A novel large conductance, nonselective cation channel in pheochromocytoma (PC12) cells

Nasal blockage is one of the most habitual symptoms in otolaryngologist office and the concha nasalis inferior hypertrophy the commonest cause of the trouble. Cases in which medical treatment is refractory, surgery could be an optional resort in accordance of a great deal of specialists. The possibility of give rise to atrophic rhinitis or even ozenatous lesions has been refraining this surgery lately. In our series are contemplated the outcomes of several surgical procedures as mode of management of the hypertrophy of the lower turbinal. In the article are assessed the results of several surgical techniques dealing with hypertrophy of concha nasalis inferior. 75 patients were operated under endoscopic control. Some underwent a submucous decompression (SD) other partial resection (PR). Results at half and long term of both techniques-the conservative one (SD) and that a little more aggressive (PR)-are compared, and so are the factors conditioning the selection of the surgical procedure done. We consider the partial resection of the inferior concha, under endoscopic vision, as the best way for improving the nose obstruction due to inferior concha hypertrophy, provided conservative measures are not wise at all. Furthermore because with our own hands we reach very good outcomes, both in breathing and nasal comfort. Only the patient's age may influence the last decision.     

Muscarinic activation of a voltage-dependent cation nonselective current in rat association cortex

The ionic mechanism underlying the acetylcholine-induced depolarization of layer V pyramidal neurons of rat prefrontal cortex was examined using whole-cell recording in in vitro rat brain slices. Consistent with previous results, pressure application of acetylcholine to layer V pyramidal neurons elicited a strong depolarization. Pharmacological analysis of this response indicated that it was mediated by the stimulation of muscarinic receptors as it was mimicked by muscarinic agonists, but not by nicotine, and was blocked by atropine. The inward current responsible for the depolarization resulted from the activation of a voltage-dependent, cation nonselective current. Thus, the amplitude of the current was critically dependent on extracellular sodium concentration but not on extracellular potassium or chloride concentration. Examination of the I-V relationship for the muscarinic current using voltage clamp revealed that the current reversed near -15 mV and exhibited a strong voltage dependence, turning off rapidly in the subthreshold range. The voltage dependence of the current led to the appearance of a current associated with a conductance decrease when examined using steady-state voltage- or current-clamp measurements. This might have led to earlier misidentification of this response as mediated by a decrease in potassium conductance. These results question the traditional interpretation that muscarinic depolarization in cortex is mediated by a decrease in potassium conductance. They indicate that the fundamental mechanism responsible for muscarinic depolarization in prefrontal cortex involves the activation of a voltage-dependent, cation nonselective current. This current might represent a previously unsuspected mechanism capable of mediating slow depolarization in the central nervous system.     

Extracellular calcium sensed by a novel cation channel in hippocampal neurons

Extracellular concentrations of Ca2+ change rapidly and transiently in the brain during excitatory synaptic activity. To test whether such changes in Ca2+ can play a signaling role we examined the effects of rapidly lowering Ca2+ on the excitability of acutely isolated CA1 and cultured hippocampal neurons. Reducing Ca2+ excited and depolarized neurons by activating a previously undescribed nonselective cation channel. This channel had a single-channel conductance of 36 pS, and its frequency of opening was inversely proportional to the concentration of Ca2+. The inhibition of gating of this channel was sensitive to ionic strength but independent of membrane potential. The ability of this channel to sense Ca2+ provides a novel mechanism whereby neurons can respond to alterations in the extracellular concentration of this key signaling ion.     

Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells

1. In A7r5 cells loaded with the Ca2+ indicator fura-2, we examined the effect of a Ca2+ channel blocker SK&F 96365 on increases in intracellular free Ca2+ concentrations ([Ca2+]i) and Mn2+ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed. 2. Higher concentrations (> or = 10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca2+]i: removal of extracellular Ca2+ abolished only the latter. A blocker of L-type voltage-operated Ca2+ channel (VOC) nifedipine at 1 microM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 microM). 3. Lower [ET-1] (< or = 1 nM) evoked only a sustained elevation in [Ca2+]i which depends on extracellular Ca2+. The elevation was partly sensitive to nifedipine but not SK&F 96365. 4. In the presence of 1 microM nifedipine, higher [ET-1] increased the rate of Mn2+ quenching but lower [ET-1] had little effect. 5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of -60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca2+ in the bath solution with Mn2+. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365. 6. In a bath solution containing only Ca2+ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn2+, whereas that at higher [ET-1] was partly sensitive to SK&F 96365. 7. These results indicate that in addition to VOC, ET-1 activates two types of Ca2+-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn2+.     

Regulation of the activity of 27 pS nonselective cation channels in excised membrane patches from rat brown-fat cells

The regulation of the activity of the approximately 30 pS nonselective cation channel (NSC channel) was studied by the patch-clamp technique in inside-out patches obtained from rat brown-fat cells. NSC channel activity was induced by excision; reduced redox state induced by dithiothreitol accelerated the kinetics in the excised state. The NSC channels were inhibited by the fenamates flufenamic acid and mefenamic acid but not by NS-1619 or SKF-96365. The channels were inhibited by purine nucleotides but not by polyamines. No evidence for protein kinase C, CaM kinase or protein kinase A activation of the NSC channel was obtained. NSC-channel activity was stimulated in a concentration-dependent manner by Ca2+ but the EC50 was very high (0.81 mM), in comparison to expected cytosolic Ca2+ levels. In the presence of ATP, even higher Ca2+ levels were necessary for comparable NSC-channel activation. The increase in Po was not associated with an increase in open-time constants. We conclude that although high Ca2+ levels can experimentally activate the NSC channel, a further mediatory step must probably be postulated in order to link alpha1-adrenergic stimulation to NSC-channel activation.     

Catecholestrogen modulation of steroid production by rat luteal cells: mechanism of action

This study investigated the mechanisms underlying 2-hydroxyestradiol (2-OHE2) effect on luteal steroidogenesis using serum-free cultures of mixed luteal cells from day 8 pseudopregnant rats. Initially, interactions between 2-OHE2 and LH or dibutyryl (db)cAMP on progesterone production were investigated. LH (250 ng/ml) and 2-OHE2 (2.5 microg/ml) had comparable effects on progesterone accumulation, while dbcAMP (5 mM) was more stimulatory. When applied together, 2-OHE2 did not synergize with LH or dbcAMP to further enhance progesterone accumulation. Furthermore, in time course experiments, the dose-dependent effect of 2-OHE2 was to reduce and eventually abolish the time-dependent increase in cAMP accumulation. In contrast LH stimulated cAMP accumulation at all times. Experiments in which cells were co-treated with 2-OHE2, 22-OH-cholesterol and cyanoketone, or with 2-OHE2 and 22-OH-cholesterol or pregnenolone indicated that 2-OHE2 not only had a stimulatory effect on the cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase enzymes, but it also appeared to inhibit the 20alpha-hydroxysteroid dehydrogenase leading to a relative increase in progesterone accumulation. Experiments with hormone antagonists suggested that the actions of 2-OHE2 were not mediated by the estrogen, alpha- or beta-adrenergic receptors. The results of this study support the concept of a physiological role for catecholestrogens in rat luteal steroidogenesis.     

Effects of anions on ATP-activated nonselective cation current in NG108-15 cells

Extracellular ATP induces a nonselective cation current and elevates intracellular Ca2+ concentration via P2Z receptors in NG108-15 cells. We found that the ATP-induced nonselective cation current became larger in methanesulfonic acid (MS-) than in Cl- external solution. We therefore examined the effects of various external anions on the ATP-induced cation current with the use of the whole cell patch-clamp technique. The concentration-response curves for ATP were obtained in different anionic external solutions. The maximum current density (Imax) and the concentration of agonist that gives 50% of maximum response (EC50) value of ATP were obtained by fitting the curves with the use of the Hill coefficient of 2. The apparent Imax decreased in the order of aspartic acid (Asp-) > MS- > F- > Cl- > Br- > or = I-. The apparent EC50 values for ATP were shifted to the right in the sequence of Asp- < F- < MS- < Br- < Cl- < I-. Thus both Imax and EC50 values were affected by anions, indicating that anions are mixed-type inhibitors of the ATP-induced current. The shift of the EC50 values of ATP indicates that anions interfere with ATP binding to the receptor. External Cl- was a noncompetitive inhibitor with respect to external Na+, a major cation carrying the ATP-induced current. We conclude that extracellular anions inhibit the ATP-induced nonselective cation current at least partly by interfering with ATP binding to the P2Z receptor, which is associated with the nonselective cation channels.     

Physiological role of Ca2+-permeable nonselective cation channel in endothelin-1-induced contraction of rabbit aorta

The alpha-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 degrees C and 55 degrees C in the presence and absence of cobalt, with an inverse relationship between alpha-amylase production and growth rate. Highest alpha-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h-1), at 40 degrees C in the absence of cobalt, while at the highest growth rate (0.35 h-1), at 55 degrees C in the presence of cobalt, alpha-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 micrograms maltose (mg biomass)-1 h-1. The pattern and levels of alpha-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.     

Properties of a slow nonselective cation conductance modulated by neurotensin and other neurotransmitters in midbrain dopaminergic neurons

1. A widespread mechanism of slow excitation throughout the nervous system involves overlapping changes in nonselective ion conductance and K+ conductance. We used whole cell patch-clamp recording to characterize such a nonselective conductance induced by neurotensin (NT) and other neurotransmitters in immunocytochemically identified dopaminergic neurons cultured from the rat ventral tegmental area (VTA). 2. The NT-induced inward current consisted of an initial peak and later "hump." The response was blocked reversibly by the nonpeptide NT-receptor antagonist SR48692, suggesting that it resulted from activation of NT receptors. 3. The channel was almost equally permeable to Na+ and K+, as determined from the reversal potential shift upon switching from Na+- to K(+)-containing external solution. The permeability of Cs+ was similar to that of Na+, as determined from the zero-current equation and average reversal potential in the 75 mM Na+ solution. Cl- was not significantly permeable. 4. In Ca(2+)-free external solution, the NT-induced current showed a fourfold increase in amplitude, and in high Mg2+ (20 mM) external solution, the NT-induced current showed an 80% decrease in amplitude, suggesting that external Ca2+ and Mg2+ could block the nonselective conductance. 5. The NT response was unaffected by loading the neurons with either the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or with 1 mM ca2+. The nonselective conductance was therefore not Ca2+ activated. 6. Loading the neurons with cyclic GMP or cyclic AMP (each with the phosphodiesterase inhibitor isobutyl-methylxanthine) did not affect the NT response. The NT-induced nonselective conductance was therefore not cyclic nucleotide-activated. 7. The latency of the NT response was long (> or = 185 ms, average 406 ms, 30 degrees C), indicating that NT did not induce the conductance through ligand-gated channels. Thus, NT activated a slow nonselective cation conductance. 8. Neurokinin B, a metabotropic glutamate agonist, and muscarine elicited responses similar to the NT response. The NT response could be elicited after desensitizing the responses to these other neurotransmitters, indicating receptor specificity in the activation of the nonselective conductance.     

Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons

We have measured the onset and recovery speed of inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion neurons by somatostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using the whole cell configuration of the patch-clamp method with 5 mM external Ca2+. With a local perfusion pipette system that changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV that elicited I(Ca). At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 microM), NE (10 microM), and oxo-M (20 microM) were 2.1, 0.7, and 1.0 s, respectively. The time constants for NE inhibition depended only weakly on the concentration, ranging from 1.2 to 0.4 s in the concentration range from 0.5 to 100 microM. Inhibition by oxo-M (20 microM) through a different G-protein pathway that uses a diffusible cytoplasmic messenger had a time constant near 9 s. The recovery rate constant from membrane-delimited inhibition was between 0.09 and 0.18 s(-1), significantly higher than the intrinsic GTPase rate of purified G protein Go, suggesting that Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel reinhibition after relief by strong depolarizing prepulses, which should reflect the kinetics of final steps in the inhibition process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating that the slowest step of inhibition must precede the binding of G protein to the channel. We propose a kinetic model for the membrane-delimited NE inhibition of Ca2+ channels. It postulates two populations of receptors with different affinities for NE, a single population of G proteins, and a single population of Ca2+ channels. This model closely simulated the time courses of onset and recovery of inhibition and reinhibition, as well as the dose-response curve for inhibition of Ca2+ channels by NE.     

Developmental expression of the rat rod photoreceptor cGMP-gated cation channel

The appearance of cGMP-gated cation channel protein in the postnatal rat retina has been studied by fluorescence immunocytochemistry of radial retinal sections and immunoblots of retinal membrane proteins. Channel immunoreactivity was first detectable with RCNGC1-7H2 monoclonal antibody at postnatal day 7 (PN7) by both methods. Immunocytochemical label in retinal sections was localized to the outer segments, and immunoreactivity increased with increasing age. We also compared the developmental appearance of the cGMP-gated cation channel to that of other phototransduction proteins and developmental markers. RET-P2, a monoclonal antibody recognizing the 39-kDa rds/peripherin disc protein, first labeled outer segments at PN7, coincident with cGMP-gated cation channel expression. Double labeling of the same section of PN7 rat retina with RET-P2 and R309 (a polyclonal antiserum against the rod cGMP-gated cation channel) revealed identical patterns of labelling. Similarly, double labeling with RCNGC1-7H2 and an antibody against the rod cGMP-phosphodiesterase gave coincident labeling, suggesting coordinate expression mechanisms of phototransduction proteins with each other and with outer segment structural proteins.     

Noradrenaline modulates the electrical activity of white adipocytes by a cAMP-dependent mechanism

The two-compartment open model is currently used to assess the glomerular filtration rate (GFR) after a single intravenous injection or a constant rate intravenous infusion. This model needs multiple blood samples from a patient, thus numerous limited sampling models have been so far developed to reduce the number of blood samples. In the present study, the three-, four- and n-compartment closed models have been developed to assess GFR after and during a constant rate intravenous infusion, which include the renal and all possible non-renal elimination pathways. Although more non-renal elimination compartments were included in the modelling, the results show it only leads to the increase in the similar analytical solutions for these compartments and the analytical solution for the blood compartment is the same as that in the two-compartment open model. Theoretically, the developed models can be used to assess GFR with a single blood sample at any sampling time with several urine samples.     

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increased to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIalpha subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions.     

Dopaminergic modulation of sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific sites in the sodium channel alpha subunit

Phosphorylation of brain Na+ channel alpha subunits by cAMP-dependent protein kinase (PKA) decreases peak Na+ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na+ channels. We have studied PKA regulation of Na+ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a concentration-dependent manner. No changes in the voltage dependence or kinetics of activation or inactivation were observed. This effect was mediated by PKA, as it was mimicked by application of the PKA activator Sp-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate(cBIMPS) and was inhibited by the specific PKA inhibitor peptide PKAI5-24. cBIMPS had similar effects on type IIA brain Na+ channel alpha subunits expressed in tsA-201 cells, but no effect was observed on a mutant Na+ channel alpha subunit in which serine residues in five PKA phosphorylation sites in the intracellular loop connecting domains I and II (LI-II) had been replaced by alanine. A single mutation, S573A, similarly eliminated cBIMPS modulation. Thus, activation of D1-like dopamine receptors results in PKA-dependent phosphorylation of specific sites in LI-II of the Na+ channel alpha subunit, causing a reduction in Na+ current. Such modulation is expected to exert a profound influence on overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.     

Anaesthetic modulation of nicotinic ion channel kinetics in bovine chromaffin cells

1. We have investigated the action of the anaesthetics methoxyflurane, methohexitone and etomidate on the nicotinic acetylcholine receptor channel of bovine adrenal chromaffin cells using the whole cell patch clamp technique. 2. Spectral analysis of macroscopic currents evoked by 25 microM carbachol revealed that each of the agents tested reduced the lifetime of the channel open state in a dose-dependent manner. The whole cell current was inhibited in a concentration-dependent fashion by each agent. 3. Channel gating parameters were calculated from single channel studies and the results used to test models explaining the modulation of nicotinic acetylcholine receptor channels by anaesthetics. 4. Each of the agents studied reduced the mean channel open time in a concentration-dependent manner. Anaesthetic concentrations reducing mean open time by 50% were: 370 microM methoxyflurane, 30 microM methohexitone or 23 microM etomidate. 5. Methohexitone and etomidate produced an increase in the number of brief closures within bursts, while no such increase was observed with methoxyflurane. Despite these inter-burst gaps, mean burst length was reduced by each of the agents tested. 6. It is concluded that a simple sequential blocking model fails to account for the action of these anaesthetics. An extended model, in which blocked channels can close, may be applicable.     

Slow closed-state inactivation: a novel mechanism underlying ramp currents in cells expressing the hNE/PN1 sodium channel

To better understand why sensory neurons express voltage-gated Na+ channel isoforms that are different from those expressed in other types of excitable cells, we compared the properties of the hNE sodium channel [a human homolog of PN1, which is selectively expressed in dorsal root ganglion (DRG) neurons] with that of the skeletal muscle Na+ channel (hSkM1) [both expressed in human embryonic kidney (HEK293) cells]. Although the voltage dependence of activation was similar, the inactivation properties were different. The V1/2 for steady-state inactivation was slightly more negative, and the rate of open-state inactivation was approximately 50% slower for hNE. However, the greatest difference was that closed-state inactivation and recovery from inactivation were up to fivefold slower for hNE than for hSkM1 channels. TTX-sensitive (TTX-S) currents in small DRG neurons also have slow closed-state inactivation, suggesting that hNE/PN1 contributes to this TTX-S current. Slow ramp depolarizations (0.25 mV/msec) elicited TTX-S persistent currents in cells expressing hNE channels, and in DRG neurons, but not in cells expressing hSkM1 channels. We propose that slow closed-state inactivation underlies these ramp currents. This conclusion is supported by data showing that divalent cations such as Cd2+ and Zn2+ (50-200 microM) slowed closed-state inactivation and also dramatically increased the ramp currents for DRG TTX-S currents and hNE channels but not for hSkM1 channels. The hNE and DRG TTX-S ramp currents activated near -65 mV and therefore could play an important role in boosting stimulus depolarizations in sensory neurons. These results suggest that differences in the kinetics of closed-state inactivation may confer distinct integrative properties on different Na+ channel isoforms.     

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.     

Voltage- and frequency-dependent modulation of L-type Ca2+ channel by MPC-1304, a novel calcium antagonist in guinea-pig hearts

The electrophysiological effects of MPC-1304, a novel calcium antagonist, were examined using the conventional microelectrode and whole-cell patch-clamp techniques in guinea-pig hearts. MPC-1304, at 100 nM or higher concentrations, produced a dose-dependent reduction in the action potential duration of guinea-pig papillary muscles, without changes in resting membrane potentials and maximum rate of rise of action potentials. In guinea-pig ventricular myocytes, MPC-1304 (1-100 nM) dose-dependently depressed the initial inward currents induced by depolarizing pulses from a holding potential of -30 mV in the external Tyrode solution, as did nifedipine, whereas the late outward current was not changed by MPC-1304. In the presence of 100 nM of MPC-1304 or 100 nM of nifedipine, the first depolarizing pulse from a holding potential of -80 mV caused a depression of the isolated L-type Ca2+ current (I(Ca)) by 29.5 % and 29.4 % of the control, respectively (tonic block), and successive pulses further suppressed I(Ca) in a use-dependent manner (use-dependent block). The degree of steady state use-dependent block of I(Ca) by 100 nM of MPC-1304 was 25.5 % at the stimulus frequency of 1 Hz and further increased to 34.0 % at 2 Hz (frequency-dependent block), which were significantly larger than those by 100 nM of nifedipine at both frequencies. The onset rate of use-dependent block by 100 nM MPC-1304 was significantly smaller than that by 100 nM nifedipine. MPC-1304 (100 nM) and nifedipine (100 nM) shifted the steady state inactivation curve of I(Ca) toward the negative potential by 3.3 mV and 9.1 mV in the mid-potential of the curve, respectively. The estimated dissociation constants of MPC-1304 were 137.7 and 49.9 nM for the resting and inactivated states of the L-type Ca2+ channel, respectively, and those of nifedipine were 113.9 and 18.1 nM, respectively. We conclude that MPC-1304 suppress the L-type Ca2+ channel with slow kinetics in a voltage- and frequency-dependent manner, which might be caused by its high affinity to the activated as well as to the inactivated state of the channel.