Biphasic Concentration-response Curve Research Articles

The Plasmodium falciparum ABC transporter ABCI3 confers parasite strain-dependent pleiotropic antimalarial drug resistance

SummaryWidespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.

Abstract 4043: SYD985 synergy with PARP inhibitors (PARPi) in human tumor cell lines and patient-derived xenografts (PDXs)

Abstract SYD985 is a HER2-targeting antibody-drug conjugate comprised of a cleavable linker and the prodrug seco-DUBA which, upon release, spontaneously rearranges to form the active toxin DUBA. DUBA covalently binds adenine in the DNA resulting in DNA damage, cell cycle perturbation and associated cytotoxicity. SYD985 induces dose-dependent cytotoxicity of human tumor cell lines and PDXs expressing HER2 on the outer cell membrane. The cytotoxicity of PARPi depends on PARP trapping, i.e. the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of clear1 and unclear origins2,3. We investigated potential synergy in cytotoxic activity of SYD985 and the PARPi niraparib and olaparib towards HER2-expressing tumor cells in vitro and in vivo. Tumor cell lines were from breast (BT-474, AU-565, UACC-893) or ovarian (TOV-112D, NIH:OVCAR-3) origin. Cytotoxicity was established by measuring DNA content and metabolic activity of living cells. For both PARPi the highest non-cytotoxic concentration for each cell line was established. This concentration was used to measure the potential potency shift of the SYD985 concentration-response curve by comparing SYD985 IC50 values in the absence and presence of PARPi. PARPi-induced fold change in IC50 for SYD985 was established in two independent experiments (N=2, n=3). Niraparib, dosed at a non-cytotoxic concentration, induced a 1.5 to 136-fold potentiation of SYD985-induced tumor cell killing. In BT-474 cells, niraparib co-treatment unveils a biphasic concentration-response curve to SYD985. This is in line with the fact that BT-474 cells express high levels of HER2 and are known to respond to naked trastuzumab at concentrations slightly above those of SYD985. Olaparib, dosed at a non-cytotoxic concentration, induced a 0.4 to 7.5-fold potentiation of SYD985-induced tumor cell killing. In a breast cancer PDX mouse clinical trial in immunodeficient mice that lack carboxylesterase 1c (Ces1c) we studied the combination of SYD985 (1 mg/kg, IV, SD) and niraparib or olaparib (50 mg/kg Q1DX21, IP). In order to detect potential additive and/or synergistic effects of SYD985 and PARPi, a suboptimal dose of SYD985 (1 mg/kg) was selected. PARPi doses were chosen known to inhibit growth of tumors with a defective DNA repair mechanism4. Data clearly show that the combination of SYD985 and PARPi are more effective in tumor volume reduction than either monotherapy alone. In conclusion, niraparib and olaparib act in synergy with SYD985 in killing human HER2-expressing tumor cells in vitro, both in high HER2 (BT-474, AU-565, UACC-893) and low HER2 (NIH:OVCAR-3; TOV-112D) expressing cells. In vivo such synergistic activity appears to translate in improved tumor growth inhibition of various breast cancer PDX models. 1Zimmermann M, Nature 559, 2018; 2Pommier Y, Sci Transl Med 8, 2016; 3Lord C, Science 355, 2017; 4Henneman L, PNAS 112, 2015 Citation Format: Wim H. Dokter, Fred A. Dijcks, Patrick G. Groothuis, Ellen Mattaar, Eline Loosveld, Daniëlle Jacobs, Monique van der Vleuten, Jan H. Schellens, Miranda van der Lee. SYD985 synergy with PARP inhibitors (PARPi) in human tumor cell lines and patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4043.

PH affects the hormesis profiles of personal care product components on luminescence of the bacteria Vibrio qinghaiensis sp. -Q67

Hormesis describes a specific phenomenon in a biphasic concentration-response curve: low concentrations stimulate a response, while high concentrations suppress it. Hormesis could be influenced by several environmental factors, e.g. pH. In this study, the concentration-response/bioluminescence inhibition profiles (CRPs) of six components in personal care products to Vibrio qinghaiensis sp.-Q67 were measured at five different pH levels. When the exposure lasted for 0.25 h, CRPs of the six components at various pH levels were S-shaped, except ascorbic acid 2-glucoside (AA2G) at pH 10.5. When it lasted for 12 h, the CRPs were J-shaped, except AA2G at pH 6.5, 7.5, and 9.5. To rationally explain these changes in hormesis expressed by J-shaped CRP, four characteristic parameters, the minimum effect (Emin) and its corresponding concentration (ECmin), the median effective concentration (EC50), and the zero effect concentration point (ZEP, where the effect is 0 and the concentration is ZEP), were used to quantify the J-shaped CRP. The results indicated that these parameters vary with pH. Additionally, ZEP showed an excellent linear relationship with EC10 (R2 = 0.9994) at all pH levels, indicating that EC10 could replace the no-observed effective concentration (NOEC) in ecological risk assessment. Furthermore, to elucidate the possible mechanism of hormesis, the binding of the components to the luciferase receptors was analyzed using molecular docking technology. The results showed that the components displaying hormesis bind more easily to the α subunit of luciferase than to the β subunit.

Effects of bioenergetics, temperature and cadmium on liver mitochondria reactive oxygen species production and consumption

A by-product of mitochondrial substrate oxidation and electron transfer to generate cellular energy (ATP) is reactive oxygen species (ROS). Superoxide anion radical and hydrogen peroxide (H2O2) are the proximal ROS produced by the mitochondria. Because low levels of ROS serve critical regulatory roles in cell physiology while excessive levels or inappropriately localized ROS result in aberrant physiological states, mitochondrial ROS need to be tightly regulated. While it is known that regulation of mitochondrial ROS involves balancing the rates of production and removal, the effects of stressors on these processes remain largely unknown. To illuminate how stressors modulate mitochondrial ROS homeostasis, we investigated the effects of temperature and cadmium (Cd) on H2O2 emission and consumption in rainbow trout liver mitochondria. We show that H2O2 emission rates increase with temperature and Cd exposure. Energizing mitochondria with malate-glutamate or succinate increased the rate of H2O2 emission; however, Cd exposure imposed different patterns of H2O2 emission depending on the concentration and substrate. Specifically, mitochondria respiring on malate-glutamate exhibited a saturable graded concentration-response curve that plateaued at 5 μM while mitochondria respiring on succinate had a biphasic concentration-response curve characterized by a spike in the emission rate at 1 μM Cd followed by gradual diminution at higher Cd concentrations. To explain the observed substrate- and concentration-dependent effects of Cd, we sequestered specific mitochondrial ROS-emitting sites using blockers of electron transfer and then tested the effect of the metal. The results indicate that the biphasic H2O2 emission response imposed by succinate is due to site IIF but is further modified at sites IQ and IIIQo. Moreover, the saturable graded H2O2 emission response in mitochondria energized with malate-glutamate is consistent with effect of Cd on site IF. Additionally, Cd and temperature acted cooperatively to increase mitochondrial H2O2 emission suggesting that increased toxicity of Cd at high temperature may be due to increased oxidative insult. Surprisingly, despite their clear stimulatory effect on H2O2 emission, Cd, temperature and bioenergetic status did not affect the kinetics of mitochondrial H2O2 consumption; the rate constants and half-lives for all the conditions tested were similar. Overall, our study indicates that the production processes of rainbow trout liver mitochondrial H2O2 metabolism are highly responsive to stressors and bioenergetics while the consumption processes are recalcitrant. The latter denotes the presence of a robust H2O2 scavenging system in liver mitochondria that would maintain H2O2 homeostasis in the face of increased production and reduced scavenging capacity.

Optimization and pharmacological characterization of receptor-mediated Gi/o activation in postmortem human prefrontal cortex.

The biochemical abnormalities in transmembrane signal transduction mediated through G protein-coupled receptors (GPCRs) have been postulated as underlying pathophysiology of psychiatric diseases such as schizophrenia and mood disorders. In the present study, the experimental conditions of agonist-induced [35 S]GTPγS binding in postmortem human brain membranes were optimized, and the responses induced by a series of agonists were pharmacologically characterized. The [35 S]GTPγS binding assay was performed in postmortem human prefrontal cortical membranes by means of filtration techniques, and standardized as to GDP concentration, membrane protein content, MgCl2 and NaCl concentrations in assay buffer, incubation period and effect of white matter contamination. Under the standard assay conditions, the specific [35 S]GTPγS binding was stimulated by the addition of 15 compounds in a concentration-dependent manner. Of these agonists, R(+)-8-OH-DPAT, UK-14,304, DAMGO and DPDPE showed apparently biphasic concentration-response curves. As for these four responses, only higher-potency site was pharmacologically characterized. The receptors involved in the responses investigated were 5-HT1A receptor (probed with R(+)-8-OH-DPAT or 5-HT), α2A -adrenoceptor (UK-14,304 or (-)-epinephrine), M2 /M4 mAChRs (carbachol), adenosine A1 receptor (adenosine), histamine H3 receptor (histamine), group II mGlu (l-glutamate), GABAB receptor (baclofen), μ-opioid receptor (DAMGO or endomophin-1), δ-opioid receptor (DPDPE or SNC-80) and NOP (nociceptin). Although dopamine also activated specific [35 S]GTPγS binding, this response was likely mediated via α2A -adrenoceptor, but not dopamine receptor subtypes. The present study provides us with fundamental aspects of the strategy for elucidation of probable abnormalities of neural signalling mediated by G proteins activated through multiple GPCRs in the brain of psychiatric patients.

Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABAA receptor subtypes indicate a different binding mode for GABA at these receptors

Ionotropic GABAA receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABAA receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABAA receptors. α4/δ-Containing GABAA receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC50=24nM) and α4β3δ (EC50=12nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC50(1)=16nM; EC50(2)=1.2μM). At α4β2δ, GABA had low micromolar activity (EC50=1μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABAA receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABAA receptors compared to their synaptic counterparts.

Sodium channel activator‐stimulated neuronal development involves BDNF‐TrkB signaling

N‐methyl D‐aspartate receptor (NMDAR) activation stimulates calcium influx and brain‐derived neurotrophic factor (BDNF) – TrkB signaling. This pathway is implicated in activity‐dependent neuronal development and synaptic plasticity. Voltage‐gated sodium channel (VGSC) activators promote neuronal development by increasing [Na+]i and upregulating NMDAR function. Here we tested the effect of the VGSC activator veratridine (VRT) on neurite outgrowth (NOG), synthesis and release of BDNF and TrkB activation in DIV1 cerebrocortical neurons. VRT enhanced NOG in a hormetic concentration‐response manner and this response was dependent on NMDAR and TrkB signaling. Inhibitors of NMDAR, TrkB, PI3K, and PLCγ inhibited VRT‐enhanced NOG. Acute treatment with VRT stimulated phosphorylation of TrkB and its downstream effectors Akt, mTOR, PLC, ERK1/2 and CREB. VRT increased BDNF synthesis and release in a concentration dependent manner; however, VRT stimulation of TrkB phosphorylation displayed a biphasic concentration‐response curve. VRT stimulation of BDNF synthesis required VGSCs and NMDARs. These data suggest that BDNF synthesis and release mediate VGSC activator‐enhanced NOG through activation of TrkB signaling.(NIH, R01NS053398)

Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells

Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl2 (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration–response curve and the current–voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil.

Inhibitory effect of 5-hydroxytryptamine on rat stomach fundus: mediated indirectly by activation of noradrenaline release.

Biphasic cumulative concentration-response curves to 5-hydroxytryptamine (5-HT) and alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) using rat stomach fundus in the presence of 50 microM pargyline suggested two sites of interaction (high and low affinity). The order of agonist potencies of 5-HT agonists for the high-affinity (contractile) response confirmed reports of a strong correlation of the 5-HT2B (contractile) receptor on fundus with reported rat brain (radioligand binding) pKd values at the 5-HT2C receptor (r = 0.94, b = 0.93 P < 0.01). 1(1-Naphthyl)-piperazine and ketanserin antagonized the high-affinity responses. The order of potencies for the low affinity (inhibitory) response was: alpha-Me-5-HT > 5-HT. Preincubation with mianserin or S-(-)propranolol, pretreatment with reserpine and removal of the mucosa layer resulted in amplification of the contractile effects and disappearance or reversal of the inhibitory effects of 5-HT. Activation of a second receptor site inducing catecholamine release may explain this effect of 5-HT on rat stomach fundus.

Information extraction from biphasic concentration-response curves for data obtained from neuronal activity of networks cultivated on multielectrode-array-neurochips

We aim to fit biphasic concentration-response curves to extract information about the effect of given biochemical substances to in-vitro neurons. Neurons extracted from embryonic mice are cultivated on multielectrode-array-neurochips (MEA-neurochip) [1]. The activity of single neurons in such networks is recorded especially the change of network activity caused by long-term application of neuroactive substances. This results in quasi-stable patterns of neuronal activity. Based on the data, different features [2] are calculated adapted from spikes and bursts and separately displayed in concentration-response curves [3]. These concentration-response curves can exhibit non sigmoid shape, then indicating that different mechanisms affect the neuronal activity. Hence, the concentration-response curves presumably include currently hidden and unused information.

Involvement of Phosphoinositide 3‐kinase‐Akt signaling pathway in sodium channel activator‐induced neurite outgrowth

Calcium influx through N‐methyl D‐aspartate receptors (NMDAR) regulates signaling cascades that underlie neuronal development and synaptic plasticity. Earlier we demonstrated that the voltage‐gated sodium channel (VGSC) activators, brevetoxin and antillatoxin, increased [Na+]i, stimulated Ca2+ influx through NMDARs and enhanced NMDAR‐mediated neurite outgrowth (Joju et al., 2009, Jabba et al., 2010). Here we tested whether VGSC activator‐induced neurite outgrowth may involve NMDAR‐dependent activation of the PI3K‐Akt pathway. Veratridine (VRT), a VGSC activator, was used in this study. VRT enhanced neurite outgrowth and dendritic arborization in immature cerebrocortical neurons and displayed a biphasic concentration‐response curve. VRT produced an elevation of [Na+]i and [Ca2+]i in a tetrodotoxin‐sensitive manner. Exposure to VRT increased Akt phosphorylation in immature cerebrocortical neurons. Either the NMDAR antagonist MK‐801, PI3K inhibitor LY294002 or calmodulin‐dependent protein kinase kinase (CaMKK) inhibitor STO‐609 abolished VRT‐induced Akt phosphorylation. VRT‐induced neurite outgrowth and dendritic arborization was abrogated by LY294002. These data suggest that VGSC activator‐enhanced neuritogenisis and dendritic arborization involves elevation of [Na+]i, and [Ca2+]i, and activation of the NMDAR‐PI3K‐Akt pathway.(NIH RO1 NS053398‐06)

Induction and inhibition of aromatase (CYP19) activity by natural and synthetic flavonoid compounds in H295R human adrenocortical carcinoma cells.

Flavonoids and related structures (e.g., flavones, isoflavones, flavanones, catechins) exert various biological effects, including anticarcinogenic, antioxidant and (anti-)estrogenic effects, and modulation of sex hormone homeostasis. A key enzyme in the synthesis of estrogens from androgens is aromatase (cytochrome P450 19; CYP19). We investigated the effects of various natural and synthetic flavonoids on the catalytic activity and promoter-specific expression of aromatase in H295R human adrenocortical carcinoma cells. Natural flavones were consistently more potent inhibitors than flavanones. IC(50) values for 7-hydroxyflavone, chrysin, and apigenin were 4, 7, and 20 microM, respectively; for the flavanones 7-hydroxyflavanone and naringenin the IC(50) values were 65 and 85 microM, respectively. The steroidal aromatase inhibitor (positive control) 4-hydroxyandrostenedione had an IC(50) of 20 nM. The inhibition by apigenin and naringenin coincided with some degree of cytotoxicity at 100 microM. The natural flavonoid derivative rotenone (IC(50) 0.3 microM) was the most potent aromatase inhibitor tested. Several synthetic flavonoid and structurally related quinolin-4-one analogs inhibited aromatase activity. The most potent inhibitor was 4'-tert-butyl-quinolin-4-one (IC(50) 2 microM), followed by two 2-pyridinyl-substituted alpha-naphthoflavones (IC(50)s 5 and >30 microM). The two 2-pyridinyl-substituted gamma-naphthoflavones consistently produced biphasic concentration-response curves, causing about 1.5-fold aromatase induction at concentrations below 1 microM and inhibition above that level (IC(50)s 7 and >30 microM). The natural flavone quercetin and isoflavone genistein induced aromatase activity 4- and 2.5-fold induction, respectively, at 10 microM. This coincided with increased intracellular cAMP concentrations and increased levels of the cAMP-dependent pII and to a lesser extent 1.3 promoter-specific aromatase transcripts. These results shed light on the structure-activity relationships for aromatase inhibition as well as mechanisms of induction in human H295R cells.

Comparison of the Pharmacological Properties of Rat NaV1.8 with Rat NaV1.2a and Human NaV1.5 Voltage-Gated Sodium Channel Subtypes Using a Membrane Potential Sensitive Dye and FLIPRR

A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPRR) have been used to characterize the pharmacological properties of rat Nav1.8 voltage-gated sodium channels (VGSC) in parallel with rat Nav1.2a and human Nav1.5 VGSC subtypes, respectively. The sensitivity of recombinant Nav1.2a-CHO, Nav1.5-293-EBNA, and Nav1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Nav1.2a-CHO and Nav1.5-293-EBNA cells with pEC50 values of 4.78 ± 0.13 and 4.84 ± 0.12, respectively (n = 3), but had negligible effect on Nav1.8-F-11 cells (pEC50 < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Nav1.8-F-11 and Nav1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Nav1.8-F-11 cells by ≥ 20-fold, in contrast to a ≤ 3-fold potentiation of the response in Nav1.2a, and Nav1.5 cells. Tetrodotoxin (TTX) inhibited VGSC activator–evoked depolarization of Nav1.8-F-11 cells with a biphasic concentration-response curve. The calculated pIC50 values were 8.05 ± 0.25 (n = 4) and 4.32 ± 0.21 (n = 4), corresponding to TTX inhibition of endogenous TTX-sensitive (TTX-S), and recombinant Nav1.8 TTX-resistant (TTX-R) VGSCs, respectively. With the exception of TTX, the potencies of a number of ion channel blockers for the Nav1.8, Nav1.2a, and Nav1.5 VGSC subtypes were similar. In summary, these high-throughput FLIPRR assays represent a valuable tool for the determination of the relative potencies of compounds at different VGSC subtypes and may prove useful for the identification of novel subtype-selective inhibitors.

Myogenic tone and reactivity of the rat ophthalmic artery.

To study and quantify myogenic behavior and reactivity of the rat ophthalmic artery to pressure and different vasoactive substances in vitro. Rat ophthalmic arteries (diameter of 217 +/- 6 microm, n = 22) were isolated, cannulated with glass pipettes in an arteriograph and pressurized in a physiological buffer. Internal diameter was continuously monitored. The effect of intraluminal pressure on the diameter was assessed and concentration-response curves to different constrictor and dilator agonists were obtained at an intraluminal pressure of 70 mm Hg. Myogenic tone developed at an intraluminal pressure of 30 to 40 mm Hg, continued to increase, and was maintained up to a pressure of 199 mm Hg in these arteries. Arteries dilated and constricted in response to 16 and 60 mM potassium, respectively. Endothelin-1 was the most potent and efficacious constrictor, with a biphasic concentration-response curve, followed by vasopressin, serotonin, U-46619 and phenylephrine. Carbachol was the most efficacious dilator, followed by isoprenaline. The peptide dilators calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) were potent but less efficacious than carbachol and isoprenaline. Histamine and adenosine were even less potent and less efficacious dilators. NG-nitro-L-arginine methyl ester (L-NAME) constricted and indomethacin dilated the arteries. This study provides the first direct evidence for myogenic autoregulatory properties and pharmacological heterogeneity in the rat ophthalmic artery.

Comparison of the Pharmacological Properties of Rat NaV1.8 with Rat NaV1.2a and Human NaV1.5 Voltage-Gated Sodium Channel Subtypes Using a Membrane Potential Sensitive Dye and FLIPRR

A novel, membrane potential sensitive dye and a fluorescence imaging plate reader (FLIPR) have been used to characterize the pharmacological properties of rat Na(v)1.8 voltage-gated sodium channels (VGSC) in parallel with rat Na(v)1.2a and human Na(v)1.5 VGSC subtypes, respectively. The sensitivity of recombinant Na(v)1.2a-CHO, Na(v)1.5-293-EBNA, and Na(v)1.8-F-11 cells to VGSC activators was subtype dependent. Veratridine evoked depolarization of Na(v)1.2a-CHO and Na(v)1.5-293-EBNA cells with pEC(50) values of 4.78 +/- 0.13 and 4.84 +/- 0.12, respectively (n = 3), but had negligible effect on Na(v)1.8-F-11 cells (pEC(50) < 4.5). Type I pyrethroids were without significant effect at all subtypes. In contrast, the type II pyrethroids deltamethrin and fenvalerate evoked direct depolarization of Na(v)1.8-F-11 and Na(v)1.5-293-EBNA cells. Deltamethrin potentiated the veratridine-evoked response in Na(v)1.8-F-11 cells by > or =20-fold, in contrast to a <or =3-fold potentiation of the response in Na(v)1.2a, and Na(v)1.5 cells. Tetrodotoxin (TTX) inhibited VGSC activator-evoked depolarization of Na(v)1.8-F-11 cells with a biphasic concentration-response curve. The calculated pIC(50) values were 8.05 +/- 0.25 (n = 4) and 4.32 +/- 0.21 (n = 4), corresponding to TTX inhibition of endogenous TTX-sensitive (TTX-S), and recombinant Na(v)1.8 TTX-resistant (TTX-R) VGSCs, respectively. With the exception of TTX, the potencies of a number of ion channel blockers for the Na(v)1.8, Na(v)1.2a, and Na(v)1.5 VGSC subtypes were similar. In summary, these high-throughput FLIPR assays represent a valuable tool for the determination of the relative potencies of compounds at different VGSC subtypes and may prove useful for the identification of novel subtype-selective inhibitors.

Neuromuscular effects of candoxin, a novel toxin from the venom of the Malayan krait (Bungarus candidus).

1 Candoxin (MW 7334.6), a novel toxin isolated from the venom of the Malayan krait Bungarus candidus, belongs to the poorly characterized subfamily of nonconventional three-finger toxins present in Elapid venoms. The current study details the pharmacological effects of candoxin at the neuromuscular junction. 2 Candoxin produces a novel pattern of neuromuscular blockade in isolated nerve-muscle preparations and the tibialis anterior muscle of anaesthetized rats. In contrast to the virtually irreversible postsynaptic neuromuscular blockade produced by curaremimetic alpha-neurotoxins, the neuromuscular blockade produced by candoxin was rapidly and completely reversed by washing or by the addition of the anticholinesterase neostigmine. 3 Candoxin also produced significant train-of-four fade during the onset of and recovery from neuromuscular blockade, both, in vitro and in vivo. The fade phenomenon has been attributed to a blockade of putative presynaptic nicotinic acetylcholine receptors (nAChRs) that mediate a positive feedback mechanism and maintain adequate transmitter release during rapid repetitive stimulation. In this respect, candoxin closely resembles the neuromuscular blocking effects of d-tubocurarine, and differs markedly from curaremimetic alpha-neurotoxins that produce little or no fade. 4 Electrophysiological experiments confirmed that candoxin produced a readily reversible blockade (IC(50) approximately 10 nM) of oocyte-expressed muscle (alphabetagammadelta) nAChRs. Like alpha-conotoxin MI, well known for its preferential binding to the alpha/delta interface of the muscle (alphabetagammadelta) nAChR, candoxin also demonstrated a biphasic concentration-response inhibition curve with a high- (IC(50) approximately 2.2 nM) and a low- (IC(50) approximately 98 nM) affinity component, suggesting that it may exhibit differential affinities for the two binding sites on the muscle (alphabetagammadelta) receptor. In contrast, curaremimetic alpha-neurotoxins have been reported to antagonize both binding sites with equal affinity.

Structural Requirements of Alkanol Interaction Sites on Human α2β4 Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes: Effects of Linear and Branched‐Chain Alkanols

Nicotine and alcohol are both drugs of abuse that are frequently used concurrently, but little is known about the interactions between these agents at the molecular level. The neuronal subgroup of nicotinic acetylcholine receptors (nAChRs) are members of a neurotransmitter-gated ion channel superfamily and have been used extensively to characterize the interactions of ethanol with these channels. Previous work has shown that both the direction and the magnitude of the modulatory effects of n-alcohols and several fluorinated analogs on nAChRs can be predicted based upon the molecular volume of the alkanol. However, size (molecular volume) is only one of several determinants of binding-site interactions; shape and flexibility of the interacting ligand and/or the site itself (i.e., structural constraints) and alkanol hydrophobicity are also likely to be involved. Two-electrode voltage-clamp electrophysiology was used to investigate the effects of alkanols on the alpha2beta4 human neuronal nicotinic acetylcholine receptor ectopically expressed in Xenopus oocytes. ACh-induced currents mediated by alpha2beta4 nAChRs were potentiated by 1- and 2-propanol, whereas 1-pentanol and several branched-chain pentanol isomers only inhibited channel activity. 1-Butanol had a biphasic concentration-response curve, producing a nearly 2-fold increase in current at lower concentrations and >50% inhibition at higher concentrations. Other four-carbon alcohols such as 2-butanol and 2-methyl-1-propanol also facilitated responses at lower concentrations and inhibited at higher concentrations, whereas 2-butene-1-ol and 2-methyl-2-propanol only inhibited responses. The effects of these alkanols on ACh responses were the result of changes in both the potency as well as the efficacy of ACh at these receptors. Although molecular volume and hydrophobicity both correlate well with the potencies of linear alkanols in modulating the activity of alpha2beta4 nAChRs, these relationships break down almost completely when comparing the structural isomers of these agents. The structure of the interacting alkanol significantly affects the potency and efficacy of alkanols at the nAChR, and this occurs independently of differences in molecular volume and hydrophobicity.

Role of G-protein availability in differential signaling by alpha 2-adrenoceptors

The impact of G-protein expression on the coupling specificity of the human α2B-adrenergic receptor (α2B-AR) was studied in Sf9 cells. The α2B-AR was shown to activate both coexpressed Gs- and Gi-proteins in a [35S]GTPγS binding assay. Noradrenaline and the synthetic agonist UK14,304 were equally potent and efficacious in stimulating Gi activation. At the effector level (adenylyl cyclase), both ligands stimulated cAMP production. In the presence of forskolin, the effects of the agonists were more complex. Noradrenaline stimulated cAMP production, while UK14,304 showed a biphasic concentration-response curve with inhibition of stimulated cAMP production at low agonist concentrations and further stimulation at high agonist concentrations. Gs coexpression caused a monophasic stimulatory response with both ligands. Coexpression with Gi resulted in a biphasic concentration-response curve for noradrenaline and a monophasic inhibition with UK14,304. Experiments with a panel of agonists demonstrated that the more efficacious an agonist is in stimulating cAMP production, the weaker is its ability to couple to inhibition of cAMP accumulation via exogenous Gi. To be able to explain the mechanistic consequences of dual G-protein coupling described above, we developed a mathematical model based on the hypothesis that an agonist induces different conformations of the receptor having different affinity for different G-proteins. The model reproduced the profiles seen in the concentration-response curves with Gs and Gi coexpression. The model predicts that the affinity of the receptor conformation for G-proteins as well as the availability of G-proteins will determine the ultimate response of the receptor.

Dual effects of muscarinic M(2) acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists.

1. Muscarinic M(2) receptors normally inhibit the production of cyclic AMP via G(i) proteins, but a stimulatory component occurs in their effect at high agonist concentrations, believed to be based on the activation of G(s) proteins. We investigated the conditions which determine the occurrence and extent of the stimulatory component in CHO cells stably expressing muscarinic M(2) receptors. 2. Biphasic concentration-response curves (decline followed by return towards control values) were obtained after 10 min incubation with carbachol, oxotremorine-M, acetylcholine, arecoline and arecaidine propargyl ester, but the upward phase was missing with oxotremorine, methylfurmethide, furmethide and pentylthio-TZTP. Shortening the incubation favoured the occurrence of the stimulatory component. Carbachol (1 mM) and oxotremorine-M (1 mM) brought about net stimulation (above 100% of control) of cyclic AMP synthesis during 2 min incubations. The stimulatory components disappeared after the density of receptors had been lowered with oxyphenonium mustard. 3. All agonists stimulated the synthesis of cyclic AMP in cells pretreated with pertussis toxin. 4. Most differences between agonists regarding the stimulatory component of their effect on cyclic AMP synthesis could be explained by differences in their efficacy and the induced receptor internalization. 5. We propose that the G(s)-mediated stimulatory component of the effect of muscarinic M(2) receptors on cyclic AMP synthesis only occurs if the density of activated receptors is high enough to saturate the G(i) proteins and proportionate to the receptors' low affinity for the G(s) proteins. It tends to be abolished by receptor internalization.

Differential effects of carvedilol on norepinephrine release in normoxic and ischemic heart.

Carvedilol is a beta-adrenoceptor antagonist with multiple actions, which may contribute to superior cardioprotection in heart failure and myocardial infarction. We hypothesized that carvedilol may modulate presynaptic norepinephrine release in the heart. Therefore, we compared the effects of carvedilol (racemate and both enantiomers) and beta1-selective as well as nonselective beta-adrenoceptor blockers on norepinephrine release in isolated perfused rat hearts under normoxic and brief ischemic conditions. Exocytotic release of endogenous norepinephrine was induced by paired electric field stimulations to compare the release before (S1) and after (S2) beta-adrenoceptor blocker application. Metoprolol, bisoprolol, and pindolol (0.1-10 microM) had essentially no effect on exocytotic norepinephrine release under normoxic and ischemic conditions. In contrast, carvedilol exerted a biphasic concentration-response curve (increase followed by suppression) on norepinephrine release. The increase in norepinephrine release was more pronounced with R-carvedilol than with S-carvedilol, indicating an effect independent from beta-receptor antagonism. During ischemia, the facilitatory effect of carvedilol on norepinephrine release was lost, resulting in a concentration-dependent suppression of the release. These results indicate that carvedilol in contrast to classic beta1-selective and -nonselective beta-adrenoceptor blockers has pronounced effects on cardiac norepinephrine release with a remarkable difference between normoxic and ischemic conditions. Whereas a facilitation of norepinephrine release prevailed in normoxia, we observed a suppression of the release in ischemia. It remains to be established whether this unique action of carvedilol on cardiac sympathetic neurotransmission is of clinical relevance.