Molecular determinants of state-dependent block of voltage-gated sodium channels by pilsicainide.

BACKGROUND AND PURPOSE: Pilsicainide, an anti-arrhythmic drug used in Japan, is described as a pure sodium channel blocker. We examined the mechanisms by which it is able to block open channels, because these properties may be especially useful to reduce hyperexcitability in pathologies characterized by abnormal sodium channel opening. EXPERIMENTAL APPROACH: The effects of pilsicainide on various heterologously expressed human sodium channel subtypes and mutants were investigated using the patch clamp technique. KEY RESULTS: Pilsicainide exhibited tonic and use-dependent effects comparable to those of mexiletine and flecainide on hNav1.4 channels. These use-dependent effects were abolished in the mutations F1586C and Y1593C within segment 6 of domain IV, suggesting that the interaction of pilsicainide with these residues is critical for its local anaesthetic action. Its affinity constants for closed channels (K(R)) and channels inactivated from the closed state (K(I)) were high, suggesting that its use-dependent block (UDB) requires the channel to be open for it to reach a high-affinity blocking site. Accordingly, basic pH, which slightly increased the proportion of neutral drug, dramatically decreased K(R) and K(I) values. Effects of pilsicainide were similar on skeletal muscle hNav1.4, brain hNav1.1 and heart hNav1.5 channels. The myotonic R1448C and G1306E hNav1.4 mutants were more and less sensitive to pilsicainide, respectively, due to mutation-induced gating modifications. CONCLUSIONS AND IMPLICATIONS: Although therapeutic concentrations of pilsicainide may have little effect on resting and closed-state inactivated channels, it induces a strong UDB due to channel opening, rendering the drug ideally suited for inhibition of high-frequency action potential firing.

Statins and fenofibrate affect skeletal muscle chloride conductance in rats by differently impairing ClC-1 channel regulation and expression.

BACKGROUND AND PURPOSE: Statins and fibrates can produce mild to life-threatening skeletal muscle damage. Resting chloride channel conductance (gCl), carried by the ClC-1 channel, is reduced in muscles of rats chronically treated with fluvastatin, atorvastatin or fenofibrate, along with increased resting cytosolic calcium in statin-treated rats. A high gCl, controlled by the Ca(2+)-dependent protein kinase C (PKC), maintains sarcolemma electrical stability and its reduction alters muscle function. Here, we investigated how statins and fenofibrate impaired gCl. EXPERIMENTAL APPROACH: In rats treated with fluvastatin, atorvastatin or fenofibrate, we examined the involvement of PKC in gCl reduction by the two intracellular microelectrodes technique and ClC-1 mRNA level by quantitative real time-polymerase chain reaction. Direct drug effects were tested by patch clamp analysis on human ClC-1 channels expressed in human embryonic kidney (HEK) 293 cells. KEY RESULTS: Chelerythrine, a PKC inhibitor, applied in vitro on muscle dissected from atorvastatin-treated rats fully restored gCl, suggesting the involvement of this enzyme in statin action. Chelerythrine partially restored gCl in muscles from fluvastatin-treated rats but not in those from fenofibrate-treated rats, implying additional mechanisms for gCl impairment. Accordingly, a decrease of ClC-1 channel mRNA was found in both fluvastatin- and fenofibrate-treated rat muscles. Fenofibric acid, the in vivo metabolite of fenofibrate, but not fluvastatin, rapidly reduced chloride currents in HEK 293 cells. CONCLUSIONS AND IMPLICATIONS: Our data suggest multiple mechanisms underlie the effect of statins and fenofibrate on ClC-1 channel conductance. While statins promote Ca(2+)-mediated PKC activation, fenofibrate directly inhibits ClC-1 channels and both fluvastatin and fenofibrate impair expression of mRNA for ClC-1.

Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC-1 channel and by increasing intracellular calcium.

BACKGROUND AND PURPOSE: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage-gated chloride channel CLC-1, in controlling muscle excitability, the availability of ligands modulating CLC-1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non-steroidal anti-inflammatory drugs (NSAID). EXPERIMENTAL APPROACH: Rat muscle Cl(-) conductance (gCl) and heterologously expressed CLC-1 currents were evaluated by means of current-clamp (using two-microelectrodes) and patch-clamp techniques, respectively. Fura-2 fluorescence was used to determine intracellular calcium concentration, [Ca(2+)](i), in native muscle fibres. KEY RESULTS: NFA inhibited native gCl with an IC(50) of 42 muM and blocked CLC-1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca(2+)](i) in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC-1. A structure-activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca-dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation. CONCLUSIONS AND IMPLICATIONS: These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC-1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium-dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side-effects.

Effects of chronic treatment with statins and fenofibrate on rat skeletal muscle: a biochemical, histological and electrophysiological study.

BACKGROUND AND PURPOSE: Skeletal muscle injury by hypolipidemic drugs is not fully understood. An extensive analysis of the effect of chronic treatment with fluvastatin (5 mgkg(-1) and 20 mgkg(-1)), atorvastatin (10 mgkg(-1)) and fenofibrate (60 mgkg(-1)) on rat skeletal muscle was undertaken. EXPERIMENTAL APPROACH: Myoglobinemia as sign of muscle damage was measured by enzymatic assay. Histological and immunohistochemical techniques were used to estimate muscle integrity and the presence of aquaporin-4, a protein controlling water homeostasis. Electrophysiological evaluation of muscle Cl(-) conductance (gCl) and mechanical threshold (MT) for contraction, index of intracellular calcium homeostasis, was performed by the two-intracellular microelectrodes technique. KEY RESULTS: Fluvastatin (20 mgkg(-1)) increased myoglobinemia. The lower dose of fluvastatin did not modify myoglobinemia, but reduced urinary electrolytes, suggesting direct effects on renal function. Atorvastatin also increased myoglobinemia, with slight effects on urinary parameters. No treatment caused any histological damage to muscle or modification in the number of fibres expressing aquaporin-4. Either fluvastatin (at both doses) or atorvastatin reduced sarcolemma gCl and changed MT. Both statins produced slight effects on total cholesterol, suggesting that the observed modifications occur independently of HMGCoA-reductase inhibition. Fenofibrate increased myoglobinemia and decreased muscle gCl, whereas it did not change the MT, suggesting a different mechanism of action from the statins. CONCLUSIONS AND IMPLICATIONS: This study identifies muscle gCl and MT as early targets of drugs action that may contribute to milder symptoms of myotoxicity, such as muscle cramps, while the increase of myoglobinemia is a later phenomenon.

Evaluation of the pharmacological activity of the major mexiletine metabolites on skeletal muscle sodium currents.

BACKGROUND AND PURPOSE: Mexiletine (Mex), an orally effective antiarrhythmic agent used to treat ventricular arrhythmias, has also been found to be effective for myotonia and neuropathic pain. It is extensively metabolized in humans but little information exists about the pharmacodynamic properties of its metabolites. EXPERIMENTAL APPROACH: To determine their contribution to the clinical activity of Mex, p-hydroxy-mexiletine (PHM), hydroxy-methyl-mexiletine (HMM), N-hydroxy-mexiletine (NHM) (phase I reaction products) and N-carbonyloxy beta-D-glucuronide (NMG) (phase II reaction product) were tested on sodium currents (I(Na)) of frog skeletal muscle fibres. Sodium currents were elicited with depolarizing pulses from different holding potentials (HP=-140, -100, -70 mV) and stimulation frequencies (0.25, 0.5, 1, 2, 5, 10 Hz) using the vaseline-gap voltage-clamp method. KEY RESULTS: All the hydroxylated derivatives blocked the sodium channel in a voltage- and use-dependent manner. The PHM, HMM and NHM metabolites were up to 10-fold less effective than the parent compound. However, HMM showed a greater use-dependent behaviour (10 Hz), compared to Mex and the other metabolites. Similar to Mex, these products behaved as inactivating channel blockers. Conjugation with glucuronic acid (NMG) resulted in almost complete abolition of the pharmacological activity of the parent compound. CONCLUSIONS AND IMPLICATIONS: Thus, although less potent, the phase I metabolites tested demonstrated similar pharmacological behaviour to Mex and might contribute to its clinical profile.

Functional alterations of mesenteric vascular bed, vas deferens and intestinal tracts in a rat hindlimb unloading model of microgravity.

1. Prolonged bed rest or exposure to microgravity may cause several alterations in autonomic nervous system response (ANSR). 2. Hindlimb unloading (HU) rats were used as an animal model of simulated microgravity to investigate ANSR changes. The experiments were carried out to investigate the effects of simulated microgravity on the autonomic nervous response of the perfused mesenteric vascular bed (MVB), vas deferens and the colon and duodenum from 2-week HU rats. 3. In MVB preparations of HU rats, the frequency-dependent increases in perfusion pressure with perivascular nerve stimulation (PNS; 8-40 Hz) were inhibited, whereas the noradrenaline (NA) concentration-dependent (1-100 microM) perfusion pressure increases were potentiated. The latter most probably reflected up-regulation of alpha-adrenergic receptor function. Relaxant responses of NA-precontracted MVB to PNS (4-30 Hz) or isoprenaline were not different between control and HU preparations, while vasodilation induced by the endothelial agonist ACh was reduced. 4. Transmural stimulation (2-40 Hz) induced frequency-dependent twitches of the vas deferens which were reduced in vas deferens of HU rats, while the sensitivity to NA-induced contraction was significantly increased. 5. In the gastroenteric system of HU rat, direct contractile responses to carbachol or tachykinin as well as relaxant or contractile responses to nervous stimulation appeared unchanged both in the proximal colon rings and in duodenal longitudinal strips. 6. In conclusion, HU treatment affects peripheral tissues in which the main contractile mediators are the adrenergic ones such as resistance vessels and vas deferens, probably by reducing the release of neuromediator. This study validates NA signalling impairment as a widespread process in microgravity, which may most dramatically result in the clinical phenotype of orthostatic intolerance.

Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative.

BACKGROUND: Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine. OBJECTIVE: To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating. METHODS: Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). RESULTS: At a holding potential (hp) of -120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak I(Na) with a potency following the order rank R1448C > WT approximately equal A1156T > G1306E. Yet, when assayed from an hp of -180 mV, TB and UDB by mexiletine were similar for the four channels. The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at -120 mV. Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential. The mexiletine-derivative, Me7 (alpha-[(2-methylphenoxy)methyl]-benzenemethanamine), behaved similarly but was approximately 5 times more potent than mexiletine. Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic I(Na). CONCLUSIONS: These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients.

Increased rigidity of the chiral centre of tocainide favours stereoselectivity and use-dependent block of skeletal muscle Na(+) channels enhancing the antimyotonic activity in vivo.

1. Searching for the structural requirements improving the potency and the stereoselectivity of Na(+) channel blockers as antimyotonic agents, new derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid alpha-proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. 2. Their ability to block Na(+) currents, elicited from -100 to -20 mV at 0.3 Hz (tonic block) and 2-10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. 3. The alpha-proline derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. 4. Contrarily to tocainide, all proline derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic derivatives markedly decreased the drug potency with respect to tocainide. 5. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. 6. Thus, constraining the chiral centre of tocainide in alpha-proline cycle leads to more potent and stereoselective use-dependent Na(+) channel blockers with improved therapeutic potential.

Carboxylic acids and skeletal muscle chloride channel conductance: effects on the biological activity induced by the introduction of an aryloxyalkyl group alpha to the carboxylic function of 4-chloro-phenoxyacetic acid.

2-(4-Chloro-phenoxy)propanoic and 2-(4-chloro-phenoxy)butanoic acids are compounds known to block chloride membrane conductance in rat striated muscle by interaction with a specific receptor. In the present study, a series of chiral analogues has been prepared and tested to evaluate the influence of a second aryloxy moiety introduced in the side-chain at a variable distance from the stereogenic centre. The results show that this chemical modification is detrimental for biological activity which, however, is increased by lengthening the alkyl chain up to three methylenic groups, then decreases to remain constant in the next analogues of the series. A possible explanation for this is proposed on the basis of steric effects and/or different approach of the molecules to the receptor.

Voltage-dependent antagonist/agonist actions of taurine on Ca(2+)-activated potassium channels of rat skeletal muscle fibers.

Emerging evidence supports the idea that taurine exerts some of its actions through inhibition of inward rectifier K(+) channels, ATP-sensitive K(+) channels, and voltage-dependent K(+) channels. However, to date not much is known about the effects of this sulfonic amino acid on Ca(2+)-activated K(+) (K(Ca(2+))) channels, which are widely expressed in various tissues, including skeletal muscle. In the present work, the effects of taurine on K(Ca(2+)) channels of rat skeletal muscle fibers were investigated using the patch-clamp technique. The application of the amino acid to the internal side of the excised macropatches induced a dose-dependent decrease in the outward K(Ca(2+)) currents recorded at positive membrane potentials in the presence of 8 to 16 microM concentrations of free Ca(2+) ions in the bath with an IC(50) of 31.9. 10(-3) +/- 1 M (slope factor = 1.2) (n = 11 patches). In contrast, at negative membrane potentials taurine caused an enhancement of the muscular inward K(Ca(2+)) currents with a DE(50) (drug concentration needed to enhance the current by 50%) of 46.7. 10(-3) +/- 2 M (slope factor = 1.3) (n = 9 patches). Single channel analysis revealed that this effect was mediated by changes in the reversal potential of the K(Ca(2+)) channel for K(+) ions with no changes in the gating properties or in the sensitivity of the channel to Ca(2+) ions. Taurine also did not affect the single channel conductance. In conclusion, taurine shows a voltage-dependent dualistic action on K(Ca(2+)) channels, being an inhibitor of the channel at positive membrane potentials and an activator at negative membrane potentials.

Carboxylic acids and skeletal muscle chloride channel conductance: effects on the biological activity induced by the introduction of methyl groups on the aromatic ring of chiral alpha-(4-chloro-phenoxy)alkanoic acids.

One or two methyl groups have been introduced on the aromatic ring of two chiral clofibric acid analogs, 2-(4-chloro-phenoxy)propanoic and 2-(4-chloro-phenoxy)butanoic acids. The biological activity of the derivatives obtained (3-6) has been evaluated on the skeletal muscle chloride conductance (gCl). The results confirm the hypothesis of two different sites modulating chloride channel function, an excitatory site that increases channel activity and an inhibitory site that produces a channel block. In fact, this chemical modification strongly reduces the blocking activity of the (R)- and (S)-enantiomers in comparison with the parent compounds, but does not markedly affect the ability of the (R)-enantiomers to increase chloride channel conductance.

Synthesis of new 2,6-prolylxylidide analogues of tocainide as stereoselective blockers of voltage-gated Na(+) channels with increased potency and improved use-dependent activity.

A series of tocainide chiral analogues were designed, synthesized, and evaluated in vitro, in pure enantiomeric form, as use-dependent blockers of skeletal muscle sodium channels to better understand the structural requirements responsible for the antimyotonic activity. The voltage clamp recordings showed a remarkable increase of both potency and use-dependent behavior with the analogue N-(2, 6-dimethylphenyl)-2-pyrrolidinecarboxamide (1a). In fact (R)-1a was 5-fold more potent than (R)-tocainide in producing the tonic block, i.e., the reduction of peak sodium current in resting conditions after application of the compound, but it was 21-fold more potent in condition of high frequency of stimulation (phasic block). Furthermore, as opposite to tocainide, this compound was also stereoselective, (S)-1a being 2-3-fold less potent than (R)-1a. The introduction in 1a of a methyl group in place of the hydrogen bonded to either the aminic nitrogen atom [N-(2, 6-dimethylphenyl)-1-methyl-2-pyrrolidinecarboxamide (2a)] or the amidic nitrogen atom [N-(2, 6-dimethylphenyl)-N-methyl-2-pyrrolidinecarboxamide (3a)] led unexpectedly to an inversion of stereoselectivity, the (S)-enantiomers being 3-fold more potent than the (R)-ones. The comparison between eutomers showed that (S)-2a and (S)-3a are almost equieffective to (R)-1a in producing a tonic block, the half-maximal concentrations being about 100 microM; however, the use-dependent behavior was remarkably decreased by the presence of the methyl group: i.e., the gain of potency observed at high frequency of stimulation amounted to 3 and 1.6 times for 2a and 3a, respectively. The replacement of both hydrogens bonded to the aminic and amidic nitrogen atoms resulted in N-(2,6-dimethylphenyl)-N, 1-dimethyl-2-pyrrolidinecarboxamide (4a) in which the (S)-isomer was still twice as potent as the (R)-one, but the absolute potency and mostly the use-dependent behavior were strongly reduced, showing therefore no clear advantages with respect to tocainide. The use-dependent behavior, which plays a pivotal role for antimyotonic activity, is strongly reduced by the presence of methyl groups on the nitrogen atoms, likely for modification of pK(a) and/or for constraint of molecular conformation.

Homologation of mexiletine alkyl chain and stereoselective blockade of skeletal muscle sodium channels.

The optical isomers (-)-(S)- and (+)-(R)-3-(2, 6-dimethylphenoxy)-2-methyl-1-propanamine (Me2), homologues of the antiarrhythmic and antimyotonic drug mexiletine (Mex), were synthesized and assayed as new potential antimyotonic agents. As observed with Mex, Me2 exhibits an enantioselective behaviour. Tests carried out on sodium currents of single muscle fibres of Rana esculenta demonstrated that (-)-(S)- and (+)-(R)-Me2 were less potent than Mex in producing tonic block, but showed a higher use-dependent block. (-)-(S)-Me2 and (-)-(R)-Mex were also used to study the excitability of muscle fibres of myotonic ADR mice, a phenotype of a recessive form of low G(Cl) myotonia. (-)-(S)-Me2 reduced spontaneous discharges and after discharges better than (-)-(R)-Mex in agreement with the use-dependent block of sodium currents.

Increased hindrance on the chiral carbon atom of mexiletine enhances the block of rat skeletal muscle Na+ channels in a model of myotonia induced by ATX.

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more efficient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. Effects of Me5 on Na+ channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 microM) reduced the maximal sodium current (INa) by 29.7+/-4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+/-4.4 % (n=6) at 1 Hz. At same concentration (10 microM), Mex was incapable of producing any effect (n=3). Me5 also shifted the steady-state inactivation curves by -7. 9+/-0.9 mV (n=6) at 0.3 Hz and -12.2+/-1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 microM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The effects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na+ channel activity shows that Me5 did not affect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.

Aging-associated down-regulation of ClC-1 expression in skeletal muscle: phenotypic-independent relation to the decrease of chloride conductance.

In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (gCl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24-29 months old) and adult (3-4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic gCl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both gCl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and gCl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal gCl and demonstrate that the decrease of gCl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.

Insulin modulation of ATP-sensitive K+ channel of rat skeletal muscle is impaired in the hypokalaemic state.

In the present work, we examined the effects of in vivo administration of insulin to rats made hypokalaemic by feeding a K+-free diet. The i.p. injection of insulin in the hypokalaemic rats provoked muscle paralysis within 3-5 h. Consistent with this observation, the skeletal muscle fibres of the paralysed rats were depolarized. In contrast, in the normokalaemic animals, insulin neither provoked paralysis nor produced significant fibre hyperpolarization. In the hypokalaemic rats, insulin almost completely abolished the sarcolemma adenosine triphosphate (ATP)-sensitive K+ currents without altering the sensitivity of the channels to ATP or glibenclamide. In contrast, in the normokalaemic rats, insulin enhanced ATP-sensitive K+ currents that became also resistant to ATP and glibenclamide. Our experiments indicate that the modulation of the sarcolemma ATP-sensitive K+ channels by insulin is impaired in the hypokalaemic state. This phenomenon appears to be related to the fibre depolarization and paralysis observed in the same animals.

Effects of HMG-CoA reductase inhibitors on excitation-contraction coupling of rat skeletal muscle.

3-Hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors currently used as cholesterol-lowering drugs produce side effects in patients, one of which is myopathy. In the present study we compared the effect of a 3-month chronic treatment with two different compounds, simvastatin and pravastatin, on the excitation-contraction coupling of rat skeletal muscle fibers, the mechanism which links membrane depolarization to the movements of cytosolic Ca2+ from intracellular stores. The voltage threshold for mechanical activation of extensor digitorum longus muscle fibers in response to depolarizing pulses of various durations was studied in vitro by the two intracellular microelectrode method in 'point' voltage clamp mode. Simvastatin (5-50 mg/kg) modified the mechanical threshold of striated fibers in a dose-dependent manner. The muscle fibers of rats treated with 10 mg/kg and 50 mg/kg of simvastatin needed significantly less depolarization to contract than did untreated fibers at each pulse duration, suggesting that levels of cytosolic Ca2+ were higher. Consequently, the rheobase voltage for fiber contraction was significantly shifted toward more negative potentials with respect to controls by 2.4 mV and 7.1 mV in the 10 mg/kg and 50 mg/kg simvastatin-treated animals, respectively. Pravastatin treatment at 100 mg/kg did not produce any alteration of excitation-contraction coupling since the rheobase voltage was similar to that of controls. The different physicochemical properties of the two drugs may underlie the different effect observed because lipophilic agents, such as simvastatin, have been shown to affect sterol biosynthesis in many tissues, whereas the hydrophilic pravastatin is hepato-selective.

Phosphorylation and IGF-1-mediated dephosphorylation pathways control the activity and the pharmacological properties of skeletal muscle chloride channels.

1. In the present study we tested the hypothesis that insulin-like growth factor-1 (IGF-1) modulates resting chloride conductance (G(Cl)) of rat skeletal muscle by activating a phosphatase and that the chloride channel, based on the activity of phosphorylating-dephosphorylating pathways, has different sensitivity to specific ligands, such as the enantiomers of 2-(p-chlorophenoxy) propionic acid (CPP). 2. For this purpose G(Cl) in EDL muscle isolated from adult rat was first lowered by treatment with 5 nM 4-beta-phorbol 12,13 dibutyrate (4-beta-PDB), presumably activating protein kinase C (PKC). The effects of IGF-1 and of the enantiomers of CPP on G(Cl) were then tested. 3. IGF-1 (3.3 nM) had no effect of G(Cl) on EDL muscle fibres in normal physiological solution, whereas it completely counteracted the 30% decrease of G(Cl) induced by 4-beta-PDB. No effects of IGF-1 were observed on G(Cl) lowered by the phosphatase inhibitor okadaic acid (0.25 microM). 4. Ceramide, reported to activate on okadaic acid-sensitive phosphatase, mimicked the effects of IGF-1. In fact, N-acetyl-sphingosine (2.5-5 microM), not very effective in control conditions, increased the G(Cl) lowered by the phorbol ester, but not the G(Cl) lowered by okadaic acid. 5. In the presence of 4-beta-PDB, G(Cl) was differently affected by the enantiomers of CPP. The S(-)-CPP was remarkably less potent in producing the concentration-dependent reduction of G(Cl), whereas the R(+)-CPP caused an increase of G(Cl) at all the concentrations tested. 6. In conclusion, the PKC-induced lowering of G(Cl) is counteracted by IGF-1 through an okadaic acid sensitive phosphatase, and this effect can have therapeutic relevance in situations characterized by excessive channel phosphorylation. In turn the phosphorylation state of the channel can modulate the effects and the therapeutic potential of direct channel ligands.