Results of a multi-institutional, randomized, non-inferiority, phase III trial of accelerated fractionation versus standard fractionation in radiation therapy for T1-2N0M0 glottic cancer: Japan Clinical Oncology Group Study (JCOG0701).

Background: We assessed the non-inferiority of accelerated fractionation (AF) (2.4 Gy/fraction) compared with standard fractionation (SF) (2 Gy/fraction) regarding progression-free survival (PFS) in patients with T1-2N0M0 glottic cancer (GC). Patients and methods: In this multi-institutional, randomized, phase III trial, patients were enrolled from 32 Japanese institutions. Key inclusion criteria were GC T1-2N0M0, age 20-80, Eastern Cooperative Oncology Group performance status of 0-1, and adequate organ function. Patients were randomly assigned to receive either SF of 66-70 Gy (33-35 fractions), or AF of 60-64.8 Gy (25-27 fractions). The primary end point was the proportion of 3-year PFS. The planned sample size was 360 with a non-inferiority margin of 5%. Results: Between 2007 and 2013, 370 patients were randomized (184/186 to SF/AF). Three-year PFS was 79.9% (95% confidence interval [CI] 73.4-85.4) for SF and 81.7% (95% CI 75.4-87.0) for AF (difference 1.8%, 91% CI-5.1% to 8.8%; one-sided P = 0.047 > 0.045). The cumulative incidences of local failure at 3 years for SF/AF were 15.9%/10.3%. No significant difference was observed in 3-year overall survival (OS) between SF and AF. Grade 3 or 4 acute and late toxicities developed in 22 (12.4%)/21 (11.5%) and 2 (1.1%)/1 (0.5%) in the SF/AF arms. Conclusion: Although the non-inferiority of AF was not confirmed statistically, the similar efficacy and toxicity of AF compared with SF, as well as the practical convenience of its fewer treatment sessions, suggest the potential of AF as a treatment option for early GC. Clinical trials registration: UMIN Clinical Trial Registry, number UMIN000000819.

Radiation Dose-escalated Chemoradiotherapy Using Simultaneous Integrated Boost Intensity-Modulated Radiotherapy for Locally Advanced Unresectable Thoracic Oesophageal Squamous Cell Carcinoma: A Single-institution Phase I Study.

AIMS: About 80% of cases of locally advanced unresectable thoracic oesophageal squamous cell carcinoma recur within the irradiation fields after chemoradiotherapy. Radiation dose escalation using advanced radiotherapy techniques is expected to improve clinical outcomes by reducing local and regional recurrence. The current study aimed to determine the recommended escalated radiation dose for these patients. MATERIALS AND METHODS: Patients with locally advanced unresectable thoracic oesophageal squamous cell carcinoma with good performance status underwent chemoradiotherapy using simultaneous integrated boost intensity-modulated radiotherapy (SIB-IMRT) with elective nodal irradiation. SIB-IMRT was delivered in five fractions per week. The radiation dose to the unresectable gross tumour was escalated from 66 Gy to a planned maximum dose of 72 Gy in 3 Gy increments in a standard 3 + 3 design. The doses to the resectable component, superficial tumours and elective nodal regions were fixed as 60, 51 and 48 Gy, respectively. Cisplatin and 5-fluorouracil were concurrently administered. Dose-limiting toxicity (DLT) was defined as acute grade 3 oesophagitis, grade 2 pneumonitis, grade 2 cardiac toxicity and a failure to complete planned radiotherapy within 60 days. Locoregional control and overall survival were estimated using the Kaplan-Meier method. Nine patients were enrolled. RESULTS: DLTs occurred in one of six and two of three patients at doses of 66 and 69 Gy, respectively. All DLTs were grade 3 oesophagitis. The recommended dose was determined as 66 Gy delivered in 30 fractions based on the predefined criteria. With a median follow-up period of 23 months, the 1-year locoregional control and overall survival rates were 67 (95% confidence interval = 19-90) and 78% (95% confidence interval = 36-94), respectively. CONCLUSION: The recommended radiation dose in chemoradiotherapy using SIB-IMRT with elective nodal irradiation was 66 Gy delivered in 30 fractions.

Wound healing effects of gingival fibroblasts cultured in animal-free medium.

OBJECTIVE: The purpose of this study was to develop a graft material made of gingival fibroblasts cultured in animal-free medium (HFDM1). METHODS: We examined the effects of human serum (HS) on cell growth and wound healing capability, demonstrated by cytokine production, of gingival fibroblasts cultured in HFDM1. Subsequently, the capability of fibroblasts cultured in HFDM1 with 2% HS to promote the healing of skin defects was evaluated using nude mice. RESULTS: The proliferation of human gingival fibroblasts was increased when HS at a concentration of 0.5-2% was added to HFDM1. Wound healing cytokines, including transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, and IL-6 produced by gingival fibroblasts were increased by adding 2% HS to HFDM1. In addition, gingival fibroblasts cultured in HFDM1 with 2% HS improved wound healing of mouse skin defects as well as those cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. CONCLUSION: Gingival fibroblasts cultured in HFDM1 with 2% HS may be useful as a graft material for reconstruction.

Influence of factors related to implant stability detected by wireless resonance frequency analysis device.

Resonance frequency analysis (RFA) was introduced as a method for measuring implant stability more than a decade ago. Implant stability quotient (ISQ) values obtained using a recently introduced wireless RFA device have made it possible to evaluate stability in a non-invasive technique; however, there are few studies of the factors that affect ISQ values determined using this device. The aim of the present study was to evaluate the association between ISQ values determined by wireless RFA and various factors related to dental implant stability using a pig cortical bone model. Dental implants (Replace) Select Tapered implants) with a length of 10 mm were placed into pig cortical bone samples, then, ISQ values were determined using wireless RFA under various conditions (probe orientation, diameter of implant, insertion torque and peri-implant bone loss). The results of this study showed that ISQ values were not affected by the direction of the probe from parallel to perpendicular to the long axis of the pig bone or to the smart peg. In addition, the diameter of the implant did not have a significant effect on the measured ISQ values. Statistically significant correlations were found between insertion torque and ISQ values (Spearman's test, P < 0.05), and lower ISQ values were observed for deeper peri-implant vertical defects (Mann-Whitney U-test, P < 0.05). A wireless RFA device appears to be useful for measuring implant stability within the limits of the present in vitro study.

Treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the effect of radiation therapy.

Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tumour radioresistance; therefore, it is recognised as an excellent target during radiation therapy. However, the inhibition of HIF-1 in unsuitable timing can suppress rather than enhance the effect of radiation therapy because its anti-angiogenic effect increases the radioresistant hypoxic fraction. In this study, we imaged changes of HIF-1 activity after treatment with radiation and/or an HIF-1 inhibitor, YC-1, and optimised their combination. Hypoxic tumour cells were reoxygenated 6 h postirradiation, leading to von Hippel-Lindau (VHL)-dependent proteolysis of HIF-1alpha and a resultant decrease in HIF-1 activity. The activity then increased as HIF-1alpha accumulated in the reoxygenated regions 24 h postirradiation. Meanwhile, YC-1 temporarily but significantly suppressed HIF-1 activity, leading to a decrease in microvessel density and an increase in tumour hypoxia. On treatment with YC-1 and then radiation, the YC-1-mediated increase in tumour hypoxia suppressed the effect of radiation therapy, whereas on treatment in the reverse order, YC-1 suppressed the postirradiation upregulation of HIF-1 activity and consequently delayed tumour growth. These results indicate that treatment regimen determines whether an HIF-1 inhibitor enhances or inhibits the therapeutic effect of radiation, and the suppression of the postirradiation upregulation of HIF-1 activity is important for the best therapeutic benefit.

Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor.

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.

Health-related quality-of-life after external beam radiation therapy for localized prostate cancer: intensity-modulated radiation therapy versus conformal radiation therapy.

We compared health-related quality-of-life (HRQL) after intensity-modulated radiotherapy (IMRT) with statuses obtained after old and new protocols of three-dimensional conformal radiation therapy (3DCRT) for localized prostate cancer. We measured the general and disease specific HRQL using the MOS 36-Item Health Survey (SF-36), and the University of California, Los Angeles Prostate Cancer Index (UCLA PCI), respectively. IMRT resulted in similar profiles of general and disease-specific HRQL to two other methods within the first year after treatment. Moreover, IMRT gave rise to comparable urinary, intestinal and sexual side effects despite the high dose of radiation applied.

An integrated Monte Carlo dosimetric verification system for radiotherapy treatment planning.

An integrated Monte Carlo (MC) dose calculation system, MCRTV (Monte Carlo for radiotherapy treatment plan verification), has been developed for clinical treatment plan verification, especially for routine quality assurance (QA) of intensity-modulated radiotherapy (IMRT) plans. The MCRTV system consists of the EGS4/PRESTA MC codes originally written for particle transport through the accelerator, the multileaf collimator (MLC), and the patient/phantom, which run on a 28-CPU Linux cluster, and the associated software developed for the clinical implementation. MCRTV has an interface with a commercial treatment planning system (TPS) (Eclipse, Varian Medical Systems, Palo Alto, CA, USA) and reads the information needed for MC computation transferred in DICOM-RT format. The key features of MCRTV have been presented in detail in this paper. The phase-space data of our 15 MV photon beam from a Varian Clinac 2300C/D have been developed and several benchmarks have been performed under homogeneous and several inhomogeneous conditions (including water, aluminium, lung and bone media). The MC results agreed with the ionization chamber measurements to within 1% and 2% for homogeneous and inhomogeneous conditions, respectively. The MC calculation for a clinical prostate IMRT treatment plan validated the implementation of the beams and the patient/phantom configuration in MCRTV.

Stereotactic body radiation therapy (SBRT) for early-stage lung cancer.

Stereotactic body radiation therapy (SBRT) is a new treatment modality for early-stage non-small-cell lung cancer, and has been developed in the United States, the European Union, and Japan. We started a feasibility study of this therapy in July 1998, using a stereotactic body frame. The eligibility criteria for primary lung cancer were: 1) solitary tumor less than 4 cm (T1-3N0M); 2) inoperable, or the patient refused operation; 3) no necessity for oxygen support; 4) performance status equal to or less than 2; 5) the peripheral tumor which dose constraints of mediastinal organs are maintained. A total dose of 48 Gy was delivered in four fractions in 2 weeks in most patients. Lung toxicity was minimal. No grade II toxicities for spinal cord, bronchus, pulmonary artery, or esophagus were observed. The 3 years overall survival for 32 patients with stage IA, and 13 patients with stage IB were 83% and 72%, respectively. Only one local recurrence was observed in a follow-up of 6-71 months. We retrospectively analyzed 241 patients from 13 Japanese institutions. The local recurrence rate was 20% when the biological equivalent dose (BED) was less than 100 Gy, and 6.5% when the BED was over 100 Gy. Overall survival at 3 years was 42% when the BED was less than 100 Gy, and 46% when it was over l00 Gy. In tumors, which received a BED of more than 100 Gy, overall survival at 3 years was 91% for operable patients, and 50% for inoperable patients. Long-term results, in terms of local control, regional recurrence, survival, and complications, are not yet evaluated. However, this treatment modality is highly expected to be a standard treatment for inoperable patients, and it may be an alternative to lobectomy for operative patients. A prospective trial, which is now ongoing, will, answer these questions.

Recombinant annexin II modulates impaired fibrinolytic activity in vitro and in rat carotid artery.

Fibrinolytic activity has been reported to be decreased in atherosclerosis. Recently, annexin II was identified as a coreceptor on endothelial cells for plasminogen and tissue plasminogen activator. In this study, we examined whether recombinant annexin II (rAN II) protein can modulate fibrinolytic activity on vascular endothelium in vitro and in vivo. The effect of rAN II on human umbilical vein endothelial cells (HUVECs) was measured. Addition of a fluorescent plasmin substrate revealed that HUVECs treated with rAN II exhibited significantly more plasmin generation than those treated with BSA. Moreover, rAN II treatment of HUVECs restored plasmin generation impaired by plasminogen activator inhibitor-1 or homocysteine pretreatment. In a rat carotid artery thrombus model, the patency of thrombosed carotid arteries was significantly enhanced by rAN II injection, in contrast to BSA injection, without systemic blood coagulation dysregulation. We found that rAN II enhanced plasmin generation on vascular endothelium in vitro and reduced thrombus formation in vivo, and concluded that enhancement of endothelial fibrinolytic activity by annexin II could modulate the hypercoagulable state of atherosclerosis. Further study of rAN II in vitro and in vivo may lead to the establishment of novel therapeutic approaches to thrombogenic vascular disease.

Comparison between tumor pH and cell sensitivity to heat in RIF-1 tumors.

We have performed a direct comparison between tumor pH and cell survival in heated RIF-1 tumors growing intradermally in thighs of C3H mice. The pH of individual tumors was measured by inserting a microelectrode into the tumor center. After pH measurements, the tumor-bearing legs were heated in a water bath (44.6 degrees C, 30 min). We then excised a small piece of tumor tissue (20-30 mg) from the area where the tip of the microelectrode had been placed and cell survival was determined by in vitro cloning. In some animals, 3 or 5 g/kg of glucose were injected i.p. 1.5 h before heating, to decrease the tumor pH. The average pH before heating was 6.83, 6.68, and 6.51, respectively, for tumors in untreated animals, those given 3 g/kg glucose, or those given 5 g/kg glucose. After heating, the average surviving fractions were 9.91 x 10(-2), 4.72 x 10(-2), and 5.43 x 10(-3), respectively. The cell yield did not differ significantly among the three treatments. As the tumor pH decreased, the surviving fraction also decreased for each treatment group. The correlation coefficient between tumor pH and log surviving fraction was highly significant for heat plus 5 g/kg glucose and all the treatment groups. The slope of the regression line obtained by a least squares method was steepest for all the treatment groups, followed by the heat plus 5 g/kg glucose and heat plus 3 g/kg glucose groups. The smallest slope of the regression line was noted for tumors treated by heat alone. The study shows that the tumor sensitivity to heat is enhanced when the tumor pH is lower and that adaptation of cells to low pH conditions may play a role in determining the relationship between pH and cell kill in RIF-1 tumors.

Induction of c-fos gene expression by exposure to a static magnetic field in HeLaS3 cells.

Effects of a static magnetic field on cell growth and c-fos oncogene expression were investigated. HeLaS3 cells exposed to the magnetic field of 0.18-0.2 T for 1-6 days were not affected in the cell growth. Exposure to the magnetic field did not enhance the effects of X-rays or heat treatment which caused the transient cell growth delay. c-fos mRNA in the HeLaS3 cells was undetectable in untreated cells, but the expression was induced in cells by the magnetic field exposure for 2-24 h. The amounts of the mRNA expression changed time dependently with a peak at a 6-h exposure. c-fos was expressed following the heat treatment at 45 degrees C for 10 and 15 min, and the expression was further enhanced by the treatment of cells with heating followed by 4 h of the magnetic field exposure. Exposure of the cells to a static magnetic field may affect some cellular metabolic events leading to the c-fos gene expression.

Relationship between heat-induced vascular damage and thermosensitivity in four mouse tumors.

The relationship between heat-induced vascular damage and thermosensitivity was studied using four mouse transplantable tumors. The tumors used were spontaneous mammary carcinoma, SCC VII carcinoma, EMT6 sarcoma, and B16 melanoma. Under cultured conditions, B16 was more thermosensitive at 43 degrees C and 44 degrees C than SCC VII or EMT6. The in vivo tumor response to heat was evaluated by the growth delay after heating at 44 degrees C for 30 min. Among the four tumors, SCC VII was the most thermosensitive in vivo followed by EMT6, whereas B16 and spontaneous mammary carcinoma were thermoresistant. Vascular damage was studied quantitatively up to 24 h after heating by using microangiography. The order of the four tumors in vascular damage was well correlated with the tumor response in vivo. Histologically, tumor vessels of spontaneous mammary carcinoma were supported by connective tissues, and those of B16 had dense endothelial cells, compared to sparse endothelial cells of SCC VII and EMT6. Our findings suggest that variability in heat sensitivity of tumors is related to variation in the histological structure of tumor vasculature. That is, tumor vasculature with perivascular connective tissues and/or dense endothelial cells is less heat labile than that composed only of sparse endothelial cells.

Histological analysis of the effect of hyperthermia on normal rabbit hepatic vasculature.

The effects of hyperthermia on rabbit hepatic vasculature were studied histologically. To investigate heat-induced vascular damage in the central veins, portal veins, and hepatic arterioles, the left lobes of rabbit liver were heated locally for 30 min in the range of 40-46 degrees C. Hyperthermia was induced by an 8-MHz radiofrequency current heating device using a needle type interstitial applicator. This device allowed application of heat to a central area of 10 x 10 mm no more than 1 degree C below the preset temperature. Within the area of 1 cm2, the percentage of damaged (ruptured or thrombosed) vessels was estimated for each type of hepatic vasculature. Vascular damage following hyperthermia continued up to 24 h after heating for the three types of hepatic vasculature. Central veins were the most thermosensitive followed by portal veins, whereas hepatic arterioles were the most thermoresistant. The temperature causing 50% vascular damage 24 h after heating was 41.5-42.5 degrees C, 42.5-43.5 degrees C, and 44-45 degrees C for central veins, portal veins, and arterioles, respectively. This differential thermal responsiveness of hepatic vasculature may be attributed to the histological structure of the vessels.

DNA replication errors produced by the replicative apparatus of Escherichia coli.

It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high fidelity and a high background level of pre-existing mutations in the single-stranded template DNA used. Using the oriC plasmid DNA replication in vitro system and the rpsL forward mutation assay, we examined the fidelity of DNA replication catalyzed by the replicative apparatus of Escherichia coli. Upon DNA synthesis by the fully reconstituted system, the frequency of rpsL-mutations in the product DNA was increased to 1.9x10(-4), 50-fold higher than the background level of the template DNA. Among the mutations generated in vitro, single-base frameshifts predominated and occurred with a pattern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the same nucleotide. Large deletions and other structural alterations of DNA appeared to be induced also during the action of the replicative apparatus.

Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells.

To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH. The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations. Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III. Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III. Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6). Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.

The effects of KNK437, a novel inhibitor of heat shock protein synthesis, on the acquisition of thermotolerance in a murine transplantable tumor in vivo.

A newly synthesized reagent, KNK437, has been found specifically to inhibit the synthesis of heat shock proteins in vitro. In this study, we investigated the effects of KNK437 on the synthesis of heat shock proteins and the induction of thermotolerance in transplantable tumors in vivo. SCC VII cells were grown in vivo and transplanted into C3H/He mice. The concentrations of KNK437 in the tumors and the sera of the mice were examined by high-performance liquid chromatography. Hsp72 synthesis was examined by Western immunoblot analysis. The response to hyperthermia was evaluated in terms of the delay in tumor growth. KNK437 had low toxicity in vivo. The concentration of KNK437 in the tumors gradually increased and reached a peak 6 h after i.p. injection. Hsp72 were synthesized 8 h after hyperthermia at 44 degrees C for 10 min, and their synthesis was inhibited by administration of KNK437 6 h before hyperthermia. At a concentration of 200 mg/kg, KNK437 alone showed no antitumor effects and did not increase the thermosensitivity of nontolerant tumors. The same dose of KNK437 enhanced the antitumor effects of fractionated heat treatment at 44 degrees C in a synergistic manner. This study strongly suggests the inhibition of thermotolerance via the inhibition of HSP72 in vivo. The inhibition of thermotolerance by KNK437 may help to improve the efficacy of clinical fractionated hyperthermia.

Effect of acetylcholine on aconitine induced delayed afterdepolarisation in the frog atrium and ventricle.

The effect of acetylcholine on the aconitine induced delayed afterdepolarisation and triggered electrical activities under nominally calcium free conditions were studied in frog atrium and ventricle. The changes in intracellular potassium activity were also examined. The aconitine induced delayed afterdepolarisation and triggered electrical activities in atrial muscles were transiently suppressed by acetylcholine. This inhibition was correlated with the time course of the development of hyperpolarisation in the resting membrane potential during acetylcholine application. Propranolol did not abolish the transient inhibition of delayed afterdepolarisation during continuous application of acetylcholine, whereas atropine completely inhibited this effect of acetylcholine. Intracellular potassium activity decreased with time after acetylcholine application, whereas the resting potential became hyperpolarised at an early stage, showing slow recovery thereafter. These results indicate that the transient inhibition of delayed afterdepolarisation by acetylcholine is associated with transient hyperpolarisation of the membrane potential, which is assumed to be caused by potassium accumulation or desensitisation of the muscarinic receptor, or both.

Ionic basis for membrane potential changes induced by hypoosmotic stress in guinea-pig ventricular myocytes.

OBJECTIVE: Causal relation between changes in action potentials and activation of several ionic currents during hypoosmotic challenge was investigated. METHODS: We recorded changes in membrane potentials and currents during hypotonic stress in guinea-pig ventricular myocytes using whole-cell patch-clamp technique. RESULTS: Exposure of ventricular myocytes to hypotonic solution (0.6 T) caused initial prolongation ( approximately 107% of control) of action potential duration at 90% repolarization (APD(90)) in 65% of examined myocytes. Later shortening (approximately 75% of control) of APD(90) and depolarization of resting potential (RP) (approximately 4 mV) developed in all cells. Initial prolongation of APD(90) in hypotonic solution was mainly caused by transient activation of Gd(3+)-sensitive non-selective cation (NSC) current. Late changes after approximately 180 s in hypotonic solution were sustained increase in slow component of delayed rectifier K(+) current (I(Ks)) in all cells, and activation of I(Clswell) in 40% of cells. Prevention of APD(90) shortening by chromanol, a selective blocker of I(Ks), was seen in about 40% of myocytes due to short APD in our experimental conditions. Application of 1 mM anthracene-9-carboxylic acid (9-AC) partially inhibited APD shortening in three of seven cells. Depolarization of RP was unaffected by the above-mentioned drugs, but was dependent on [K(+)](o). CONCLUSIONS: Initial prolongation followed by later shortening of APD in hypotonic solution are mostly caused by different sequences of NSC, I(Ks) and I(Clswell) currents activation. Depolarization of RP in hypotonic solution is probably due to dilution of subsarcolemmal K(+) concentration and/or change in permeability ratio for Na(+) and K(+).

External pH modifies sodium channel block by mexiletine in guinea pig ventricular myocytes.

OBJECTIVE: The aim was to examine the kinetics and the degree of Na+ channel block by mexiletine under different values of external pH to produce a fractional variation of charged and uncharged forms of mexiletine. METHODS: The Na+ current (INa) was recorded using the patch clamp technique of whole cell configuration in guinea pig ventricular myocytes. RESULTS: In the presence of 20 microM mexiletine, INa block developed with a single exponential when depolarising pulse trains of 5 ms were applied to -30 mV from a holding potential of -100 mV at a diastolic interval of 500 ms. At the 30th pulse, the degree of use dependent block of INa was 12.3(SEM 0.41)% (n = 4) of the 1st pulse. When the duration of pulses was prolonged to 200 ms, the degree of use dependent block was increased to 25.6(4.7)%, which had the block development of two exponentials with fast and slow rate constants. The slow component at the 200 ms pulses was comparable to the single exponential component at the 5 ms pulses. Drug binding to the inactivated channel was increased by external alkalinisation, which increased the uncharged form of mexiletine, whereas the block at the 5 ms pulses was not affected by the change of external pH. CONCLUSIONS: Uncharged mexiletine caused use dependent block with a fast rate constant having affinity for the inactivated state of the Na+ channel, while the charged form produced a slow component of the block having affinity for the activated state. Therefore the fraction of the uncharged to the charged form of mexiletine could be one of the important determinants in the development and mechanism of INa block.