Biomedical subjects

Diana Conte Camerino

Publications and source records attributed to Diana Conte Camerino.

At least 19 recordsLinked to original sources

PubMed2006

Overactivity of exercise-sensitive cation channels and their impaired modulation by IGF-1 in mdx native muscle fibers: beneficial effect of pentoxifylline.

Cell-attached patch-clamp recordings on native striated myofibers from adult dystrophic mdx mice revealed a higher occurrence and open probability compared to non-dystrophic wild-type myofibers of a 30 pS voltage-insensitive Ca2+-permeable channel, inhibited by Gd3+, streptomycin and ruthenium red. Myofibers from in vivo exercised animals had higher channel occurrence and/or open probability. Insulin-like growth factor 1 (3.3 nM) induced and/or enhanced channel activity, via PI3 kinase, in wild-type but not in mdx myofibers. Interestingly, in both genotypes the current was silenced by db-cAMP or pentoxifylline, a phosphodiesterase inhibitor. The channel activity/occurrence in pentoxifylline-treated exercised mdx (50 mg/kg/day i.p. for 4-8 weeks) overlapped that of exercised wild-type mice. Thus, a growth factor-sensitive current, likely due to a TRP channel, is activated in vivo by exercise in native striated fibers; its deregulation in the absence of dystrophin may contribute to Ca2+ homeostasis alteration. The possibility to pharmacologically counteract abnormal channel activity discloses important therapeutic application.

Animals↗

PubMed2006

Hybrid assemblies of ATP-sensitive K+ channels determine their muscle-type-dependent biophysical and pharmacological properties.

ATP-sensitive K(+) channels (K(ATP)) are an octameric complex of inwardly rectifying K(+) channels (Kir6.1 and Kir6.2) and sulfonylurea receptors (SUR1 and SUR2A/B), which are involved in several diseases. The tissue-selective expression of the subunits leads to different channels; however, the composition and role of the functional channel in native muscle fibers is not known. In this article, the properties of K(ATP) channels of fast-twitch and slow-twitch muscles were compared by combining patch-clamp experiments with measurements of gene expression. We found that the density of K(ATP) currents/area was muscle-type specific, being higher in fast-twitch muscles compared with the slow-twitch muscle. The density of K(ATP) currents/area was correlated with the level of Kir6.2 expression. SUR2A was the most abundant subunit expressed in all muscles, whereas the vascular SUR2B subunit was expressed but at lower levels. A significant expression of the pancreatic SUR1 was also found in fast-twitch muscles. Pharmacological experiments showed that the channel response to the SUR1 agonist diazoxide, SUR2A/B agonist cromakalim, SUR1 antagonist tolbutamide, and the SUR1/SUR2A/B-antagonist glibenclamide matched the SURs expression pattern. Muscle-specific K(ATP) subunit compositions contribute to the physiological performance of different muscle fiber types and determine the pharmacological actions of drugs modulating K(ATP) activity in muscle diseases.

ATP-Binding Cassette Transporters↗

PubMed2005

Carbonic anhydrase inhibitors ameliorate the symptoms of hypokalaemic periodic paralysis in rats by opening the muscular Ca2+-activated-K+ channels.

Carbonic-anhydrase inhibitors are effective in channelopathies possibly by opening the Ca2+-activated-K+ channels. However, the in vivo effects of these drugs in K+-deficient rats, the animal model of familial hypokalaemic periodic paralysis(hypokalaemic-PP), are currently unknown. Measures of insulin-responses, serum electrolytes levels and patch-clamp experiments were therefore performed in K+ -deficient rats treated in vivo with dichlorphenamide (DCP), ethoxzolamide (ETX), hydrochlorthiazide (HCT), methazolamide (MTZ), bendroflumethiazide (BFT) and acetazolamide (ACTZ). Ten days treatments of K+-deficient rats with DCP, BFT, ETX and ACTZ (5.6 mg/kg per day) restored the serum [K+] to control values and prevented the insulin-induced paralysis. In ex vivo experiments, the carbonic-anhydrase inhibitors enhanced the activity of Ca2+-activated-K+ channels with the order of efficacy: ACTZ>BFT>ETX>DCP. In contrast, HCT and MTZ failed to stimulate the Ca2+-activated-K+ channels and to prevent the hypokalaemia and paralysis. At the concentration of 1mg/kg per day, all these drugs failed to ameliorate the hypokalaemic-PP symptoms. The activation of Ca2+-activated-K+ channel in addition to the mild diuretic effect explained the efficacy of ACTZ and DCP in K+ -deficient rats and in familial hypokalaemic-PP.

Adenosine Triphosphate↗

PubMed2005

Activation and inhibition of kidney CLC-K chloride channels by fenamates.

CLC-K Cl(-) channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 microM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartter's syndrome.

Animals↗

PubMed2005

Fiber type-related changes in rat skeletal muscle calcium homeostasis during aging and restoration by growth hormone.

The mechanisms by which aging induces muscle impairment are not well understood yet. We studied the impact of aging on Ca2+ homeostasis in the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL) muscles of aged rats by using the fura-2 fluorescent probe. In both muscles aging increases the resting cytosolic calcium concentration ([Ca2+]i). This effect was independent on calcium influx since a reduced resting permeability of sarcolemma to divalent cations was observed in aged muscles likely due to a reduced activity of leak channels. Importantly the effects of aging on resting [Ca2+]i, fiber diameter, mechanical threshold and sarcolemmal resting conductances were less pronounced in the soleus muscle, suggesting that muscle impairment may be less dependent on [Ca2+]i in the slow-twitch muscle. The treatment of aged rats with growth hormone restored the resting [Ca2+]i toward adult values in both muscles. Thus, an increase of resting [Ca2+]i may contribute to muscle weakness associated with aging and may be considered for developing new therapeutic strategies in the elderly.

Aging↗

PubMed2005

Recovery of the soleus muscle after short- and long-term disuse induced by hindlimb unloading: effects on the electrical properties and myosin heavy chain profile.

The hindlimb unloading (HU) rat is a model of muscle disuse characterized by atrophy and slow-to-fast phenotype transition of the postural muscles, such as the soleus. We previously found that the resting sarcolemmal chloride conductance (gCl) that is typically lower in slow-twitch myofibers than in fast ones increased in soleus fibers following 1 to 3 weeks of HU in accord with the slow-to-fast transition of myosin heavy chain (MHC) isoforms. Nevertheless, the gCl already raised after a 3-day HU, whereas no change in MHC expression was detected. The present work evaluates the ability of soleus muscle to recover on return to normal load after a short (3 days) or long (2 weeks) disuse period. The changes observed after a 2-week HU were slowly reversible, since 3-4 weeks of reloading were needed to completely recover gCl, fiber diameter, MHC expression pattern, as well as the mechanical threshold Rheobase, an index of calcium homeostasis. After 3-day HU, the gCl increased homogeneously in most of the soleus muscle fibers and gCl recovery was rapidly completed after 4-day reloading. These results suggest different induction mechanisms for gCl augmentation after the short and long HU periods, as well as a possible role for gCl in the slow muscle adaptation to disuse.

Adaptation, Physiological↗

PubMed2005

Phenotype-dependent functional and pharmacological properties of BK channels in skeletal muscle: effects of microgravity.

We investigated the involvement of calcium-activated potassium channel (BK) in skeletal muscle phenotype determination and response to acetazolamide, a BK opener. The BKs of slow-twitching soleus (SOL) and fast-twitching flexor digitorum brevis (FDB) muscles of the rat were investigated by patch-clamp technique. The changes of BK properties following muscle disuse were investigated in the hindlimb-unloaded (HU) rat, an animal model of disuse/microgravity. Two functionally different BKs were found in skeletal muscle. The BK of FDB was sensitive to calcium and to acetazolamide, in contrast the BK of SOL was less sensitive to calcium and was resistant to acetazolamide. After 3-14 days HU, in parallel with the slow-to-fast phenotype transition of the fibers, the BK of SOL acquired properties similar to those of FDB. In skeletal muscle, the BK plays muscle-specific roles contributing to the calcium-dependent phenotype determination/adaptation to disuse. The phenotype specificity of acetazolamide has implications for drug-based therapy of neuromuscular disorders associated to disuse.

Acetazolamide↗

PubMed2005

A multidisciplinary evaluation of the effectiveness of cyclosporine a in dystrophic mdx mice.

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.

Animals↗

PubMed2004

Molecular determinants of differential pore blocking of kidney CLC-K chloride channels.

The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.

2-Methyl-4-chlorophenoxyacetic Acid↗

PubMed2004

Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K+-deficient rats.

Carbonic-anhydrase (CA) inhibitors are used in the treatment of hypokalaemic periodic paralysis (hypoPP) and related channelopathies but their mechanism of action is unknown. Patch-clamp experiments and molecular modeling investigations were performed to evaluate the mechanism of actions of CA inhibitors on skeletal muscle Ca2+-activated-K+ (BK) channel of K+-deficient rats used as animal model of hypoPP. CA inhibitors showing different degree of CA inhibition such as acetazolamide (ACTZ), dichlorphenamide (DCP), hydrochlorthiazide (HCT), etoxzolamide (ETX), methazolamide (MTZ), and bendroflumethiazide (BFT), which lacks inhibitory effects on CA enzymes, were tested in vitro on BK channels. The application of ACTZ, BFT, ETX, and DCP to excised patches activated the BK channel with potency: ACTZ(DE50=7.3x10(-6)M)>BFT(DE50=5.93x10(-5)M)>ETX(DE50=1.17x10(-4)M)>>DCP. In contrast, MTZ and HCT failed to activate the BK channel. Molecular modeling studies showed that the capability of CA inhibitors to open the BK channel was related to the presence in their structures of an intra-molecular hydrogen bond with calculated inter-atomic distances ranging between 1.82 A degrees and 3.01 A degrees and of an aromatic ring poor of electrons. ACTZ, BFT, ETX, and DCP showed these pharmacofores, while MTZ and HCT did not. Our data indicate that the activation of BK channel is a property of CA inhibitors that interact with the channel subunit/s and that this effect is not related to their capability to inhibit the CA enzymes.

Animals↗

PubMed2004

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation.

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.

Animals↗

PubMed2004

New potent mexiletine and tocainide analogues evaluated in vivo and in vitro as antimyotonic agents on the myotonic ADR mouse.

The antimyotonic activity of chiral derivatives of mexiletine and tocainide, selected as potent use-dependent blockers of skeletal muscle sodium channels, was evaluated in vivo acutely in myotonic ADR mice. The compounds had either aromatic (Me4 and Me6) or branched isopropyl groups (Me5 and To1) on the asymmetric centre, or had this latter one methylene apart from the amino group (Me2). Therapeutic doses of mexiletine (5-10 mg/kg) and tocainide (7-20 mg/kg) significantly reduced the long time of righting reflex (TRR), typical of ADR mice. Me4, Me5 and Me6 were 2-fold more potent than mexiletine. To1 fully normalised the TRR at 7 mg/kg. The electromyographic analysis confirmed a muscle-based activity for drug effectiveness on TRR. All the compounds reduced the myotonic hyperexcitability of intercostal muscle fibres when tested in vitro by current-clamp recordings, with a potency correlated with their action on sodium channels. On stimulus-evoked firing, the isopropyl analogues were 2-4-fold more potent than parent compounds, while the aromatic analogues were about 10-fold more potent than mexiletine. Patch-clamp recordings confirmed a normal-like pharmacological sensitivity of sodium channels of native ADR muscle fibres. Finally, the in vivo antimyotonic activity is due to the block of sodium channels and divergences with in vitro potency can be related to structure-based changes in drug pharmacokinetics.

Action Potentials↗

PubMed2004

Taurine and skeletal muscle disorders.

Taurine is abundantly present in skeletal muscle. We give evidence that this amino acid exerts both short-term and long-term actions in the control of ion channel function and calcium homeostasis in striated fibers. Short-term actions can be estimated as the ability of this amino acid to acutely modulate both ion channel gating and the function of the structures involved in calcium handling. Long-term effects can be disclosed in situations of tissue taurine depletion and are likely related to the ability of the intracellular taurine to control transducing pathways as well as homeostatic and osmotic equilibrium in the tissue. The two activities are strictly linked because the intracellular level of taurine modulates the sensitivity of skeletal muscle to the exogenous application of taurine. Myopathies in which ion channels are directly or indirectly involved, as well as inherited or acquired pathologies characterized by metabolic alterations and change in calcium homeostasis, are often correlated with change in muscle taurine concentration and consequently with an enhanced therapeutic activity of this amino acid. We discuss both in vivo and in vitro evidence that taurine, through its ability to control sarcolemmal excitability and muscle contractility, can prove beneficial effects in many muscle dysfunctions.

Aging↗

PubMed2004

Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-Chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers.

CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na(+) reabsorption. The identification of barttin as an essential beta-subunit of CLC-K channels allowed performance of a pharmacologic characterization of wild-type CLC-K1 expressed in Xenopus oocytes. To this end, a series of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives were screened using the two-microelectrode voltage-clamp technique. Several chemical modifications regarding the phenoxy group of the side chain (elimination of the oxygen atom or of methylenic groups, substitutions of the chlorine atom) did not alter the drug blocking activity, with five different derivatives showing a similar potency. Among these, a derivative of CPP carrying a benzyl group on the chiral center in the place of the methyl group represented the minimal structure for blocking CLC-K1. It inhibited the channel from the extracellular side with an affinity in the 150 micro M range. The blocking potency of this compound is fourfold increased by lowering the extracellular chloride concentration, suggesting that the drug interacts with the channel pore. Concomitantly, the effect of some "classical" Cl(-) channel blockers (9-anthracenecarboxylic acid, 2-(phenylamino)benzoic acid, iminodibenzoic acid, niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt, and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt) was screened. 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt was the only one capable of blocking CLC-K1 with a potency similar to the CPP derivative, although in an irreversible manner. The newly identified substances provide a useful tool to investigate the biophysical and physiologic role of these renal channels and a starting point for the development of therapeutic drugs with diuretic action.

2-Methyl-4-chlorophenoxyacetic Acid↗

PubMed2003

Optically active mexiletine analogues as stereoselective blockers of voltage-gated Na(+) channels.

Optically active mexiletine analogues were synthesized and evaluated in vitro as use-dependent blockers of skeletal muscle sodium channels. The mexiletine analogues were obtained by replacing either the methyl group on the stereogenic center of mexiletine [1-(2,6-dimethylphenoxy)propan-2-amine] with a phenyl group or modifying the phenoxy moiety (by removal of one or both of the methyl groups, or introducing a chlorine atom), or both. The voltage clamp recordings showed that, regardless of the substitution pattern of the aryloxy moiety, all the compounds bearing a phenyl group on the stereogenic center (3a-f) were more active than mexiletine both in tonic and phasic block. This observation was in contrast with what was observed for mexiletine, where the removal of both methyls from the aryloxy moiety caused a dramatic reduction of potency. The most potent congener, (R)-2-(2-methylphenoxy)-1-phenylethanamine [(R)-3b], was 27-fold more potent than (R)-mexiletine in producing a tonic block, i.e., the reduction of peak sodium current in resting conditions after application of the compound. (R)-3b maintained a use-dependent behavior, being 23-fold more potent in condition of high frequency of stimulation (phasic block). Despite what was observed with mexiletine, the stereoselectivity held in phasic block conditions. Stereoselectivity indexes were generally low, ranging from 1 to 4, but except for that of the 2,6-xylyloxy congener 3c, they were higher for the congeners bearing a phenyl ring on the stereogenic center than for mexiletine and its strictly related analogue 1-methyl-2-phenoxyethanamine (1). This finding was in agreement with Pfeiffer's rule. The introduction of a chlorine atom in the 4-position of the aryloxy moiety caused a reduction of potency and a reversal of stereoselectivity as well. On the basis of the model to date accepted for the sodium channel local anesthetic-like molecule receptor, some possible explanations of our observations will be proposed.

Animals↗

PubMed2003

Decrease in resting calcium and calcium entry associated with slow-to-fast transition in unloaded rat soleus muscle.

Using fura-2 and the manganese quenching technique, we show here that sarcolemmal permeability to cations (SP-Ca) of slow-twitch muscles is greater than that of fast-twitch ones. This appears to be related to a higher expression and/or activity of stretch-activated channels, whereas leak channel activities are similar. During hindlimb suspension (HU), we found highly correlated decreases in SPCa and resting calcium of soleus muscle toward values of extensor digitorum longus (EDL) muscle. This was significant as soon as 3 days of suspension, contrary to soleus muscle caffeine sensitivity and responsiveness that were not modified after this HU period. After 14 days of HU, SP-Ca, resting calcium, and caffeine response of soleus muscle became similar to that normally observed in EDL muscle. These results demonstrate that the correlated decreases in SP-Ca and resting calcium precede most functional changes due to HU. Given the known shortening of HU soleus muscle, we proposed that this could induce a decrease of SP-Ca and a consequent reduction of resting calcium. According to the crucial role of resting cytosolic free calcium in the maintenance and the adaptation of muscle phenotype, our results suggest that slow-to-fast transition of HU soleus muscle is calcium dependent.

Animals↗

PubMed2003

Involvement of 3Na+/2K+ ATP-ase and Pi-3 kinase in the response of skeletal muscle ATP-sensitive K+ channels to insulin.

The modulation of ATP-sensitive K+ channel (K(ATP)) by insulin plays a role in neuromuscular disorders associated to altered K+ homeostasis. However, the mechanisms by which insulin modulates K(ATP) channels are not known. Here, the insulin-dependent 3Na+/2K+ ATP-ase and Pi-3 kinase pathways were explored by using patch-clamp techniques. High and low affinity inhibition of K(ATP) channels by ouabain was observed in the insulin-stimulated and resting fibers, respectively. The 9A5 antibody directed against the alpha1-subunit of the pump inhibited the K(ATP) channel in the resting fibers but fails to inhibit it in the insulin-stimulated fibers. In contrast, the RT2NKATPabr, an alpha2-subunit specific antibody, inhibited the K(ATP) channels in the insulin-stimulated fibers failing to inhibit it in the resting fibers. The insulin-dependent stimulation of K(ATP) channel was prevented by Pi-3 kinase inhibitors Wortmannin and LY294002. In conclusion, insulin stimulating the 3Na+/2K+ ATP-ase activates K(ATP) channels through a membrane-delimited interaction thus controlling the K+ homeostasis. The Pi-3 kinase is the intracellular insulin signal linking the glucose homeostasis to the K(ATP) channel.

Androstadienes↗

PubMed2003

Dualistic actions of cromakalim and new potent 2H-1,4-benzoxazine derivatives on the native skeletal muscle K ATP channel.

1 New 2H-1,4-benzoxazine derivatives were synthesized and tested for their agonist properties on the ATP-sensitive K(+) channels (K(ATP)) of native rat skeletal muscle fibres by using the patch-clamp technique. The novel modifications involved the introduction at position 2 of the benzoxazine ring of alkyl substituents such as methyl (-CH(3)), ethyl (-C(2)H(5)) or propyl (-C(3)H(7)) groups, while maintaining pharmacophore groups critical for conferring agonist properties. 2 The effects of these molecules were compared with those of cromakalim in the presence or absence of internal ATP (10(-4) M). In the presence of internal ATP, all the compounds increased the macropatch K(ATP) currents. The order of potency of the molecules as agonists was -C(3)H(7) (DE(50)=1.63 x 10(-8) M) >-C(2)H(5) (DE(50)=1.11 x 10(-7) M)>-CH(3) (DE(50)=2.81 x 10(-7) M)>cromak-slim (DE(50)= 1.42 x 10(-5) M). Bell-shaped dose-response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. 3 In contrast, in the absence of internal ATP, all molecules including cromakalim inhibited the K(ATP) currents. The order of increasing potency as antagonists was cromakalim (IC(50)=1.15 x 10(-8) M)> or =-CH(3) (IC(50)=2.6 x 10(-8) M)>-C(2)H(5) (IC(50)=4.4 x 10(-8) M)>-C(3)H(7) (IC(50)=1.68 x 10(-7) M) derivatives. 4 These results suggest that the newly synthesized molecules and cromakalim act on muscle K(ATP) channel by binding on two receptor sites that have opposite actions. Alternatively, a more simple explanation is to consider the existence of a single site for potassium channel openers regulated by ATP which favours the transduction of the channel opening. The alkyl chains at position 2 of the 2H-1,4-benzoxazine nucleus is pivotal in determining the potency of benzoxazine derivatives as agonists or antagonists.

Adenosine Triphosphate↗