Bell-shaped Dose-response Curve Research Articles

A Mathematical Model of the Effect of Immunogenicity on Therapeutic Protein Pharmacokinetics

A mathematical pharmacokinetic/anti-drug-antibody (PK/ADA) model was constructed for quantitatively assessing immunogenicity for therapeutic proteins. The model is inspired by traditional pharmacokinetic/pharmacodynamic (PK/PD) models, and is based on the observed impact of ADA on protein drug clearance. The hypothesis for this work is that altered drug PK contains information about the extent and timing of ADA generation. By fitting drug PK profiles while accounting for ADA-mediated drug clearance, the model provides an approach to characterize ADA generation during the study, including the maximum ADA response, sensitivity of ADA response to drug dose level, affinity maturation rate, time lag to observe an ADA response, and the elimination rate for ADA-drug complex. The model also provides a mean to estimate putative concentration-time profiles for ADA, ADA-drug complex, and ADA binding affinity-time profile. When simulating ADA responses to various drug dose levels, bell-shaped dose-response curves were generated. The model contains simultaneous quantitative modeling and provides estimation of the characteristics of therapeutic protein drug PK and ADA responses in vivo. With further experimental validation, the model may be applied to the simulation of ADA response to therapeutic protein drugs in silico, or be applied in subsequent PK/PD models.

Optimisation and Use of Humanised RBL NF-AT-GFP and NF-AT-DsRed Reporter Cell Lines Suitable for High-Throughput Scale Detection of Allergic Sensitisation in Array Format and Identification of the ECM–Integrin Interaction as Critical Factor

We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 μg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose–response curve, and correlated (R 2 = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format.

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine‐binding site of N‐methyl‐D‐aspartate receptor

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.

Buprenorphine

Buprenorphine

Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin

The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml−1, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.

The Influence of IgE on Cultured Human Mast Cells

PurposeThe mast cell plays a pivotal role in the human immune response. Crosslinking of 2 IgE molecules bound to the high affinity IgE receptor (FcεRI) on the surface of the mast cell results in mast cell degranulation and the release of several proinflammatory mediators. Patients with type-I allergy have increased levels of IgE in the blood compared to healthy individuals.MethodsIn a 6-week culture system of stem cells to human mast cells we investigated the effect of the concentration of IgE. The mast cells were cultured with different concentrations of IgE for the last 10 days of the maturation period. It was observed how the IgE concentration affects the histamine release, FcεRI density on the mast cell surface and the concentration of other mediators.ResultsA clear correlation between IgE concentration in culture medium and the release of histamine upon activation was observed. It showed a bell-shaped dose response curve, with maximal response around an IgE-concentration of 250 ng/mL. Furthermore, the sensitivity of the mast cells and surface density of FcεRI on mast cell surface was also influenced by the IgE concentration in the culture medium.ConclusionsIgE in the culture medium during the last 10 days of mast cell maturation influences the release of the preformed mediator histamine after mast cell activation and the density of FcεRI on the mast cell surface. The release of the de novo synthetized mediator prostaglandin D2 and the expression of chymase and tryptase are not influenced by IgE in culture medium.

Agonism and Antagonism at the Insulin Receptor

Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed.

Donepezil in low micromolar concentrations modulates voltage-gated potassium currents in pyramidal neurons of rat hippocampus

Donepezil is a cholinesterase inhibitor widely used for the treatment of Alzheimer’s disease. Voltage-gated K+-channels are discussed as possible targets for the drug, but the results obtained by different authors are contradictory. In the present study performed on pyramidal cells isolated from rat’s hippocampus, we investigated the effect of donepezil on delayed rectifier K+-current (IK(DR)) and transient outward K+-current (IK(A)) using patch-clamp technique. The inhibitory effect of donepezil on IK(DR) was found in all the cells tested, but its strength varied in different cells. Two groups of neurons were differing in their sensitivity to donepezil: more sensitive (IC50=8.9μM) and less sensitive (IC50=114.9μM). The effect of the drug on IK(DR) was rapid, reversible and voltage-dependent, increasing with depolarization. Donepezil modulated IK(A) in two different ways: in some cells it suppressed the current with the IC50 value of 23.4μM, while in other cells it augmented the current with a bell-shaped dose–response curve. Maximal (about twofold) enhancement of IK(A) amplitude was caused by 10μM donepezil. Augmentation of IK(A) increased with membrane depolarization. Our results show for the first time that voltage-dependent potassium channels in mammals’ neurons are effectively modulated by low micromolar concentrations of donepezil.

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders

Cannabidiol (CBD) is a major phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ(9)-tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1A-mediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamide-mediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed with CBD. Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARγ receptors agonism, intracellular (Ca(2+)) increase, etc.), on CBD behavioural effects.

Autoinactivation of the Stargazin–AMPA Receptor Complex: Subunit-Dependency and Independence from Physical Dissociation

Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This “autoinactivation” has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1–A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

Local overexpression of GH and GH/IGF1 effects in the adult mouse hippocampus

GH therapy improves hippocampal functions mainly via circulating IGF1. However, the roles of local GH and IGF1 expression are not well understood. We investigated whether transgenic (TG) overexpression in the adult brain of bovine GH (bGH) under the control of the glial fibrillary acidic protein (GFAP) promoter affected cellular proliferation and the expression of transcripts known to be induced by systemic GH in the hippocampus. Cellular proliferation was examined by 5-bromo-2'-deoxyuridine immunohistochemistry. Quantitative PCR and western blots were performed. Although robustly expressed, bGH-Tg did not increase either cell proliferation or survival. However, bGH-Tg modestly increased Igf1 and Gfap mRNAs, whereas other GH-associated transcripts were unaffected, i.e. the GH receptor (Ghr), IGF1 receptor (Igf1r), 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp), ionotropic glutamate receptor 2a (Nr2a (Grin2a)), opioid receptor delta (Dor), synapse-associated protein 90/postsynaptic density-95-associated protein (Sapap2 (Dlgap2)), haemoglobin beta (Hbb) and glutamine synthetase (Gs (Glul)). However, IGF1R was correlated with the expression of Dor, Nr2a, Sapap2, Gs and Gfap. In summary, although local bGH expression was robust, it activated local IGF1 very modestly, which is probably the reason for the low response of previous GH-associated response parameters. This would, in turn, indicate that hippocampal GH is less important than endocrine GH. However, as most transcripts were correlated with the expression of IGF1R, there is still a possibility for endogenous circulating or local GH to act via IGF1R signalling. Possible reasons for the relative bio-inactivity of bGH include the bell-shaped dose-response curve and cell-specific expression of bGH.

Synthetic Allergen Design Reveals the Significance of Moderate Affinity Epitopes in Mast Cell Degranulation

This study describes the design of a well-defined homotetravalent synthetic allergen (HTA) system to investigate the effect of hapten-IgE interactions on mast cell degranulation. A library of DNP variants with varying affinities for IgE(DNP) was generated (K(d) from 8.1 nM to 9.2 μM), and 8 HTAs spanning this range were synthesized via conjugation of each DNP variant to the tetravalent scaffold. HTAs with hapten K(d) < 235 nM stimulated degranulation following a bell-shaped dose response curve with maximum response occurring near the hapten K(d). HTAs with hapten K(d) ≥ 235 nM failed to stimulate degranulation. To mimic physiological conditions, the percent of allergen specific IgE on cell surface was varied, and maximum degranulation occurred at 25% IgE(DNP). These results demonstrated that moderate hapten-IgE affinities are sufficient to trigger mast cell degranulation. Moreover, this study established the HTA design as a well-defined, controllable, and physiologically relevant experimental system to elucidate the mast cell degranulation mechanism.

P2‐084: T‐type voltage‐gated calcium channel activated by ST101 is a novel molecular target as cognitive enhancer

We have developed a novel cognitive enhancer, ST101 (spiro[imidazo[1,2- a]pyridine-3,2-indan]-2(3 H)-one; previously coded as ZSET1446, Sonexa Therapeutics, Inc. San Diego, CA). We previously reported that ST101 improves the impairment in hippocampal long-term potentiation (LTP) via activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) in olfactory bulbectomized (OBX) mice (Han et al., 2008). As yet, the mechanisms underlying the activation of memory-related protein kinases are not known. Rat somatosensory cortical and hippocampal slices were incubated with 0.01 to 100 nM ST101 in the perfusion chamber with or without voltage-gated calcium channel (VGCC) inhibitors and performed immunoblotting and electrophysiological analyses. Treatment of the cortical slices with a range of ST101 concentrations significantly increased CaMKII autophosphorylation with a bell-shaped dose-response curve, with the maximal effective concentration at 0.1 nM ST101. The enhanced CaMKII autophosphorylation with 0.1 nM ST101 treatment was significantly inhibited by pretreatment with 1 m M mibefradil, a T-type VGCC inhibitor, but not with N-type (w -conotoxin), P/Q-type (w -agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated LTP, which was inhibited by 1 m M mibefadil treatment in cortical slices. Finally, whole-cell patch-clamp analysis of Neuro2A cells overexpressing recombinant human Ca V 3.1 (a 1G) T-channels confirmed that T-type VGCC currents are stimulated with 0.1 nM ST101 (1). These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutics.(1) Moriguchi et al. Journal of Neurochemistry (2012) The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: Enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices.(doi: 10.1111/j.1471-4159.2012.07667.x.)

Genotoxicity and cellular uptake of cyclotides: Evidence for multiple modes of action

Cyclotides are a family of ultra-stable, head-to-tail cyclic mini-proteins from plants, with each member comprising about 30 amino acid residues. Their stability derives from the unique structural topology where the cyclic backbone and two disulfide bonds make up an embedded ring, which is knotted by a third disulfide bond. The cyclotides find potential applications in the pharmaceutical industry as stable peptide-based scaffolds for unstable drugs, and also as medicinal agents, due to the wide range of their inherent pharmacological activities. However, there is a lack of fundamental toxicological studies on this type of compound. The current study determined the possible DNA-damaging effects of three cyclotides, i.e., cycloviolacin O2, vaby D, and kalata B1, in human lymphoma cells by use of the alkaline version of the comet assay. The three cyclotides induced massive DNA fragmentation at lethal concentrations. At a sub-lethal concentration, cycloviolacin O2 and vaby D gave a bell-shaped dose–response curve for their DNA-damaging effect. Kalata B1 caused no significant DNA damage at sub-cytotoxic concentrations. Single-cell micro-autoradiography was carried out on tritium-labeled cycloviolacin O2 in order to understand the mechanism behind the dose–response curve. The results revealed that the peptide is taken up into the cell, both at cytotoxic and at low concentrations. Most biological effects of the cyclotides have been taken to follow from the disruption of cell membranes, but even if the intracellular mechanisms and targets still remain unknown, the current study has unequivocally demonstrated that cyclotides also must have other dose-dependent modes of action.

Difficulties and perspectives of immunomodulatory therapy with mistletoe lectins and standardized mistletoe extracts in evidence based medicine

Viscum album preparations are aqueous mistletoe plant extracts used in complementary and alternative medicine as immunomodulators in cancer therapy. However, evidence of immunological efficacy of mistletoe extracts (MEs) used in clinical trials is often lacking. Mechanisms involved in anti-tumor properties of ME and mistletoe lectins (MLs) modify both innate and adaptive immune systems, according to animal model experiments. In the background of these effects, a selective binding of ML on CD75 ganglioside receptors of interleukin 12 (IL-12)-producing macrophages or dendritic cells can play an important role. Immunological effects of ME correlate with their lectin activity, showing a bell-shaped dose-response curve of efficacy. Therefore, a correct determination of MLs for the standardization of commercial ME is essential. However, plant MLs exhibit heterogeneity, which most likely results from post-translational processing. In addition, amino acid analysis of ML has revealed numerous conservative substitutions along their amino acid sequence. Consequently, ML research needs new perspectives, and the advantages and disadvantages of purified and biologically better defined ML preparations are also discussed in this article.

Cannabidiol, a non-psychotropic component of cannabis, attenuates vomiting and nausea-like behaviour via indirect agonism of 5-HT(1A) somatodendritic autoreceptors in the dorsal raphe nucleus.

To evaluate the hypothesis that activation of somatodendritic 5-HT(1A) autoreceptors in the dorsal raphe nucleus (DRN) produces the anti-emetic/anti-nausea effects of cannabidiol (CBD), a primary non-psychoactive cannabinoid found in cannabis. The potential of systemic and intra-DRN administration of 5-HT(1A) receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT(1A) receptors and to modify the ability of the 5-HT(1A) agonist, 8-OH-DPAT, to stimulate [(35) S]GTPγS binding in rat brainstem membranes. CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg(-1) , but not 40 mg·kg(-1) )-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose-response curve) at enhancing the ability of 8-OH-DPAT to stimulate [(35) S]GTPγS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping. These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT(1A) autoreceptors in the DRN. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Fine-tuning of defensive behaviors in the dorsal periaqueductal gray by atypical neurotransmitters

This paper presents an up-to-date review of the evidence indicating that atypical neurotransmitters such as nitric oxide (NO) and endocannabinoids (eCBs) play an important role in the regulation of aversive responses in the periaqueductal gray (PAG). Among the results supporting this role, several studies have shown that inhibitors of neuronal NO synthase or cannabinoid type 1 (CB1) receptor agonists cause clear anxiolytic responses when injected into this region. The nitrergic and eCB systems can regulate the activity of classical neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that control PAG activity. We propose that they exert a ‘fine-tuning’ regulatory control of defensive responses in this area. This control, however, is probably complex, which may explain the usually bell-shaped dose-response curves observed with drugs that act on NO- or CB1-mediated neurotransmission. Even if the mechanisms responsible for this complex interaction are still poorly understood, they are beginning to be recognized. For example, activation of transient receptor potential vanilloid type-1 channel (TRPV1) receptors by anandamide seems to counteract the anxiolytic effects induced by CB1 receptor activation caused by this compound. Further studies, however, are needed to identify other mechanisms responsible for this fine-tuning effect.

The T‐type voltage‐gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long‐term potentiation and CaMKII autophosphorylation in rat cortical slices

In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepared rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiological analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concentration. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice preparations. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 μM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole-cell patch-clamp analysis of Neuro2A cells over-expressing recombinant human Ca(V) 3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.

Bell-shaped and ultrasensitive dose-response in phosphorylation-dephosphorylation cycles: the role of kinase-phosphatase complex formation

BackgroundPhosphorylation-dephosphorylation cycles (PDCs) mediated by kinases and phosphatases are common in cellular signalling. Kinetic modelling of PDCs has shown that these systems can exhibit a variety of input-output (dose-response) behaviors including graded response, ultrasensitivity and bistability. In addition to proteins, there are a class of lipids known as phosphoinositides (PIs) that can be phosphorylated. Experimental studies have revealed the formation of an antagonistic kinase-phosphatase complex in regulation of phosphorylation of PIs. However, the functional significance of this type of complex formation is not clear.ResultsWe first revisit the basic PDC and show that partial asymptotic phosphorylation of substrate limits ultrasensitivity. Also, substrate levels are changed one can obtain non-monotonic bell-shaped dose-response curves over a narrow range of parameters. Then we extend the PDC to include kinase-phosphatase complex formation. We report the possibility of robust bell-shaped dose-response for a specific class of the model with complex formation. Also, we show that complex formation can produce ultrasensitivity outside the Goldbeter-Koshland zero-order ultrasensitivity regime through a mechanism similar to competitive inhibition between an enzyme and its inhibitor.ConclusionsWe conclude that the novel PDC module studied here exhibits new dose-response behaviour. In particular, we show that the bell-shaped response could result in transient phosphorylation of substrate. We discuss the relevance of this result in the context of experimental observations on PI regulation in endosomal trafficking.

Donepezil in a Narrow Concentration Range Augments Control and Impaired by Beta-Amyloid Peptide Hippocampal LTP in NMDAR-Independent Manner

Acetylcholinesterase (AChE) inhibitor donepezil is widely used for the treatment of Alzheimer's disease (AD). The mechanisms of therapeutic effects of the drug are not well understood. The ability of donepezil to reverse a known pathogenic effect of β-amyloid peptide (Abeta), namely, the impairment of hippocampal long-term potentiation (LTP), was not studied yet. The goal of the present study was to study the influence of donepezil in 0.1-10 μM concentrations on control and Abeta-impaired hippocampal LTP. Possible involvement of N-methyl-D: -aspartate receptors (NMDARs) into mechanisms of donepezil action was also studied. LTP of population spike (PS) was studied in the CA1 region of rat hippocampal slices. Change of LTP by donepezil treatment had a bell-shaped dose-response curve. The drug in concentrations of 0.1 and 1 μM did not change LTP while in concentration of 0.5 μM significantly increased it, and in concentration of 5 and 10 μM suppressed LTP partially or completely. Abeta (200 nM) markedly suppressed LTP. Addition of 0.1, 0.5 or 1 μM donepezil to Abeta solution caused a restoration of LTP. N-methyl-D: -aspartate (NMDA) currents were studied in acutely isolated pyramidal neurons from CA1 region of rat hippocampus. Neither Abeta, nor 0.5 μM donepezil were found to change NMDA currents, while 10 μM donepezil rapidly and reversibly depressed it. Results suggest that donepezil augments control and impaired by Abeta hippocampal LTP in NMDAR-independent manner. In general, our findings extend the understanding of mechanisms of therapeutic action of donepezil, especially at an early stage of AD, and maybe taken into account while considering the possibility of donepezil overdose.