European Journal of Pharmacology (v.510, #3)

Cellular electrophysiological effect of terikalant in the dog heart by Péter Biliczki; Károly Acsai; László Virág; László Tálosi; Norbert Jost; András Biliczki; Julius Gy. Papp; András Varró (161-166).
The cellular mechanism of action of terikalant, an investigational antiarrhythmic agent known to block the inward rectifier and other potassium currents, has not yet been fully clarified. The aim of the present study was therefore to analyse the in vitro electrophysiological effects of terikalant in canine isolated ventricular muscle and Purkinje fibers by applying the standard microelectrode technique. The effects of terikalant on the duration of action potential at a stimulation cycle length of 1000 ms and on the maximum upstroke velocity of the action potential in right ventricular papillary muscle were examined at 1, 2.5, 10, and 20 μM concentrations. Terikalant significantly prolonged the action potential duration measured both at 50% and 90% of repolarization in concentration-dependent manner. The maximum upstroke velocity of the action potential was unaffected at 1 and 2.5 μM concentrations. However, this parameter was significantly reduced at 10 and 20 μM concentrations of terikalant. In Purkinje fibers terikalant (2.5 μM) also produced a marked action potential lengthening effect. Frequency dependence (cycle length of 300–5000 ms) of the action potential lengthening effect of terikalant was studied at a concentration of 2.5 μM. Prolongation of the duration of action potential occurred in a reverse frequency-dependent manner both in papillary muscle and Purkinje fibers, with a more pronounced frequency-dependence observed in Purkinje fibers. The onset kinetics of the terikalant (10 μM) induced block of the maximum upstroke velocity of the action potential was rapid (0.6±0.1 beat−1, n=6) like that of Class I/B antiarrhythmics, and the offset (recovery) kinetics of the drug (2956±696 ms, n=6) best resembled that of Class I/A antiarrhythmic drugs. It was concluded that terikalant, unlike pure Class III antiarrhythmic drugs, has combined mode of action by lengthening repolarization and blocking the inward sodium current in a use-dependent manner.
Keywords: Terikalant; Cellular electrophysiology; Antiarrhythmic action; Multichannel block;

We investigated the effects of branched-chain amino acids on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Of the branched-chain amino acids, only leucine (10−5–10−3 M) induced hepatocyte DNA synthesis and proliferation in a time- and dose-dependent manner. The addition of valine or isoleucine on its own had no significant effects on the hepatocyte DNA synthesis and proliferation. When combined, isoleucine competitively antagonized leucine-stimulated hepatocyte mitogenesis. U73122 (10−6 M), AG1478 (10−7 M), wortmannin (10−7 M), PD98059 (10−6 M) and rapamycin (10 ng/ml) inhibited the ability of leucine to stimulate the hepatocyte DNA synthesis and proliferation, suggesting that phospholipase C, tyrosine kinase, phosphatidylinositol 3-kinase, mitogen-activated protein (MAP) kinase, and p70 S6 kinase are involved in leucine signaling. The mitogenic effects of leucine are completely abolished by the addition of anti-transforming growth factor-α (TGF-α) antibody to the culture medium. Furthermore, leucine stimulated TGF-α secretion into the culture medium and the leucine effect was inhibited by U73122. Isoleucine alone had no significant effect on TGF-α secretion but this agent blocked leucine-induced TGF-α secretion. The results suggest that leucine triggers TGF-α secretion through a putative leucine receptor. The secreted TGF-α then stimulates hepatocyte DNA synthesis and proliferation through activation of TGF-α receptor to induce tyrosine kinase/MAP kinase activity and other downstream growth-related signal transducers.
Keywords: Leucine; DNA synthesis; Proliferation; Hepatocyte;

Aldosterone stimulates active Na+ transport in rabbit urinary bladder by both genomic and non-genomic processes by Timothy J. Burton; Dermot M.F. Cooper; Bryony Dunning-Davies; Dina Mansour; Nanako Masada; Douglas R. Ferguson (181-186).
The ability of aldosterone to stimulate Na+ transport in a range of epithelial tissues has been known for many years. Early work suggested that aldosterone had a delayed action operating by transcriptional up-regulation of proteins such as the epithelial Na+ channel. However more recent data has suggested that the hormone has a short-term non-genomic action. In this paper we investigate short and long-term actions of aldosterone on Na+ transport in the rabbit urinary bladder. We have shown that aldosterone stimulates epithelial Na+ channel activity, as measured by the amiloride-sensitive short-circuit current over a 3.75 h period and that this action is potentiated by cAMP. Using reverse transcriptase-polymerase chain reaction we have shown that aldosterone and forskolin in combination up-regulate mRNA synthesis for the β- and γ-subunits of the epithelial Na+ channel. Using Western blotting we have shown in the case of the β-subunit that a corresponding increase in channel protein occurs. We have also demonstrated that aldosterone in the presence of inhibitors of phosphodiesterase can stimulate the short-circuit current across rabbit bladder epithelium over a 20 min period. An explanation for the synergistic interaction between aldosterone and cAMP is provided. We have shown that aldosterone can increase cAMP levels within urothelial cells over a 4 min period. We propose that this represents a non-genomic action of the steroid hormone.
Keywords: Urinary bladder; Epithelial Na+ channel; Aldosterone;

Activation of apical CFTR and basolateral Ca2+-activated K+ channels by tetramethylpyrazine in Caco-2 cell line by Jin Xia Zhu; Gui Hong Zhang; Ning Yang; Dewi Kenneth Rowlands; Hau Yan Connie Wong; Lai Ling Tsang; Yiu Wa Chung; Hsiao Chang Chan (187-195).
We have previously demonstrated that tetramethylpyrazine (TMP) could stimulate colonic and pancreatic anion secretion. The present study investigated the signaling pathways and cellular mechanisms underlying the effect of TMP using human colonic Caco-2 cells, with permeabilized apical or basolateral membranes, in conjunction with Ussing chamber technique, intracellular cAMP and Ca2+ measurements as well as competitive RT-PCR for mRNA expression of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-dependent Cl channels (CACC). Basolateral addition of TMP induced a short circuit current (I SC) response, which could be mimicked by forskolin and 3-isobutyl-1-methylxanthine (IBMX). Adenylate cyclase inhibitor, MDL12330A, and intracellular Ca2+ chelator, BAPTA-AM, significantly inhibited the TMP-induced I SC. In basolateral membrane-permeabilized cells, TMP, as well as forskolin and IBMX, induced an I SC response, which was sensitive to MDL-12330A, H89, and specific channel blocker CFTRinh-172, but insensitive to apical application of 4-4′-didsothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and basolateral pretreatment with BAPTA-AM. In apical membrane-permeabilized cells, TMP, similar to forskolin and IBMX, produced a very small current increase, which was sensitive to K+ channel blockers, BaCl2 and tetraethylammonium (TEA), but not Chromanol 293B and charybdotoxin (ChTX), alone or combined. However, in intact Caco-2 monolayers, the TMP-induced I SC could be partially inhibited by ChTX. TMP (5 mM) could stimulate intracellular cAMP production. Intracellular Ca2+ was also increased by TMP (5 mM) in both Ca2+-containing and Ca2+-free bathing solutions. RT-PCR showed that the expression of CFTR in Caco-2 cells was 5.2 fold higher than that of Ca2+-activated Cl channel (CACC). In conclusion, TMP stimulates Cl secretion by activating cAMP and [Ca2+]i signaling pathways leading to subsequent activation of apical CFTR and basolateral K+ channels.
Keywords: Tetramethylpyrazine; Cystic fibrosis transmembrane conductance regulator; cAMP; K+ channel; Membrane permeabilization; Caco-2 cell line;

Tetrodotoxin-resistant and tetrodotoxin-sensitive Na+ channels contribute to the abnormal spontaneous firing in dorsal root ganglion neurons associated with neuropathic pain. Effects of the anti-nociceptive agent ralfinamide on tetrodotoxin-resistant and tetrodotoxin-sensitive currents in rat dorsal root ganglion neurons were therefore investigated by patch clamp experiments. Ralfinamide inhibition was voltage-dependent showing highest potency towards inactivated channels. IC50 values for tonic block of half-maximal inactivated tetrodotoxin-resistant and tetrodotoxin-sensitive currents were 10 μM and 22 μM. Carbamazepine, an anticonvulsant used in the treatment of pain, showed significantly lower potency. Ralfinamide produced a hyperpolarising shift in the steady-state inactivation curves of both currents confirming the preferential interaction with inactivated channels. Additionally, ralfinamide use and frequency dependently inhibited both currents and significantly delayed repriming from inactivation. All effects were more pronounced for tetrodotoxin-resistant than tetrodotoxin-sensitive currents. The potency and mechanisms of actions of ralfinamide provide a hypothesis for the anti-nociceptive properties found in animal models.
Keywords: Dorsal root ganglion; Sensory neuron; Tetrodotoxin-resistant sodium channel; Tetrodotoxin-sensitive sodium channel; Ralfinamide; NW-1029;

Effects of natriuretic peptides on ventricular myocyte contraction and role of cyclic GMP signaling by Qihang Zhang; Jacob Moalem; James Tse; Peter M. Scholz; Harvey R. Weiss (209-215).
Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) act through different receptors and at different potencies to affect cardiac myocyte function. We tested the hypothesis that these three peptides would differentially reduce cardiomyocyte function through their effects on the cyclic GMP signaling pathway. Rabbit ventricular myocytes were isolated and stimulated by electrical field stimulation. Cell function was measured using a video edge detector. ANP BNP or CNP at 10−9, 10−8, 10−7 M were added to the myocytes. Intracellular cyclic GMP was determined using a radioimmunoassay in the absence or presence of ANP, BNP or CNP. All natriuretic peptides decreased myocyte contractility in a similar concentration dependent manner. Myocyte percentage shortening was significantly decreased with all peptides at 10−7 M compared with baseline (ANP from 5.4±0.4 to 3.9±0.2%; BNP from 5.0±0.2 to 3.5±0.1%; CNP from 5.6±0.3 to 4.0±0.3%). Maximum rate of shortening and relaxation were also decreased similarly and significantly. Intracellular cyclic GMP was significantly increased in myocytes treated with ANP, BNP or CNP (Baseline 1.0±0.2, ANP 2.1±0.2, BNP 2.3±0.3, CNP 2.0±0.2 pmol/105 myocytes). Furthermore, inhibition of the cyclic GMP protein kinase with KT5823 caused a reversal in the functional effects of CNP. We concluded that all natriuretic peptides had similar negative effects on ventricular myocyte function and their effects were accompanied by increased cyclic GMP. Blockade the effect of CNP by a cyclic GMP protein kinase inhibitor demonstrated that effects were mediated through the cyclic GMP signaling pathway.
Keywords: Atrial natriuretic peptide; Brain natriuretic peptide; C-type natriuretic peptide; Cyclic GMP protein kinase; (Rabbit);

Capacitative Ca2+ entries and mRNA expression for TRPC1 and TRPC5 channels in human epidermoid carcinoma A431 cells by Junko Yoshida; Takaharu Ishibashi; Noriko Imaizumi; Tsutomu Takegami; Matomo Nishio (217-222).
In human epidermoid carcinoma A431 cells, capacitative Ca2+ entries in response to intracellular Ca2+ store depletion with thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhibitor, and uridine 5′-triphosphate, a phospholipase C-linked agonist, were inhibited by trivalent cations such as Gd3+ and La3+, and by the store-operated Ca2+ channel inhibitor, 2-aminoethoxydiphenyl borate. Of the seven types of canonical transient receptor potential (TRPC) channels as molecular candidates for store-operated Ca2+ channels, mRNAs for TRPC1 and TRPC5 were detected in the cells with the reverse transcription-polymerase chain reaction. Western blotting confirmed the protein expressions of TRPC1 and TRPC5 in A431 cells. The present results suggest that TRPC1 and/or TRPC5 channels serve as store-operated Ca2+ channels in A431 cells, and may function as regulators for intracellular Ca2+ signaling.
Keywords: TRPC1; TRPC5; Capacitative Ca2+ entry; Human epidermoid carcinoma A431 cell; Thapsigargin; Uridine 5′-triphosphate;

The effects of nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist JTC-801 on allodynia and hyperalgesia were examined in rats in order to explore the involvement of N/OFQ system in these pathological pain states. Tactile allodynia induced by L5/L6 spinal nerve ligation was reversed by both systemic (3–30 mg/kg) and spinal (22.5 and 45 pg) JTC-801 in a dose-dependent manner. Concerning hyperalgesia induced by formalin injection into the hindpaw, JTC-801 dose-dependently suppressed the second phase, but not the first phase, of the licking behavior. Furthermore, systemic JTC-801 reduced Fos-like immunoreactivity in the dorsal horn of the spinal cord (laminae I/II). In conclusion, N/OFQ receptor antagonist JTC-801 exerted anti-allodynic and anti-hyperalgesic effects in rats, suggesting that N/OFQ system might be involved in the modulation of neuropathic pain and inflammatory hyperalgesia.
Keywords: N/OFQ (nociceptin/orphanin FQ); NOP receptor antagonist; JTC-801; Allodynia; Hyperalgesia;

Contrasting effects of estradiol and 17β-aminoestrogens on blood clotting time in rats and mice by Cristina Lemini; Yanira Franco; Ma. Estela Avila; Ruth Jaimez (229-233).
Estrogens have been associated with thromboembolic events. Our group has described the anticoagulant effect of 17β-aminoestrogens in rodents, potentially new alternative estrogenic agents without thrombogenic risk. This work compares the contrasting effects of estradiol and the 17β-aminoestrogens (prolame, butolame, and pentolame) on blood clotting time. Ovariectomized CD1 mice received a single injection of 17β-aminoestrogens, estradiol (20 to 80 mg/kg), or vehicle. Estradiol decreased blood clotting time from −10% to −25% (48 h; P<0.01) and 17β-aminoestrogens increased it, differing in latency (∼12 h; +48%, P<0.01) and duration (∼72 h +58%, P<0.01). In male Wistar rats, similar effects (pentolame +45%; estradiol −31%; P<0.01) were observed 48 h after five consecutive daily injections of 1000 μg/animal/day. The maximum procoagulant effect of estradiol was obtained after 72 h with 10 μg/animal/day (−45%; P<0.01). 17β-Aminoestrogens always produced opposite effects to those of estradiol on blood coagulation.
Keywords: 17β-Aminoestrogen; Estradiol; Anticoagulant effect; Thrombogenic effect;

Estrogenic effects of 17β-aminoestrogens assessed in uteri of rats and mice by Cristina Lemini; Yanira Franco; Ma. Estela Avila; Ruth Jaimez (235-239).
Administration of exogenous estrogens has been associated with an increase of thromboembolic events. The 17β-aminoestrogens produce anticoagulant effects contrasting with the procoagulant effects of the natural occurring estradiol in rodents. This work compares the estrogenic effects induced by 17β-aminoestrogens prolame, butolame, pentolame, and estradiol in vivo models. Dose–response curves were performed using immature CD1 mice and Wistar rats. The animals were injected with estradiol or 17β-aminoestrogens (0.01 to 1000 μg/kg), or vehicle. The uterine wet and dry weights were determined. The 17β-aminoestrogens increased uterine weight in a dose-dependent manner. The uterotrophic effect produced by estradiol induced lower ED50 (6.5 and 4 μg/kg) and higher E max values (+523–350%) in mice as compared with those from the rat, indicating more susceptibility of the mice model. The 17β-aminoestrogens are partial estrogenic agonists with a relative uterotrophic effect of estradiol (100%) from 9–86%. Only the ED50 values of 17β-aminoestrogens in CD1 mice showed a direct correlation to the length of the amine group substitution in C-17 since their efficacy and potency were in the order: prolame>butolame>pentolame.
Keywords: 17β-aminoestrogen; Estradiol; Uterotrophic effect; Estrogenic effect;

Author index (241-243).

Keyword index (245-249).