced mortality rate, anesthesiologists get more chances to encounter patients with a previous history of malignant hyperthermia and knowledge to provide proper anesthetic management become essential. We present a case in which malignant hyperthermia was detected in a 67 year old female patient with gastric cancer and a thyroid mass during the first operation and successfully treated with promptly initiated supportive measures based on capnography finding without dantrolene which was not available at the time and the same patient rescheduled for subsequent gastrectomy in which we chose non-triggering agents in adjunct to epidural anesthesia without provoking malignant hyperthermia.

4-Aminopyridine (4-AP) augments Ca(2+)-dependent action potential and changes oscillatory firing patterns in rat cerebellar Purkinje cells

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.

Effects of t-butyl hydrogen peroxide on single SR calcium release channels

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.

Mechanical and Electrophysiological Effects of Mepivacaine on the Direct Myocardial Depression on the 1solated Ventricular Myocardium / 대한마취과학회지

BACKGROUND: The effects of various concentration (20, 50, 100? M) of mepivacaine were studied in isolated guinea pig and rat right ventricular papillary muscles by measuring the effects on myocardial contractility and electrophysiological parameters. METHODS: Isometric force of isolated guinea pig ventricular papillary muscle was studied in modified normal and 26 mM K+ Tyrode's solution. Rat papillary muscle was used to evaluate the effect on Ca2+ release from the sarcoplasmic reticulum (SR) at low stimulation rates. Normal and slow action potentials (APs) were evaluated by using conventional microelectrode technique. Rapid cooling contractures (RCCs), an index of SR Ca2+ content, which are known to be activated by Ca2+ released from the SR were performed. RESULTS: Mepivacaine caused dose-dependent depression of peak force from 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) at all concentration ranges. In rat, ~20% depression of peak force was shown at rested state contraction. Shortening of AP duration and rate-dependent depression of dV/dt max could be observed at 100 M mepivacaine. In 26 mM K+ Tyrode's solution, 50 and 100 M mepivacaine caused dose-dependent depression of early and late force development. In slow APs, neither shortening of AP duration nor changes of dV/dtmax were not shown at 100 M mepivacaine. ~30% depression of RCC after 2 Hz stimulation rate was shown at 100 M mepivacaine. CONCLUSION: It may be concluded that the direct myocardial depressant effects of mepivacaine may partly be related to inhibition of Ca2+ release from the SR. Shortening of AP duration in normal APs seems to be partly related by blockade of TTX-sensitive ""window"" Na+ current.

The effects of changes in intracellular Ca2+ activity of osteoblast-like cell on fracture healing / 대한정형외과학회잡지

Bone formation by osteoblast may be closely related to the increase in intracellular Ca2+ activity of osteoblast. In order to study the effects of changes in Ca2+ activity of osteoblast-like cell on fracture healing, we changed intracellular Ca2+ activity of osteoblast-like cells by vanadate and verapamil. And the process of fracture healing was observed after injection of the treatment osteoblast-like cells into the fracture site by hematoxylin-eosin (H-E) stain and bromodeoxyuridine (BrdU) stain. The results were as follow: 1) The most effective range of concentration which could facilitate bone formation was 10-6 to 10-5 M. 2) H-E stain showed more abundant osteoblast and osteoid tissues, more active mitotic division of osteoblast, and earlier appearance of chondroblast and chondroid tissue, making the maturation of woven bone faster in the vanadate-treated group than in the control group. The opposite was true in the verapamil-treated group compared with the control group. 3) BrdU labeling index showed more active osteoblastic proliferation in the vanadate-treated than in the control group. The opposite was observed in the verapamil-treated group compared with the control group. From these results, the fracture healing appears to be facilitated and decelerated by vanadate which apparently increase intracellular Ca2+ activity of osteoblast and verapamil which decreases it, repectively. Therefore the change of intracellular Ca2+ activity of osteoblast can be considered to be one of fracture healing mechanisms and expected to be applied for clinical purpose.

Fast and slow gating types of SR ryanodine receptor/channel purified from canine latissimus dorsi muscle

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.

Intracellular acidosis decreases the outward Na(+)-Ca2+ exchange current in guinea pig ventricular myocytes

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.

Myocardial Depressant Effects of Sevoflurane: A comparative Study with Isoflurane on Mechanical and Electrophysiologic Effects In Vitro / 대한마취과학회지

Dose-related depression of left ventricular function or cardiac output has been reported in humans and in vivo animal studies with sevoflurane (SEVO) anesthesia and myocardial depressant effect of SEVO appeared to be comparable to that produced by isoflurane (ISO). This study was designed to determine the mechanical and electrophysiologic mechanism of the direct negative inotropic effects of SEVO. The effects of SEVO were comprared to those produced by equipotent concentration of ISO in the same isolated myocardial preparations. Isometric force of isolated guinea pig ventricular papillary muscle was studied in normal and 26 mM K+ Tyrode's solution. Rat papillary muscle was also used to evaluate the effect on Ca2+ release from the sarcoplasmic reticulum (SR) at low stimulation rates. Muscles were bathed at 36-37 degrees C in normal K Tyrode's solution bubbled with 95% O2/ 5% CO2 (pH 7.4) and were electrically stimulated following rest and at rates up to 3 Hz. Normal and slow action potentials were evaluated by using conventional microelectrodes. Muscles were also subjected to rapid cooling (from 37 degrees C to 2 degrees C) in order to elicit a transient rapid cooling contracture (RCC) known to be activatel by Ca2+ content released from the SR. RCCs were elicited after 2 Hz stimulation, which produced an RCC tension similar to that of the preceding contraction in control. SEVO and ISO were administered by dial setting in each vaporizer at 1.7 (1 MAC) and 3.4% (2 MAC), and 1.15 (1 MAC) and 2.3% (2 MAC), respectively. 20% and 40% depression of contractility was shown at 1.7 and 3.4% concentration of SEVO and the extent of depression was similar to equipotent concentration of ISO from rested state up to 3Hz stimulation rates. 1 and 2 MAC concentrations of SEVO (1.7 and 3.4%) or ISO (1.15% and 2.3%) in normal K+ Tyrode's solution caused dose-related depression of peak force at low stimulation rates (RS, 0.1, and 0.5 Hz). Although the normal action potential (AP) amplitude or Vmax were not changed, APD50 and APD90 were prolonged characteristically at 2 MAC of both anesthetics. Whereas no contractile depression was shown at RS and 0.1 Hz stimulation rates in rat papillary muscles, significant depression was noted from 0.5 to 3 Hz in 3.4% SEVO or 2.3% ISO. In the partially depolarized (26 mM K+ Tyrode's solution) beta-adrenergically stimulated myocardium, 2 MAC concentration of both anesthetics caused selective depression of late peak in the biphasic contraction without changing early peak. In slow AP, 3.4% SEVO or 2.3% ISO did not cause any change in AP amplitude and Vmax whereas APD50 and APD90 were prolonged as in Normal APs. Rapid cooling preceded by 15 min rest showed little contractile force and marked prolongation of the time to peak contracture with almost complete absence of contracture after 2Hz stimulation rates following 3.4% SEVO or 2.3% ISO. Although complete recovery of peak force could be observed, little restoration of RCC was shown after washout for 15 minutes at 2 MAC concentration of both anesthetics characteristically. The effect of SEVO on isolated myocardial contraction was similar to that of ISO. While neither anesthetic depressed the rapid initial Ca+ release from the SR, the depression of RCC and late tension suggest an alteration in some SR pathway. The direct myocardial depressant effects of SEVO and ISO are likely to be related to depressed Ca2+ influx through the cardiac memebrane, while AP prolongation may be due to actions on K+ currents.

Cardioprotective drugs decrease the Na+ background current

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.

Effects of Na+ and Ca2+ concentration in cardioplegic and reperfusion solutions on the intracellular Ca2+ of cardiac muscle cells

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.