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The most common diseases are of polygenic origin. Since early last century, these are being treated with drugs that act downstream of the genetic hurdle. This strategy has saved millions of lives. The advent of genetics revealed the molecular basis of diseases of monogenic origin, including the neuromuscular ones. It was tempting to believe that the defective genes can be repaired with novel chemicals, with a cure being “just down the road”.

Experimental pharmacology is undergoing fundamental changes. This article describes the challenges and opportunities associated with these changes from the perspective of the Swiss Society of Pharmacology (SSEP), the society which aims to advance experimental pharmacology in Switzerland and abroad.

Background and Purpose Chronic elevation in intracellular Ca2+ concentration participates in death of skeletal muscle from mdx mice, a model for Duchenne muscular dystrophy (DMD). Candidate pathways mediating this Ca2+ overload involve store-operated channels (SOCs) and stretch-activated channels (SACs), which are modulated by the Ca2+-independent form of PL A2 (iPLA2). We investigated the effect of doxorubicin (Dox), a chemotherapeutic agent reported to inhibit iPLA2 in other systems, on the activity of this enzyme and on the consequences on Ca2+ handling and muscle function in mdx mice. Experimental Approach Effects of Dox on iPLA2 activity, reactive oxygen species production and on Ca2+ influx were investigated in C2C12 and mdx myotubes. The mechanism of Dox-mediated iPLA2 inhibition was evaluated using purified 6x histidine-tagged enzyme. Aequorin technology was used to assess Ca2+ concentrations underneath the plasma membrane. Isolated muscles were exposed to fatigue protocols and eccentric contractions to evaluate the effects of Dox on muscle function. Key Results Dox at 1–30 μM inhibited iPLA2 activity in cells and in the purified enzyme. Dox also inhibited SAC- but not SOC-mediated Ca2+ influx in myotubes. Stimulated elevations of Ca2+ concentrations below the plasmalemma were also blocked. Exposure of excised muscle to Dox was not deleterious to force production and promoted recovery from eccentric contractions. Conclusions and Implications Dox showed efficacy against targets known to play a role in the pathology of DMD, namely iPLA2 and SAC. The potent SAC inhibitory effect of Dox is a novel finding that can explain partly the cardiomyopathy seen in chronic anthracycline treatment.

Fluoroquinolone antibiotics cause rare, but clinically important, adverse events including hyperglycaemia and hypoglycaemia. The present study focuses on the possible effect of levofloxacin and moxifloxacin on the cardiovascular functions of rats with type I diabetes. Both antibiotics caused bradycardia. Levofloxacin but not moxifloxacin caused hypoglycaemia in diabetic rats and an increase in amplitude of the ST segment revealed by electrocardiogram (ECG) analysis of isolated hearts. In pressurized mesenteric arteries, levofloxacin did not affect the endothelium-derived hyperpolarising factor (EDHF) pathway or its main components, the small-conductance Ca2+ activated potassium (SKCa) and intermediate-conductance Ca2+ activated potassium (IKCa) channels. In moxifloxacin-treated rats, an increase in the EDHF response was observed, which was largely attributed to SKCa-activation. In conclusion, levofloxacin and moxifloxacin use appeared to vary but with no evidence of impairment of the cardiovascular function. However, it is still possible that these antibiotics may produce different effects if there are co-morbidities and therefore their use must be with care.

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (−)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients.

Background and purpose: Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by the absence of the cytoskeletal protein dystrophin. This leads to muscle cell death accompanied by chronic inflammation. Cyclosporin A (CsA) is a powerful immunosuppressive drug, which has been proposed for DMD treatment. CsA also directly regulates the mitochondrial permeability transition pore (mPTP), which participates in cell death pathways through the inhibition of cyclophilin D. Here, we evaluated whether Debio 025, a cyclophilin inhibitor with no immunosuppressive activity, improves the dystrophic condition in a mouse model of DMD, through regulation of mPTP. Experimental approach: The potency of Debio 025 to protect mouse dystrophic cells against mitochondria-mediated death was assessed by caspase-3 activity and calcium retention capacity assays. Mdx5Cv mice (3-week-old) were treated daily by gavage for 2 weeks with Debio 025 (10, 30 or 100 mg kg−1), CsA (10 mg kg−1) or placebo. The effects on muscle necrosis and function were measured. Key results: In vitro investigations showed protective effect of low concentrations of Debio 025 against cell death. Histology demonstrated that Debio 025 partially protected the diaphragm and soleus muscles against necrosis (10 and 100 mg kg−1, respectively). Hindlimb muscles from mice receiving Debio 025 at 10 mg kg−1 relaxed faster, showed alteration in the stimulation frequency-dependent recruitment of muscle fibres and displayed a higher resistance to mechanical stress. Conclusions and implications: Debio 025 partially improved the structure and the function of the dystrophic mouse muscle, suggesting that therapies targeting the mPTP may be helpful to DMD patients. British Journal of Pharmacology (2008) 155, 574–584; doi:10.1038/bjp.2008.285; published online 21 July 2008

The introduction of highly active antiretroviral therapy has led to significant changes in disease progression and mortality of human immunodeficiency virus (HIV) type 1 (HIV-1) infection and its sequela, AIDS. HIV-1 infection has been turned into a chronic condition that can be treated and that may be manageable over many years (90). However, a significant proportion of patients still fail to have a complete response to treatment over a prolonged time and are at risk of virological rebound, which may lead to the emergence of drug-resistant virus variants (20). The 21 anti-HIV-1 drugs used at present target only three steps in the viral replication cycle, namely, virus fusion, reverse transcription, and the proteolytic processing of viral proteins (22). An inhibitor directed against a fourth step, envelope glycoprotein binding to the CCR5 coreceptor (24, 99), and an inhibitor of a fifth step, integration of viral DNA in the cellular genome (34), were recently approved by FDA. Further inhibitors of the last two steps are in clinical evaluation (23, 86). Thus, the four virus-coded proteins, the envelope glycoprotein, reverse transcriptase (RT), protease, and integrase, are the molecular targets of anti-HIV-1 chemotherapy in the clinic at present. This limits the number of possible combinations that may be used, because cross-resistance of virus strains against drugs targeted to the same viral molecule is common (61). Therefore, novel anti-HIV drugs directed against other steps in the viral replication cycle are highly needed. Cyclophilin A (CypA) was established more than a decade ago to be a valid target in anti-HIV-1 chemotherapy (69, 70). The cellular protein CypA fulfills an essential function early in the HIV-1 replication cycle. It was found to bind specifically to the HIV-1 Gag polyprotein (48). A defined amino acid sequence around G89 and P90 of capsid protein p24 (CA) was identified as the binding site for CypA (17, 31). The affinity of CypA for CA promotes the incorporation of CypA into the virion particles during assembly (10, 30, 87). Experimental evidence indicates that the CypA-CA interaction is essential for HIV-1 replication; inhibition of this interaction impairs HIV-1 replication in human cells (10, 37, 81, 82). The step in the viral replication cycle where CypA is involved was demonstrated to be an event after penetration of the virus particle and before integration of the double-stranded viral DNA into the cellular genome (13, 51, 82). CypA is a member of the immunophilin class of proteins. These ubiquitous cellular proteins possess cis-trans prolyl isomerase (PPIase) activities (27) and are assumed to be involved in protein folding and to function as chaperones in intracellular transport (78). Cyclophilins are also known to be the intracellular receptor molecules for cyclosporines (35), a class of cyclic undecapeptides produced by Trichoderma polysporum (25, 71). Binding of cyclosporines to cyclophilins leads to the blockade of the isomerase activity. The most prominent representative of this class of compounds is cyclosporine (Cs), a potent inhibitor of T-cell activation widely used in the clinic as an immunosuppressant in organ transplantation (11). When Cs is bound to CypA it forms a ternary complex with calcineurin. This binding inhibits the phosphatase activity of calcineurin, which is crucial for signal transduction in the activation cascade of T cells. Inhibition of calcineurin function is thus the molecular basis of the immunosuppressive action of Cs (8). The structure of Cs bound to its ligands has been elucidated: two separate domains in the undecapeptide Cs that are involved in binding to CypA and calcineurin, respectively, can be distinguished (40, 43, 66, 94, 98). The immunosuppressive capacity of Cs can therefore be separated from its affinity to CypA by chemical modification. The anti-HIV-1 activity of Cs was first reported in 1988 (91). Evaluation of this drug and many derivatives for inhibition of HIV-1 replication revealed that nonimmunosuppressive Cs analogs had anti-HIV-1 activities equal to or even superior to those of immunosuppressive analogs (7, 10). A clear correlation was evident, however, between their antiviral activities and the ability of cyclosporines to bind to their cellular receptor protein, CypA. Modification of amino acids in the calcineurin binding domain of Cs, in particular, substitution of the undecapeptide at position 4, was shown to abolish the formation of the ternary complex with calcineurin and, thus, led to the loss of its immunosuppressive capacity (62, 96, 105). Binding to CypA and anti-HIV-1 activity were not impaired by such modifications. (Methyl Ile4)cyclosporine (NIM811), a nonimmunosuppressive cyclosporine with a high affinity for binding to CypA and also potent and selective anti-HIV-1 activity, was characterized in more detail (68). Mode-of-action studies revealed that NIM811 inhibited the CypA-CA interaction in a dose-dependent manner (10, 87) and that an early step in the replication cycle of HIV-1 was impaired in the presence of the compound (10, 51, 82). NIM811 was produced by fermentation of the fungus Tolypocladium niveum, followed by extraction and purification (89). Chemical derivatization procedures for the production of a large variety of Cs derivatives are well established. A large amount of knowledge exists about the structure-activity relationships regarding immunosuppressive capacity, CypA binding, and anti-HIV-1 activity (7, 10, 41, 96, 105). We set out to synthesize a series of novel cyclosporines using Cs as a starting material. The goal was to produce by chemical derivatization nonimmunosuppressive cyclosporines with increased affinities for cyclophilin, which was expected to yield derivatives with improved anti-HIV-1 activities. The most promising compound in this series of derivatives turned out to be (d-MeAla3-EtVal4)cyclosporine (Debio-025; where Me and Et are methyl and ethyl, respectively) (97). The in vitro pharmacological profile of this novel CypA-blocking compound and, in particular, its anti-HIV-1 potential are described in this report.

Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.

1. Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA). 2. The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS. 3. We show that CsA (1 -- 10 microM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-dichlorofluorescin diacetate. 4. Using cytochrome c as substrate, we show that CsA (10 microM) did not inhibit NADPH cytochrome P-450 reductase in microsomes prepared from rat liver, kidney or RASMC. 5. CsA (10 microM) did not uncouple the electron flow from NADPH via NADPH cytochrome P-450 reductase to the CYP enzymes because CsA did not inhibit the metabolism of substrates selective for several CYP enzymes that do not metabolize CsA in rat liver microsomes. 6. CsA (10 microM) did not generate more radicals in CYP 3A4 expressing immortalized human liver epithelial cells (T5-3A4 cells) than in control cells that do not express CYP 3A4. 7. Neither diphenylene iodonium nor the CYP 3A inhibitor ketoconazole were able to block ROS formation in rat aortic smooth muscle or T5-3A4 cells. 8. These results demonstrate that CYP enzymes do not contribute to CsA-induced ROS formation and that CsA neither inhibits NADPH cytochrome P-450 reductase nor the electron transfer to the CYP enzymes.

1. The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. 2. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. 3. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. 4. Receptor internalization studies revealed that CsA (10(-6) M) did not significantly alter AVP receptor trafficking. 5. V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time-course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. 6. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter-luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin-calcineurin pathway. 7. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half-life of V1AR mRNA about 2 fold. 8. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

Glucocorticoids, namely alpha-methylprednisolone (PDN) and deflazacort, are the only drugs reported to have a beneficial effect on the degenerative course of Duchenne muscular dystrophy (DMD). Increased cytosolic calcium concentrations ([Ca2+]c) have been implicated as one of the pathological events responsible for the degeneration of dystrophic skeletal muscles. In previous studies, we have demonstrated that PDN treatment of both normal and dystrophic murine skeletal muscle cells was able to normalize elevated [Ca2+]c and improved myogenesis. Here we have investigated the mechanism underlying the effects of glucocorticoids on cellular Ca2+ influx into C2C12 skeletal muscle cells. Long-term incubation of C2C12 myocytes with PDN was necessary to observe a reduction of 45Ca2+ influx. PDN was most effective in inhibiting 45Ca2+ uptake when added for 4 days (at the time of fusion of myoblasts into myotubes) and to a lesser extent, when added after fusion. It was ineffective when added to C2C12 cells at the myoblast stage. Short PDN incubation times, at the time of fusion were insufficient to elicit a response. Several steroids were tested for their ability to inhibit 45Ca2+ influx in C2C12 myocytes. All four glucocorticoids examined were able to reduce Ca2+ influx, dexamethasone being the most potent (IC50 3.14+/-0.34 x 10(-8) M). Mineralocorticoids (aldosterone and 11-deoxycorticosterone) were also able to reduce Ca2+ influx. The vitamin E-derived lazaroid U-83836E and the glucocorticoid-derived lazaroid U-74389G also elicited a decrease in Ca2+ influx, but higher concentrations were necessary. Because both glucocorticoids and lazaroids display antioxidant properties, but U-83836E is devoid of glucocorticoid activity, the reduction in Ca2+ influx was suspected to be triggered via an antioxidant mechanism. To test this hypothesis, we assessed the action of several antioxidants, such as vitamin E, vitamin C, 2-tert.-butyl-4-methoxyphenol (BHA), 2,6-di-tert.-butyl-4-methyl-phenol (BHT) and nordihydroguaiaretic acid (NDGA), on 45Ca2+ influx. None of these agents had an effect on 45Ca2+ influx. In addition, several oxidants were tested (either acutely or chronically) for their ability to elicit 45Ca2+ influx in C2C12 myocytes and were found to be inactive. The involvement of the glucocorticoid receptor on the modulation of Ca2+ influx was investigated. The glucocorticoid receptor antagonist mifepristone (code name RU38486, 10(-6) M) caused a shift of two orders of magnitude of the PDN response. However, neither actinomycin D nor cycloheximide affected the response to PDN. Results with the phospholipase A2 inhibitor, manoalide, suggest that glucocorticoid-induced protein synthesis (e.g. enhanced stimulation of lipocortin) does not play a role in the reduction of calcium influx. Our results suggest that steroids elicit a decrease in calcium influx in C2C12 skeletal muscle cells. This decrease is not due to an antioxidant mechanism or to a mechanism which requires gene expression. Since mineralocorticoids and U-83836E also had similar effects, the mechanism could belong to the non-genomic effects of corticoids (e.g. membrane stabilization). The beneficial effect of glucocorticoids in DMD could be attributed to a reduction of the pathological increase in Ca2+ influx via an effect on the sarcolemma.

The immunosuppressive drug cyclosporin A (CsA) depresses neutrophil oxidative burst which may lead to an increased susceptibility to infection in transplant patients. Using specific CsA analogues we investigated the mechanism of inhibition of the oxidative burst and evaluated short and long-term effects of CsA on dimethylsulphoxide-differentiated HL-60 neutrophils. A biphasic pattern was observed: a 4 h pre-treatment with CsA (1 μM) diminished the fMLP induced [Ca2+]c rise, reactive oxygen species (ROS) production, and β-glucuronidase release by about 40%, whereas a 20 h pre-treatment increased these responses by about 1.5 fold. [MeVal4]CsA, which binds with high affinity to cyclophilin but inhibits the interaction of the CsA-cyclophilin complex with calcineurin, blocked the stimulation observed with CsA after a 20 h incubation but did not alter the CsA effects after a 4 h pre-treatment. PSC 833 (1 μM), a potent multi drug resistance transporter (MDR) inhibitor, diminished ROS production to the same extent as a 4 h CsA incubation but was ineffective after a 20 h pre-treatment. An involvement of MDR as a basis for CsA or PSC 833 action was ruled out based on the results of the calcein retention assay. [3H]CsA uptake showed that CsA and [MeVal4]CsA, but not CsH or PSC 833 were strongly taken up and retained by the cells. In conclusion, the reduction of the responses after 4 h appear to be due to a primary reduction of calcium signalling, while the enhanced responses after 20 h may be due to calcineurin inhibition. British Journal of Pharmacology (1998) 124, 1774–1780; doi:10.1038/sj.bjp.0702020

The use of the immunosuppressive drug cyclosporin A (CsA) is limited by two major side effects, nephrotoxicity and hypertension, which are caused by drug-induced local vasoconstriction. We have recently shown that CsA potentiates the contraction of isolated resistance arteries to vasoconstrictor hormones and increases the calcium response to these agents in vascular smooth muscle cells (VSMC). The goal of the present study was to investigate further the molecular mechanism(s) involved in these effects. Stimulation of VSMC with [Arg]8vasopressin (AVP) induced a concentration-dependent increase in total inositol phosphates (InsP) and cellular calcium response (as measured by 45Ca2+ efflux). Preincubation of VSMC with CsA increased both InsP formation and 45Ca2+ efflux. The potentiating effect of CsA on AVP-elicited InsP formation and 45Ca2+ efflux was inhibited by co-incubation with the protein synthesis inhibitors actinomycin D and cycloheximide, indicating that CsA acted on gene expression. Binding experiments with [3H]-AVP on VSMC showed that CsA increased the number of AVP receptors by about two fold without affecting receptor affinity. Actinomycin D completely blocked this increase. These results demonstrate for the first time that incubation of VSMC with CsA increases the expression of AVP receptors, resulting in a potentiation of InsP formation and calcium response upon stimulation with AVP. This effect of CsA is likely to occur with other vasoconstrictor hormone receptors as well and could be a key mechanism in the induction of vasoconstriction, and subsequent drug-induced nephrotoxicity and hypertension. British Journal of Pharmacology (1997) 121, 248–252; doi:10.1038/sj.bjp.0701102

1. In Duchenne muscular dystrophy (DMD) dysregulation of cytosolic calcium appears to be involved in the degeneration of skeletal muscle fibres. Therefore, we have studied the regulation of the free cytosolic calcium concentration ([Ca2+]c) under specific stress conditions in cultured myotubes isolated from the hind limbs of wild-type (C57BL10) and dystrophin-deficient mutant mdx mice. [Ca2+]c in the myotubes was estimated by the use of the Ca(2+)-sensitive fluorescent dye, fura-2. 2. Resting [Ca2+]c was similar in mdx and normal myotubes (35 +/- 9 nM and 38 +/- 11 nM, respectively). However, when mdx myotubes were exposed to a high extracellular calcium concentration ([Ca2+]c) of 40 mM, the [Ca2+]c was elevated to 84 +/- 29 nM, compared to 49 +/- 7 nM in normal myotubes. 3. Lowering the osmolarity of the superfusion solution from 300 mOsm to 100 mOsm resulted also in a rise in [Ca2+]c which was about two times higher for mdx (243 +/- 65 nM) than for C57BL10 (135 +/- 37 nM). Replacing extracellular Ca2+ by EGTA (0.2 mM) prevented the rise in [Ca2+]c in both mdx and normal myotubes when exposed to the low osmolarity solution. 4. Gadolinium ion (50 microM), an inhibitor of Ca2+ entry, antagonized the rise in [Ca2+]c of myotubes superfused with 40 mM [Ca2+]c by 20-40% for both mdx and C57BL10 cells, but did not significantly reduce the rise in [Ca2+]c when the cells were exposed to the hypo-osmotic buffer (100 mOsm). 5. Incubation of the cell culture for 3-5 days from the onset of induction of myotube formation with the membrane permeable protease inhibitor, calpeptin (50 microM) abolished the rise in [Ca2+]c in mdx myotubes upon exposure to hypo-osmotic shock. 6. Treatment of the cell culture for 3-5 days with alpha-methylprednisolone (PDN, 10 microM) attenuated the rise in [Ca2+]c following hypo-osmotic stress for both normal and mdx myotubes by about 50%. 7. The results described here suggest an increased permeability of mdx myotubes to Ca2+ under specific stress conditions. The ameliorating effect of PDN on [Ca2+]c could explain, at least partly, the beneficial effect of this drug on DMD patients.

1. Increased calcium (Ca2+) influx has been incriminated as a potential pathological mechanism in the chronic skeletal muscle degeneration exhibited by Duchenne muscular dystrophy (DMD) patients. We have studied the influence of the glucocorticoid alpha-methylprednisolone (PDN), the only drug known to have a beneficial effect on the degenerative course of DMD, on Ca2+ handling in the C2 skeletal muscle cell line. 2. PDN, when added 3 days (when myoblasts start to fuse into myotubes) after cell seeding, led to a 2 to 4 fold decrease in cellular Ca2+ uptake. This decrease was independent of the extracellular Ca2+ concentration applied to cells. The effect took at least 24 h in order to become established (PDN of 10(-5) M) and took longer for lower PDN concentrations (EC50 of ca. 10(-6) M at day 5, 10(-6.5) M at day 7 and 10(-7.5) M at day 9 in culture). 3. Cellular calcium accumulation was also decreased in PDN-treated myotubes exposed to 45Ca(2+)-containing medium for 1 to 6 days. 4. No effect of PDN was seen on 45Ca2+ efflux; a decrease in the amount of 45Ca2+ released was observed due to the reduction of cellular 45Ca2+ loading. 5. PDN treatment led to an approximately 2 fold decrease in basal cytosolic Ca2+ concentration. 6. Three antioxidant drugs (lazaroids), previously shown to enhance in vitro skeletal muscle cell differentiation to the same extent as PDN, induced a similar decrease in Ca2+ influx. 7. Our results suggest that long-term incubation of C2 cells with PDN leads to a decrease of the size of the cellular Ca2+ pools and to reduced resting cytosolic Ca2+ levels. Part of the beneficial effect of PDN in DMD patients could be attributed to a reduction of Ca2+ influx and of the size of Ca2+ pools in dystrophic muscle fibres.

The effects of the new inotropic agent, SDZ 218-135 [(+)-(S)-4-[3-(4-diphenyl-methyl-1-piperazinyl)-2-hydroxy-propyl]- 6-(2-hydroxyethyl)-5-methyl-1,2,4-triazolo-[1,5-a]pyrimidin-7(4H)-one], were investigated using in vitro and in vivo techniques. In isolated rat atria, SDZ 218-135 elicited a dose-dependent increase in contractile force (+50% at 10 microM), which was paralleled by an increase in functional refractory period. In anesthetized rats SDZ 218-135 enhanced left ventricular (+)dP/dtmax by 100% at 10 mg/kg without influencing heart rate, arterial blood pressure, and cardiac output. In contrast to its predecessor, DPI 201-106, cardiac relaxation remained essentially unimpaired. The positive inotropic action was also maintained in a rabbit model of depressed heart function after myocardial infarction, where SDZ 218-135 increased peak acceleration of blood in the aorta. The prolongation of the effective refractory period in rat atria suggested possible antiarrhythmic effects. Indeed, SDZ 218-135 showed a dose-dependent marked reduction in reperfusion arrhythmias after coronary artery occlusion in rats. This effect was most likely due to a Class III action, since SDZ 218-135 significantly increased action potential duration (+10% at 10 microM/l) of the isolated guinea pig papillary muscle. In conclusion, SDZ 218-135 is a novel positive inotropic agent with an interesting profile of action. It does not impair cardiac relaxation and shows antiarrhythmic effects in a model of reperfusion-induced arrhythmias. The in vivo and in vitro data are consistent with a mechanism of action via sodium channel agonism.

Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution.

In the context of atherosclerosis, calcium antagonists should be evaluated from two points of view: their hemodynamic action on the atherosclerotic cardiovascular system, and their preventive action on atherosclerosis. Contraction of atherosclerotic vessels in response to vasoconstrictors, especially serotonin and acetylcholine, is enhanced mainly because of endothelial dysfunction. Due to their antivasoconstrictor effect, calcium antagonists partly compensate for this endothelial dysfunction. Agents which show a pronounced 'use-dependence', such as isradipine, are of particular interest as their action increases with the intensity of contraction. Therefore, their action is most prominent where it is most needed. Nevertheless, experiments in atherosclerotic animals indicate that the dose has to be chosen more cautiously than for young animals, as the dose-response curve for several effects is bell-shaped. Calcium antagonists also interfere with the process of atherosclerosis. In cholesterol-fed rabbits, lesion development is reduced, and regression after reverting to a normal diet is modestly enhanced. Calcium antagonists do not lower blood lipid levels. However, they attenuate proliferative lesions following endothelial damage due to a balloon catheter or electrical stimulation. The mechanism of these effects is still not clear. Blockade of the potential-sensitive L-type calcium channel is probably the important mechanism. Many types of cells (macrophages, other leukocytes and platelets, smooth muscle cells, connective tissue cells, endothelium, etc.) are involved in the genesis of atherosclerotic plaques, but it is not known which of these are the important targets of calcium antagonists. Calcium antagonists are most effective when administered at the onset of atherosclerosis, as they probably inhibit events occurring during the initiation of the atherosclerotic process.(ABSTRACT TRUNCATED AT 250 WORDS)

Duchenne muscular dystrophy is a frequent muscular disorder caused by mutations in the gene encoding dystrophin, a cytoskeletal protein that contributes to the stabilization of muscle fiber membrane during muscle activity. Affected individuals show progressive muscle wasting that generally causes death by age 30. In this study, the dystrophic mdx 5Cv mouse model was used to investigate the effects of green tea extract, its major component (−)-epigallocatechin gallate, and pentoxifylline on dystrophic muscle quality and function. Three-week-old mdx 5Cv mice were fed for either 1 or 5 wk a control chow or a chow containing the test substances. Histological examination showed a delay in necrosis of the extensor digitorum longus muscle in treated mice. Mechanical properties of triceps suræ muscles were recorded while the mice were under deep anesthesia. Phasic and tetanic tensions of treated mice were increased, reaching values close to those of normal mice. The phasic-to-tetanic tension ratio was corrected. Finally, muscles from treated mice exhibited 30–50% more residual force in a fatigue assay. These results demonstrate that diet supplementation of dystrophic mdx 5Cv mice with green tea extract or (−)-epigallocatechin gallate protected muscle against the first massive wave of necrosis and stimulated muscle adaptation toward a stronger and more resistant phenotype.

Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring 45Ca2+ uptake into cultured rat aortic smooth muscle cells. Wash solution (0 degrees C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular 45Ca2+ retention more effectively than EGTA (0.2 mM). Basal Ca2+ entry was 1.45 x 10(9) Ca2+ x cell(-1) x min(-1). This translated to approximately 250 micromol(-1) x min(-1) given cell volumes of 4-15 pl as determined by 3-D image reconstruction. Gadolinium (100 microM) blocked 80% of the leak and exhibited a biphasic concentration-response relation (IC50s=1 microM and 2 mM). Organic ion channel blockers also inhibited approximately 80% of the leak; 45% by nifedipine (10 microM), 7% was exclusively blocked by SKF 96365 (1-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole) (50 microM) and 23% was exclusively sensitive to 2-aminoethoxy-diphenylborate (2-APB, 75 microM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca2+ entry is mainly due to a basal open probability of excitable Ca2+ -channels.

A preclinical screening for prompt-to-use drugs that are safer than steroids and beneficial in Duchenne muscular dystrophy was performed. Compounds able to reduce calcium-induced degeneration (taurine or creatine 10% in chow) or to stimulate regeneration [insulin-like growth factor-1 (IGF-1); 50 or 500 μg/kg s.c.] were administered for 4 to 8 weeks to mdx mice undergoing chronic exercise on a treadmill, a protocol to worsen dystrophy progression. α-Methyl-prednisolone (PDN; 1 mg/kg) was used as positive control. The effects were evaluated in vivo on forelimb strength and in vitro electrophysiologically on the macroscopic chloride conductance (gCl), an index of degeneration-regeneration events in mdx muscles, and on the mechanical threshold, a calcium-sensitive index of excitation-contraction coupling. The exercise produced a significant weakness and an impairment of gCl, by further decreasing the already low value of degenerating diaphragm (DIA) and fully hampering the increase of gCl typical of regenerating extensor digitorum longus (EDL) mdx muscle. The already negative voltage threshold for contraction of mdx EDL was also slightly worsened. Taurine > creatine > IGF-1 counteracted the exercise-induced weakness. The amelioration of gCl was drug- and muscle-specific: taurine was effective in EDL, but not in DIA muscle; IGF-1 and PDN were fully restorative in both muscles, whereas creatine was ineffective. An acute effect of IGF-1 on gCl was observed in vitro in untreated, but not in IGF-1-treated exercised mdx muscles. Taurine > PDN > IGF-1, but not creatine, significantly ameliorated the negative threshold voltage values of the EDL fibers. The results predict a potential benefit of taurine and IGF-1 for treating human dystrophy.

Elevation of intracellular Ca2+, excessive ROS production and increased phospholipase A2 activity contribute to the pathology in dystrophin-deficient muscle. Moreover, Ca2+, ROS and phospholipase A2, in particular iPLA2, are thought to potentiate each other in positive feedback loops. NADPH oxidases (NOX) have been considered as a major source of ROS in muscle and have been reported to be overexpressed in muscles of mdx mice. We report here on our investigations regarding the effect of diapocynin, a dimer of the commonly used NOX inhibitor apocynin, on the activity of iPLA2, Ca2+ handling and ROS generation in dystrophic myotubes. We also examined the effects of diapocynin on force production and recovery ability of isolated EDL muscles exposed to eccentric contractions in vitro, a damaging procedure to which dystrophic muscle is extremely sensitive. In dystrophic myotubes, diapocynin inhibited ROS production, abolished iPLA2 activity and reduced Ca2+ influx through stretch-activated and store-operated channels, two major pathways responsible for excessive Ca2+ entry in dystrophic muscle. Diapocynin also prevented force loss induced by eccentric contractions of mdx muscle close to the value of wild-type muscle and reduced membrane damage as seen by Procion orange dye uptake. These findings support the central role played by NOX-ROS in the pathogenic cascade leading to muscular dystrophy and suggest diapocynin as an effective NOX inhibitor that might be helpful for future therapeutic approaches.

We thank V. Ullrich and T. Payne for critically reading this manuscript. This research was supported by grants (No. 31-47294.96 and 31-56877.99) from the Swiss National Science Foundation.

The use of the immunosuppressant cyclosporin A (CsA) is frequently associated with hypertension. Drug-induced local vasoconstriction appears to be responsible for this effect. Using fura-2 and 45Ca2+ efflux techniques, we have examined variations in the cytosolic calcium concentration ([Ca2+]c) in rat aortic smooth muscle cells and have shown that increases in [Ca2+]c after [Arg8]vasopressin, serotonin, endothelin-1 or angiotensin II stimulation were potentiated after preincubation of cells with CsA. This effect was independent of cyclophilin or calcineurin inhibition by CsA. Measurements of inositol phosphates (InsPn) after agonist stimulation showed that CsA also potentiated their formation. As for 45Ca2+ efflux this effect was not related to cyclophilin or calcineurin inhibition. Direct stimulation of G proteins with aluminium tetrafluoride induced an increase in InsPn formation and 45Ca2+ efflux. Neither of these responses was potentiated by CsA. These results indicate that CsA acts on a target upstream of G protein activation, possibly at the receptor level, resulting in a potentiation of InsPn formation and subsequent calcium increase.

1. The full therapeutic potential of the main immunosuppressive drug, cyclosporin A (CsA), is limited because of its side effects, namely nephrotoxicity and hypertension. Several lines of evidence suggest that the origin of both side effects could be CsA-induced vasoconstriction. However, the underlying molecular mechanisms are not well understood. 2. Diameter measurements of rat isolated mesenteric arteries showed an increase in noradrenaline- and [Arg]8vasopressin-induced vasoconstriction when arteries were pretreated with CsA. 3. Measurements in cultured vascular smooth muscle cells (VSMC) of either cytosolic calcium concentration or of 45Ca2+ efflux showed that CsA potentiated the calcium influx to several vasoconstrictor hormones: [Arg]8vasopressin, angiotensin II, endothelin-1 and 5-hydroxytryptamine. On the other hand, 45Ca2+ efflux in response to thapsigargin, which depletes calcium from intracellular pools, was not potentiated by CsA. 45Ca2+ uptake was not altered by CsA or by any of the analogues tested. 4. Time-course studies in cultured VSMC showed that maximal CsA-induced Ca2+ potentiation occurred after ca. 20 h and this effect was reversed over approximately the next 20 h. 5. To investigate the possible role played by the known intracellular targets of CsA, namely cyclophilin and calcineurin, CsA derivatives with variable potencies with respect to their immunosuppressive activity, were tested on the calcium influx to [Arg]8vasopressin. Derivatives devoid of immunosuppressive activity (cyclosporin H, PSC-833) potentiated calcium signalling, while the potent immunosuppressant, FK520, a close derivative of FK506, and MeVal4CsA, an antagonist of the immunosuppressive effect of CsA did not. The latter compound was unable to reverse the calcium potentiating effect of CsA. 6. Our results show that CsA increases the calcium influx to vasoconstrictor hormones in smooth muscle cells, which presumably increases vasoconstriction. Loading of the intracellular calcium pools appears not to be involved. Experiments with derivatives of CsA and FK520 suggest that interactions with cyclophilins and calcineurin are not the mechanism involved. This indicates, for the first time, that the immunosuppressive activity can be dissociated from the calcium potentiating effect of CsA in vascular smooth muscle.

Vascular selective calcium antagonists (CAs) show an improved tolerance and a reduced incidence of adverse cardiac effects, especially in treatment of hypertension. The effects of seven well-known CAs on contractions of single isolated rat myocytes were studied and compared with their effects on stimulated 45Ca2+ uptake of rat aortic smooth muscle cells (A7r5 cell line). In the latter test system, the order of potency to inhibit 45Ca2+ uptake was as follows (pIC25, -logM): isradipine (9.2), felodipine (8.7), nifedipine (8.5), nisoldipine (8.5), nicardipine (8.1), verapamil (6.7), and diltiazem (6.5). The potencies for inhibition of ventricular myocyte contraction at 0.5 Hz were (pIC25): isradipine (6.9), nisoldipine (6.7), felodipine (6.6), nicardipine (6.5), nifedipine (6.5), verapamil (5.3), and diltiazem (4.8). Thus, the order of vascular selectivity (i.e., the ratios of IC25 cardiocytes/IC25 A7r5 cells) was: isradipine (184), felodipine (128), nifedipine (107), nisoldipine (63), diltiazem (48), nicardipine (43), and verapamil (23). When ventricular cells were stimulated at 1 Hz, the order of selectivity was changed: Diltiazem was the least selective. Verapamil, diltiazem, and felodipine showed a highly frequency-dependent negative inotropic effect, whereas the effects of the other dihydropyridines were less affected by the frequency of stimulation. CAs show different degrees of vascular selectivity and different frequency-dependent profiles, and vascular selectivities are also dependent on experimental conditions. Selectivity is thus not necessarily related to chemical classes of drugs (e.g., dihydropyridines) or to different binding sites at the channel protein but could instead be due to varying dissociation rates from the respective binding sites at the channel in its different voltage-dependent states.

Single adult cardiac ventricular cells were prepared by collagenase perfusion of a rat heart. They were stimulated electrically in a perfusion chamber and their length changes were followed under a microscope. The motion was followed via a video camera and by a TV-line counting device and was recorded on-line by a personal computer. The program RECORD was used to calculate peak amplitude, base line drift and peak width at different peak heights allowing the determination of a number of variables of the cellular motion. The method was applied to drugs affecting the amplitude of contractions and the speed of relaxation. Results of beta-adrenergic stimulation, muscarinic inhibition and of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) are shown. Besides its stimulatory effect on length, the beta-adrenergic agonist isoprenaline concentration-dependently shortened relaxation time. Carbachol reversed the increase in cellular shortening caused by isoprenaline in a concentration-dependent manner without fully reversing the shortened relaxation. CPA prolonged the return to diastole, presumably due to its inhibition of Ca(2+)-reuptake into the sarcoplasmatic reticulum.

The pharmacologic properties of isradipine (PN200-110), its enantiomers, and its major metabolites occurring in human blood are reviewed. Isradipine is a dihydropyridine derivative with a high specific and a low nonspecific affinity to the dihydropyridine binding site of the L-type calcium channel. The nonspecific receptor-operated calcium channel of rabbit aorta is not blocked, but receptor-operated channels in resistance vessels are blocked, which implies that they resemble L-channels. In the heart, isradipine acts selectively on the sinus node but not on the atrioventricular node. Also, its cardiodepressant effect is minimal. The cause of this selectivity is not known; differences between the channels, or a different electrophysiologic behavior of these tissues, might account for it. The pharmacodynamic activity resides in the (+)enantiomer that is on the order of 150 times more active than the (−)enantiomer. The major metabolites found in human blood were tested for evidence of calcium channel blockade. None of them had a relevant calcium antagonist-like activity in vitro. Four of the five metabolites were also tested for their effect on the cardiovascular system in anesthetized cats, and all four were found to be inactive. Pharmacodynamic effects of isradipine are therefore attributable entirely to isradipine itself.

The extracellular Ca2+ dependence of agonist stimulation of vascular smooth muscle (VSM) has been investigated in rat cultured aortic smooth muscle cells (SMCs) and isolated mesenteric resistance vessels (MRVs). Agonists such as [Arg8]vasopressin (AVP), angiotensin II (Ang II), and adenosine-5'-triphosphate (ATP) stimulated 45Ca2+ entry into the SMCs that was (a) independent of the extent to which the membranes were polarized, and (b) was not inhibited by organic Ca2+ channel antagonists. Measuring the intracellular Ca2+ concentration [( Ca2+]i) after stimulation with agonists revealed a rapid increase of [Ca2+]i, which was followed by a sustained rise that was insensitive to Ca2+ antagonists. In Ca2+-free medium, only the initial peak of [Ca2+]i was still observed, but the sustained response to the agonists disappeared completely. This observation indicates that the sustained elevation seen in Ca2+-containing medium was the consequence of agonist-induced Ca2+ entry. In MRVs, a corresponding Ca2+-antagonist-insensitive, agonist (norepinephrine and AVP)-induced tonic tension was also identified. Moreover, agonists were able to induce sustained tension in the MRVs regardless of whether the membrane was normally polarized or was previously depolarized (80 mM K+) upon their administration. The agonist-stimulated 45Ca2+ entry in the SMCs could be blocked by the multivalent cations La3+, Cd2+, Mn2+, Co2+, Ni2+, and Mg2+ (in this order of potency). Depolarization-induced 45Ca2+ influx was inhibited by these cations in the same order of potency, but was significantly more sensitive to Cd2+ and significantly less sensitive to La3+ than that stimulated by agonists. Treatment with 2-nitro-4-carboxyphenyl-N,N-diphenyl-carbamate (NCDC, a proposed inhibitor of phospholipase C) reduced both the agonist-induced 45Ca2+ influx and the sustained elevation of [Ca2+]i in the SMCs. NCDC also abolished both contraction and depolarization induced by agonists in the MRVs. The kinase C stimulator phorbol-12-myristate-13-acetate (PMA) inhibited the agonist-induced 45Ca2+ influx and sustained increase in [Ca2+]i in the SMCs, whereas the kinase C inhibitor staurosporine had no effect. In the MRVs, in contrast, PMA had no influence on agonist-induced contractions. Staurosporine (1 microM), however, completely prevented these contractions, as did NCDC, but, unlike NCDC, it did so without affecting the agonist-induced depolarization. These data support an important role of receptor-operated Ca2+-permeable channels in VSM activation by agonists and suggest that these channels may be controlled by intracellular enzymic pathways and second messenger systems.

The stereoisomers of the new dihydropyridine derivative 202-791 [isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3 -pyridinecarboxylate] were synthesized separately and tested on isolated rabbit aortic rings for effects on depolarization-induced contraction and depolarization-stimulated uptake of 45Ca2+. The racemic mixture enhanced contraction of rabbit aortic rings at low levels of depolarization but inhibited contraction and 45Ca2+ uptake at high levels of depolarization. The IC50 values were 2.0 X 10(-7) and 1.7 X 10(-7) M, respectively. The R enantiomer inhibited contraction and 45Ca2+ uptake with IC50 values of 3.2 X 10(-8) and 4.3 X 10(-8) M, respectively. This compound showed no stimulant activity. By contrast, the S enantiomer of 202-791 shifted the concentration-response curve for depolarization-induced contraction in an almost parallel fashion to the left, thus enhancing contraction. The EC50 value for this effect at a KCl concentration of 16 mM was 1.8 X 10(-7) M. This compound enhanced 45Ca2+ uptake concentration dependently at all levels of depolarization tested. Thus, the stereoisomers of a dihydropyridine derivative may behave as a calcium entry blocker or a calcium entry enhancer on vascular smooth muscle, depending only on the stereochemistry. If asymmetric compounds elicit effects suggesting a dualistic action at the calcium channel, then the stereoisomers should be prepared.

The effects of endothelin-1 (ET-1) on tension and membrane potential in rat isolated mesenteric resistance vessels (MRVs) and on 45Ca influx, 45Ca efflux, inositol-1,4,5-triphosphate (IP3) production, and cytoplasmic Ca2+ concentration ([Ca2+]1) in cultured aortic smooth muscle cells were compared with those of other agonists. ET-1 induced contractions of the MRVs, which were slow in onset, but reached a similar maximum amplitude (at 10 nM ET-1) as that seen with norepinephrine (NE, 10 microM) or [arg8]vasopressin (AVP, 0.1 microM). The EC50 for ET-1 was 1.3 +/- 0.1 nM. Removal of extracellular Ca2+ reduced ET-1-induced contractions to 11 +/- 3% of those in Ca2+-containing medium. With NE, the same procedure reduced contractions to 47 +/- 7% of those in Ca2+-containing medium, while with AVP, the reduction was similar in magnitude to that induced by ET-1 (11 +/- 5% of those in Ca2+-containing medium). Relaxation of ET-1-induced and NE-induced contractions by diltiazem was not complete (maximal at 58 +/- 6% with 10 microM diltiazem after 6 nM ET-1, and at 70 +/- 3% after 0.1 microM NE), in contrast to that of 80 mM K+-induced contractions, which were potently (IC50 = 0.2 microM) and completely reversed (100% relaxation at 10 microM diltiazem). ET-1 (6 nM) caused a small but significant depolarization of the MRVs (approximately 7 mV), the magnitude of which was only about one-third of that induced by equieffective contractile concentrations of NE and AVP. The voltage-sensitive Ca2+ channel agonist Bay K 8644 (1 microM), in contrast to ET-1, NE, and AVP, produced a small contraction (30 +/- 2% of the maximum response to NE), but no further depolarization when added in the presence of 15 mM K+ (which elicited approximately 12 mV depolarization but no contraction).(ABSTRACT TRUNCATED AT 250 WORDS)

The enantiomers of PN 200-110 (PN), a highly potent calcium antagonist, were synthesized by a stereoselective synthesis. Cross-contamination was less than 0.2% for the (S)-enantiomer and 0.5% for the (R)-enantiomer. Both isomers inhibited depolarization-induced contraction of rabbit aorta, the (S)-enantiomer being much more active: The pD'2 values were 9.1 and 6.9 for the (S)- and the (R)-enantiomer, respectively. This activity of (R)-PN on potential-operated channels can be attributed to the contamination with 0.5% of (S)-PN. In anesthetized cats, the (S)-enantiomer lowered blood pressure and heart rate dose-dependently (0.3-10 micrograms/kg i.v.). The (R)-enantiomer had almost no effect on heart rate (HR) and blood pressure (BP) at doses up to 300 micrograms/kg. However, both enantiomers increased cardiac output and blood flow to the heart and the brain. The effects on regional blood flow were tissue-dependent, the (R)-enantiomer being surprisingly potent in the subendocardium of the left ventricle. The qualitative and quantitative differences between the activities of the two enantiomers suggest that the results obtained cannot merely be explained by traces of the more active enantiomer contaminating the less active enantiomer. These results, together with those found with the enantiomers of a nitro-substituted dihydropyridine, suggest that calcium channels appear to be able not only to discriminate between enantiomers but also to respond differently to them.

The effects of the vasoconstrictive dihydropyridine, CGP 29392, upon tension development and 45Ca2+ uptake by vascular smooth muscle were studied using isolated rabbit aorta. CGP 28392 did not elicit contractile responses when administered alone but lowered the threshold for the contractile response to elevated extracellular K+. CGP 28392 also increased the magnitude of the contractions elicited by submaximal concentrations of K+. This effect was competitively antagonized by PY 108-068, a Ca antagonist of the dihydropyridine class. Similarly, the relaxing effects of PY 108-068 upon KCl-constricted rabbit aorta were competitively antagonized by CGP 28392. The calcium content of unstimulated tissues was only minimally increased by CGP 28392 but the 45Ca2+ uptake stimulated by depolarizing levels of K+ was markedly augmented by this agent. Further analysis of the data indicated that CGP 28392 did not alter the relationship between tension development and 45Ca2+ uptake. In contrast, CGP 28392 was much less effective in augmenting the contractile response and 45Ca2+ uptake elicited by noradrenaline. These results thus support the hypothesis that CGP 28392 predominantly facilitates Ca2+ entry through potential-operated Ca2+ channels.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8

The effects of noradrenaline (NA, 10(-5) M) and [arginine8]vasopressin (AVP, 10(-7) M) on tension in Ca2+-free medium and on membrane potential, and the inhibition of NA- and AVP-induced contractions by isradipine, have been compared in mesenteric resistance vessels (MRVs) from Wistar-Kyoto (WKY) rats. The release of intracellular Ca2+ by AVP contributed significantly less to its tension development than does that by NA. Nonetheless, the concentration-response curves for inhibition by isradipine of NA- and AVP-induced tonic tension were nearly identical. Similarly, these two agonists produced the same degree of membrane depolarization. In addition, both agonists were able to stimulate large contractions in vessels previously depolarized by 80 mM K+. AVP also stimulated 45Ca influx into rat cultured aortic smooth muscle cells. In contrast to the stimulation of 45Ca influx by KCl depolarization, the agonist-stimulated 45Ca influx was insensitive to inhibition by organic Ca2+ antagonists. It is concluded that Ca2+ entry through receptor-operated Ca2+-permeable channels (ROCs) may contribute to agonist-induced activation of rat aortic and MRV smooth muscle.