The slope parameter of concentration-response curves used as a touchstone for the existence of spare receptors

The present work was stimulated by findings of a large reserve of presynaptic α₂-autoreceptors in rat neocortex by different investigators and our own group, using classical models of receptor agonism. The mathematical background of these classical models seems erroneous since the asymmetry that spare receptors introduce into concentration-response curves is not considered appropriately. This asymmetry leads to a steepening of curve fits based on the logistic function. Therefore, the slope parameter c of a logistically fitted concentration-response curve can be used as a touchstone for the existence of spare receptors. Spare receptors induce a c > 1. Concentration-response data of the α₂-autoreceptor-mediated inhibition of evoked [³H]-noradrenaline release in rat neocortex slices were re-analysed. The estimates of the slope parameter c of logistically fitted concentration-response curves obtained after treatment of rats with either vehicle or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to achieve an irreversible inactivation of α₂-autoreceptors, were not compatible with the existence of a large receptor reserve. A model for nonlinear regression analysis developed under the a priori assumption of spare receptors confirmed the absence of spare receptors. Received: 13 December 1996 / Accepted: 30 May 1997     

Mathematical modelling and quantification of the autoinhibitory feedback control of noradrenaline release in brain slices

Summary Concentration-response curves, reflecting ₂-autoreceptor-mediated inhibition of [³H]-noradrenaline release by exogenous noradrenaline in rat cerebral cortex and rabbit hippocampus slices, were analysed in order to test the usefulness of a mathematical model describing the relation between the independent variable, exogenous noradrenaline, and the dependent variable, inhibition of release. This model was based on the assumption of direct proportionality between receptor occupation and response, implying that there is correspondence between the shape of a concentration-binding curve and a concentration-response curve. The experimental concentration-response curves were obtained by different approaches: noradrenaline release from brain slices prelabelled with [³H]-noradrenaline was elicited electrically either by pseudo-one-pulse (POP) stimulation or by stimulation with 36 pulses applied with a frequency of 3 Hz. POP stimulation avoids autoinhibition by released noradrenaline and, therefore, was a suitable touchstone for the applied mathematical model which evaluates by nonlinear regression analysis two primary parameters: the dissociation constant between noradrenaline and the ₂-adrenoceptor and the biophase concentration of noradrenaline which reflects the extent of autoinhibition and should be zero under POP conditions. In rat cerebral cortex tissue, the corresponding biophase concentration of endogenous noradrenaline was indeed estimated to be zero and the dissociation constant was K _d = 10^{–7.62±0.14} mol/l. With 3 Hz stimulation, the biophase concentration was 10^{–7.80±0.05} mol/l, which has to be interpreted with respect to a simultaneously estimated K _d of 10^{–7.63±0.12} mol/l. Since the K _d-values under POP or 3 Hz conditions were similar, the biophase concentration obviously had no influence on the estimate of the other primary parameter, K _d. With rabbit hippocampus, however, the main prerequisite of the mathematical model; i.e. direct proportionality between receptor occupation and response, was not established since the slope of the POP concentration-response curve (estimated as slope parameter c) did not correspond to that of a concentration-binding curve.In conclusion, mathematical modelling by nonlinear regression analysis of the autoinhibitory circuit of noradrenaline release allows the estimation of a parameter c of this feedback regulation which supports or rejects the assumption of direct proportionality between receptor occupation and functional response. When the given requirement of direct proportionality is shown to hold, this analysis allows the feedback circuit to be described quantitatively in terms of the affinity of noradrenaline for, and of the biophase concentration of noradrenaline at, the presynaptic ₂-adrenoceptors.Correspondence to T. J. Feuerstein     

Nicotine exerts a permissive role on NMDA receptor function in hippocampal noradrenergic terminals

The coexistence of nicotinic cholinergic receptors (nAChRs) and of N-methyl-D-aspartate (NMDA) receptors on the same noradrenergic axon terminals and the nAChR/NMDA receptor cross-talk were investigated by monitoring the release of noradrenaline (NA) evoked in superfused rat hippocampal synaptosomes by (-)-nicotine and NMDA alone or in combination. In medium containing a physiological concentration (1.2 mM) of Mg2+, the release of [3H]NA was very slightly increased by NMDA plus glycine, whereas it was significantly enhanced by (-)-nicotine. The (-)-nicotine/NMDA combination elicited supraadditive release which was totally abolished by the nAChR blocker mecamylamine and partly prevented by selectively blocking NMDA receptors. Supraadditive [3H]NA release was also observed by exposing synaptosomes to veratrine, but not to ionomycin. The supraadditive release elicited by the (-)-nicotine/NMDA or the veratrine/NMDA combination was sensitive to the protein kinase A/C inhibitor staurosporine and the selective protein kinase A inhibitor H89, but insensitive to the protein kinase C inhibitor Ro 31-8220. It is concluded that (i) release-modulating nAChRs and NMDA receptors coexist on hippocampal noradrenergic axon terminals; and (ii) nicotine permits NMDA receptor activation in the presence of Mg2+, possibly because the nicotine-induced influx of Na+ depolarizes the nerve ending membrane sufficiently to remove the Mg2+ block.     

Biochemical evidence that 2-phenyl-4[(4-piperidinyl) ethyl]quinoline, a quinoline derivative with pure anticonflict properties, is a partial agonist of benzodiazepine receptors

The atypical profile of 2-phenyl-4[2-(4-piperidinyl) ethyl]quinoline (PK 8165), a quinoline derivative with pure anticonflict properties, seems to be due to the fact that this compound is a partial agonist of benzodiazepine receptors. The drug PK 8165 is a competitive inhibitor of benzodiazepine binding sites with a Hill coefficient near unity. Opposite to 3-methyl-6-(3-trifluoromethylphenyl)2,4-triazolo(4,5-b)pyridazine (CL 218,872) it was unable to discriminate between BZ1 and BZ2 receptors in sections of brain. However, modulation by gamma-aminobutyric acid (GABA) and the effect of photolabelling by flunitrazepam on the affinity of PK 8165 indicated that GABA or photolabelling shifts of PK 8165 were between full agonists and antagonists. By itself PK 8165 was unable to modify the levels of cGMP in the cerebellum, but potentiated the lowering of levels of cGMP by diazepam and did not present antagonistic properties of this effect.     

新型戒烟药瓦伦尼克林

瓦伦尼克林(varenicline)是一种α_4β_2烟碱乙酰胆碱受体选择性部分激动剂,对α_4β_2神经元烟碱乙酰胆碱受体具有高度亲和力,能够选择性与之结合,阻断烟碱激活α_4β_2受体的作用,从而阻断了反复吸烟后成瘾的中枢性机制,临床用于戒烟的治疗。现综述其作用机制、药动学、临床研究、药物相互作用及不良反应。     

Polymyxin B,a selective inhibitor of protein kinase C,diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

Summary Slices of the rabbit hippocampus were labelled with ³H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.Abbreviations PKC protein kinase C - PDB phorbol 12,13-dibutyrate - TPA 12-O-tetradecanoyl 13-acetate     

Cytisine, a partial agonist of high-affinity nicotinic acetylcholine receptors, has antidepressant-like properties in male C57BL/6J mice

The nicotine in tobacco is thought to modulate neuronal systems regulating mood. Moreover, it appears possible that blockade rather than activation of beta2-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) may lead to antidepressant-like effects. We used cytisine, a partial agonist of alpha4/beta2*nAChRs and a full agonist at alpha3/beta4*nAChRs, in several tests of antidepressant efficacy. Further, we used c-fos expression to identify potential neurobiological correlates of the antidepressant-like effects of cytisine. Cytisine had antidepressant-like effects in several animal models of antidepressant efficacy. In addition, immunohistochemical analyses indicated that cytisine could reduce c-fos immunoreactivity in the basolateral amygdala by approximately 50%. These data show that cytisine acts like classical antidepressants in rodent models of antidepressant efficacy. In addition, cytisine's ability to block alpha4/beta2*nAChRs may be responsible for its antidepressant-like properties, and these may be mediated through a reduction of neuronal activity in the basolateral amygdala. These studies also suggest that both antagonists and partial agonists of alpha4/beta2*nAChRs would be interesting targets for the development of novel antidepressant drugs.     

Rolofylline,an adenosine A1 receptor antagonist,inhibits osteoclast differentiation as an inverse agonist

BACKGROUND AND PURPOSE

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A₁ receptors (A₁R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A₁R agonists neither affect basal osteoclastogenesis nor do they reverse A₁R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A₁R antagonist-mediated inhibition, and, when we saw no effect, determined whether A₁R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.

EXPERIMENTAL APPROACH

Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand–macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.

KEY RESULTS

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC₅₀ = 20–70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.

CONCLUSIONS AND IMPLICATIONS

Based on these findings, we hypothesize that the A₁R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A₁R antagonists act as inverse agonists to release A₁R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A₁R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.     

Syntheses of dual‐radioisotope‐labeled CP‐I,a GABAA receptor partial agonist

CP‐I is a potent subtype‐selective GABA _A receptor partial agonist. Owing to its significant metabolic cleavage at C₈ observed in preliminary biotransformation studies with non‐radiolabeled CP‐I, the syntheses of CP‐I labeled at the right or left hand side with ¹⁴C or labeled with 3H at the right hand side were required. The two compounds labeled with ¹⁴C at the left or right hand side were synthesized in 2 and 5 radio‐synthetic steps using [¹⁴C]2‐chloroacetyl chloride and [¹⁴C]NaCN as starting radiolabeled materials, respectively. CP‐I was labeled with tritium at the right hand side by a tritium de‐halogenation method. Batches of radiolabeled CP‐I were mixed to give dual‐radioisotope‐labeled CP‐I. An efficient approach to [¹⁴C]fluoropyridinyl imidazole was developed, and a short synthesis of iodo‐substituted fluoropyridinyl imidazole was also achieved. The details of these syntheses are discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Maternal deprivation and handling modify the effect of the dopamine D3 receptor agonist,BP 897 on morphine-conditioned place preference in rats

Rationale Maternal deprivation and handling can lead to a vulnerability to opiate dependence. However, the involvement of the dopamine D3 receptors has not been investigated. Objectives This study analysed the effects of a selective partial D3 receptor agonist, BP 897, on morphine-conditioned place preference (CPP) in deprived and handled rats. Materials and methods The effects of BP 897 were studied on the expression and the extinction of morphine CPP. Quantitative autoradiography of D2, D3 receptors and immunoautoradiography of dopamine transporter were performed in some saline- and morphine-treated rats 24 h after the place preference test. Results Morphine (5 mg/kg) induced a more prolonged morphine CPP in deprived and handled rats than in control animals. BP 897 (0.5 or 2 mg/kg) enhanced the expression of morphine conditioning in control rats. Same doses did not change morphine conditioning in deprived rats. BP 897 (2 mg/kg) suppressed morphine CPP in handled rats. An increase in basal D2 receptor density in the mesencephalon of handled rats, which was suppressed after morphine CPP, was observed. A decrease in D2 receptor levels in morphine-treated deprived rats occurred in the nucleus accumbens. Conclusions This study shows that maternal deprivation and handling induced a prolonged morphine CPP, and different changes of D2/D3 receptor functioning revealed after morphine CPP. Early manipulations of infant–mother relationships may have different consequences on the balance of opioidergic and dopaminergic neurotransmission and may be of interest to reveal pharmacological properties of dopamine receptor partial agonists or antagonists potentially useful for therapeutic applications.     

The partial dopamine D2-like receptor agonist terguride functions as an agonist in preweanling rats after a 5-day reserpine regimen

Rationale Treating children and adolescents with partial D₂-like agonists is becoming increasingly common, although few developmental animal studies have assessed the psychopharmacology of this class of drug. Contrary to results from adult rat studies, it has been reported that partial D₂-like agonists may not induce agonist-like behavioral effects in preweanling rats during states of low dopaminergic tone. Objective The purpose of the present study was to determine whether a partial D₂-like agonist would act as an agonist in preweanling rats after a 5-day regimen of the dopamine-depleting agent reserpine or the tyrosine hydroxylase inhibitor α-methyl-dl-p-tyrosine (AMPT). Methods: Sprague–Dawley rats were pretreated with reserpine (1 mg kg^-1 per day) or AMPT (3×200 mg kg^-1 per day) on postnatal day (PD) 16–PD 20. Either 2 h (AMPT) or 5 h (reserpine) after the last pretreatment injection, rats were treated with saline, the partial D₂-like agonist terguride, or the full D₂-like agonist R(−)-propylnorapomorphine (NPA). Distance traveled and repetitive motor movements were measured for 60 min. Results After repeated reserpine treatment, both terguride and NPA increased the distance-traveled scores of preweanling rats; however, only NPA, but not terguride, increased distance-traveled scores after a 5-day regimen of AMPT or an acute injection of reserpine. Conclusions It is now apparent that partial D₂-like agonists are capable of inducing agonist-like behavioral effects in preweanling rats during a state of low dopaminergic tone. For agonistic actions to be observed, the pretreatment regimen must result in substantial and prolonged dopamine depletion.     

The profile of sabcomeline (SB-202026), a functionally selective M1 receptor partial agonist,in the marmoset

1. Sabcomeline (SB-202026, 0.03 mg kg⁻¹, p.o.), a potent and functionally selective M₁ receptor partial agonist, caused a statistically significant improvement in the performance of a visual object discrimination task by marmosets. No such improvement was seen after RS86 (0.1 mg kg⁻¹, p.o.).
2. Initial learning, which only required an association of object with reward and an appropriate response to be made, was not significantly affected. Reversal learning, which required both the extinction of the previously learned response and the acquisition of a new response strategy, was significantly improved after administration of sabcomeline (0.03 mg kg⁻¹, p.o.).
3. Sabcomeline (0.03 and 0.1 mg kg⁻¹, p.o.) had no significant effect on mean blood pressure measured for 2 h after administration in the conscious marmoset.
4. Sabcomeline (0.03 mg kg⁻¹, p.o.) caused none of the overt effects such as emesis or behaviours often seen after the administration of muscarinic agonists, e.g. face rubbing and licking.
5. This is the first study to demonstrate cognitive enhancement by a functionally selective M₁ receptor partial agonist in a normal (i.e. non-cognitively impaired) non-human primate and this effect was seen at a dose which did not cause side effects.
6. Perseverative behaviour and deficient acquisition of new information are seen in patients with Alzheimer''s disease (AD). Therefore the data suggest that sabcomeline might be of therapeutic benefit in the treatment of AD.

     

Adenosine but not an adenine nucleotide mediates tonic purinergic inhibition,as well as inhibition by glutamate,of noradrenaline release in rabbit brain cortex slices

Summary A possible contribution of adenine nucleotides to the endogenous purinergic, A₁-receptor-mediated inhibition of noradrenaline release was studied in rabbit occipito-parietal cortex slices. The slices were preincubated with [³H]-noradrenaline and then superfused and stimulated electrically, in most experiments by trains of 6 pulses/100 Hz. A few experiments were carried out in rat occipito-parietal cortex slices. The A₁-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1–100 nmol/l) as well as the enzyme adenosine deaminase (0.1–10 U/ml) increased the electrically evoked overflow of tritiated compounds. The maximal increase was by about 85% for both DPCPX and adenosine deaminase. The increases obtained with maximally effective concentrations of DPCPX and adenosine deaminase were not additive. The ₁-adrenoceptor-selective agonist methoxamine (10 but not 1 mol/l) reduced the evoked overflow. Its effect was antagonized by yohimbine 1 mol/l but then not attenuated further by DPCPX100 nmol/l.L-Glutamate (300 mol/l–2.3 mmol/l) also reduced the evoked overflow of tritium. Its effect was not changed by yohimbine 1 mol/l but greatly, and to the same extent, attenuated by DPCPX 100 mol/l and adenosine deaminase 3 U/ml. Neither the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine nor omission of Mg⁺⁺ changed the inhibition by glutamate. Glutamate did not alter the basal efflux of tritium from rabbit cortex slices under any experimental condition. In contrast, glutamate (100 mol/l and 1 mol/l) caused an immediate, marked and transient acceleration of tritium outflow from rat occipitoparietal cortex slices (medium without Mg⁺⁺). It is concluded that adenosine but not an adenine nucleotide mediates the tonic purinergic presynaptic inhibition of noradrenaline release in rabbit brain cortex. The marked degree of disinhibition by DPCPX and adenosine deaminase underscores the potential physiological role of this inhibition. The purinergic inhibitory tone is reinforced by glutamate, indicating that glutamate releases adenyl compounds in rabbit brain cortex. Again adenosine but not an adenine nucleotide mediates the indirect inhibition by glutamate of the release of noradrenaline. The noradrenaline-releasing effect that glutamate exerts in rat occipito-parietal cortex does not occur in rabbit occipito-parietal cortex. Methoxamine depresses the release of noradrenaline in rabbit brain cortex directly at presynaptic ₂-adrenoceptors rather than by release of purines.Correspondence to I. von Kügelgen at the above address     

Selective enhancement by an adenosine A1 receptor agonist of agents inducing contraction of the rat vas deferens

The adenosine analogue N⁶-cyclopentyladenosine (CPA), acting via postjunctional A₁ receptors, has been shown to enhance contractions of the rat vas deferens induced by adenosine 5-triphosphate (ATP), the sympathetic cotransmitter in this tissue. The aim of the present study was to examine the ability of CPA to enhance contractions induced by other contractile agents. CPA (0.01–0.3 M) enhanced contractions induced by exogenous ATP (10 M), 5-hydroxytryptamine (5-HT) (3 M), tyramine (10 M), 2-methyl-5-hydroxytryptamine (2-Me-5-HT) (10 M) and KCl (35 mM) and this enhancement was blocked by an A₁-selective concentration (3 nM) of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). CPA failed to enhance contractions induced by exogenous noradrenaline (NA) (1 M or 10 M), bradykinin (0.1 M), phenylephrine (3 M) or carbachol (10 M).The contractions induced by ATP (10 M), 5-HT (3 M), 2-Me-5-HT (10 M) and KCl (35 mM) were unaffected by tetrodotoxin (1 M) as well as by desensitisation of the P_2x-purinoceptors with the ATP analogue adenosine 5-(, -methylene) triphosphonate. The contractions induced by tyramine (10 M) and 2-Me-5-HT (10 M) were blocked by prazosin (100 nM) or by imipramine (1 M). Ketanserin (10 nM) antagonised the response to 5-HT giving a dose-ratio of 12.9 corresponding to an apparent pA₂ of 9.1.In conclusion, the A₁-mediated effect was clearly selective for certain contractile agents and not due to a non-specific increase in contractility of the tissue. CPA enhanced contractions induced by both ATP and indirect sympathomimetics which release endogenous NA, and this enhancement of the two sympathetic cotransmitters may have a functional significance, and demonstrates the complexity of the neuromodulatory effects of adenosine in the rat vas deferens.     

L-687,414, a low efficacy NMDA receptor glycine site partial agonist in vitro,does not prevent hippocampal LTP in vivo at plasma levels known to be neuroprotective

1. N-methyl-D-aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long-term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK-801 and glycine-site antagonism by 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414).
2. In vitro experiments using rat cortical slices revealed L-687,414 to be ∼3.6 fold more potent than its parent analogue, R(+)HA-966 at antagonizing NMDA-evoked population depolarizations (apparent K_bs: 15 μM and 55 μM, respectively).
3. Whole-cell voltage-clamp experiments using rat cultured cortical neurones revealed L-687,414 to shift to the right the concentration-response relationship for NMDA-evoked inward current responses (pK_b=6.2±0.12). L-687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration-inhibition curves, pKi=6.1±0.09. In the latter experiments, L-687,414 and R(+)HA-966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity=∼10 and 20% of glycine, respectively).
4. L-687,414 and MK-801 were compared for their effects on NMDA receptor-dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg⁻¹ followed by 0.0298 ml min⁻¹), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK-801 dosing regimen (0.12 mg kg⁻¹ i.v. followed by 1.8 μg kg⁻¹ h⁻¹) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L-687,414 treatment (28 mg kg⁻¹ i.v. followed by 28 mg kg⁻¹ h⁻¹).
5. These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor-dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L-687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of `normal'' synaptic transmission whilst curtailing excessive receptor activation.

     

ATP,a partial agonist for the P2Z receptor of human lymphocytes

1. Although extracellular adenosine 5′-triphosphate (ATP) is the natural ligand for the P2Z receptor of human lymphocytes it is less potent than 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion channel, which conducts a range of permeants including Ba²⁺ and ethidium⁺. We have quantified the influx of ethidium⁺ into lymphocytes produced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATPγS, studied competition between ATP and BzATP and investigated the effects of KN-62, a new and potent inhibitor of the P2Z receptor.
2. BzATP and ATP stimulated ethidium⁺ influx with EC₅₀ values of 15.4±1.4 μM (n=5) and 85.6±8.8 μM (n=5), respectively. The maximal response to ATP was only 69.8±1.9% of that for BzATP. Hill analysis gave n_H of 3.17±0.24 (n=3) and 2.09±0.45 (n=4) for BzATP and ATP, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z receptor-operated ion channel.
3. A rank order of agonist potency of BzATP>ATP=2MeSATP>ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP>ATP>2MeSATP>ATPγS.
4. Preincubation with 30–50 μM oxidized ATP (ox-ATP), an irreversible P2Z inhibitor, reduced the maximal response but did not change the steepness of the Ba²⁺ influx-response curve produced by BzATP (n_H 3.2 and 2.9 for 30 and 50 μM ox-ATP, respectively (n=2)).
5. ATP (300–1000 μM) added simultaneously with 30 μM BzATP (EC₉₀) inhibited both ethidium⁺ and Ba²⁺ fluxes to a maximum of 30–40% relative to the values observed with BzATP alone. Moreover, ATP (300 μM) shifted the concentration-response curve to the right for BzATP-stimulated Ba²⁺ influx, confirming competition between ATP and BzATP.
6. KN-62, a new and powerful inhibitor of the lymphocyte P2Z receptor, showed less potency in antagonizing BzATP-mediated fluxes than ATP-induced fluxes when maximal concentrations of both agonists (BzATP, 50 μM; ATP, 500 μM) were used.
7. These data suggest that the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a more efficacious agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes.

     

Pharmacokinetics of cytisine,an α4β2 nicotinic receptor partial agonist,in healthy smokers following a single dose

Cytisine, an α₄β₂ nicotinic receptor partial agonist, is a plant alkaloid that is commercially extracted for use as a smoking cessation medication. Despite its long history of use, there is very little understanding of the pharmacokinetics of cytisine. To date, no previous studies have reported cytisine concentrations in humans following its use as a smoking cessation agent. A high performance liquid chromatography‐ultraviolet (HPLC‐UV) method was developed and validated for analysis of Tabex® and nicotine‐free oral strips, two commercial products containing cytisine. A sensitive liquid chromatography‐mass spectrometry (LC‐MS) method was developed and validated for the quantification of cytisine in human plasma and for the detection of cytisine in urine. Single‐dose pharmacokinetics of cytisine was studied in healthy smokers. Subjects received a single 3 mg oral dose administration of cytisine. Cytisine was detected in all plasma samples collected after administration, including 15 min post‐dose and at 24 h. Cytisine was renally excreted and detected as an unchanged drug. No metabolites were detected in plasma or urine collected in the study. No adverse reactions were reported. Copyright © 2014 John Wiley & Sons, Ltd.     

Pharmacological profile of the alpha4beta2 nicotinic acetylcholine receptor partial agonist varenicline, an effective smoking cessation aid

The preclinical pharmacology of the alpha4beta2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline, a novel smoking cessation agent is described. Varenicline binds with subnanomolar affinity only to alpha4beta2 nAChRs and in vitro functional patch clamp studies in HEK cells expressing nAChRs show that varenicline is a partial agonist with 45% of nicotine's maximal efficacy at alpha4beta2 nAChRs. In neurochemical models varenicline has significantly lower (40-60%) efficacy than nicotine in stimulating [(3)H]-dopamine release from rat brain slices in vitro and in increasing dopamine release from rat nucleus accumbens in vivo, while it is more potent than nicotine. In addition, when combined with nicotine, varenicline effectively attenuates the nicotine-induced dopamine release to the level of the effect of varenicline alone, consistent with partial agonism. Finally, varenicline reduces nicotine self-administration in rats and supports lower self-administration break points than nicotine. These data suggest that varenicline can reproduce to some extent the subjective effects of smoking by partially activating alpha4beta2 nAChRs, while preventing full activation of these receptors by nicotine. Based on these findings, varenicline was advanced into clinical development and recently shown to be an effective and safe aid for smoking cessation treatment.     

Three-dimensional structure of the Y1 receptor agonist [Leu31, Pro34]NPY as determined by NMR and molecular modeling

The solution structure of the Y1 receptor agonist, porcine [Leu³¹, Pro³⁴]NPY, has been investigated by two-dimensional NMR and molecular modeling. A complete assignment of the NMR resonances was achieved and 201 inter-residue nuclear Overhauser enhancement spectroscopy (NOESY) connectivities could be identified, comprising several connectivities between the N- and C-terminal segments. A molecular model was calculated by distance geometry, simulated annealing and conjugate gradients energy minimization using the NOE constraints. The results indicate that, like NPY and other peptides of the family, [Leu³¹, Pro³⁴]NPY adopts a folded hairpin structure with the terminal segments in close proximity. Analysis of the secondary chemical shifts for the CHα's and of the temperature dependence of the NH chemical shifts combined with the NOE constraints indicates a tendency toward helix structure for the segment 18-30 and the presence of turn structure for the C-terminal segment (residues 31-36). Native NPY and [Leu³¹, Pro³⁴]NPY have most of their structures in common but differ slightly in their C-terminal portion. Based on the structures of NPY and of its specific agonists, [Leu³¹, Pro³⁴]NPY and NPY 13-36, conclusions can be drawn about the structural requirements for binding to the Y1 and Y2 receptor subtypes.     

Evidence for mediation of nociception by injection of the NK-3 receptor agonist,senktide, into the dorsal periaqueductal gray of rats

Rationale  Ultrasound vocalizations (USVs) at approximately 22 kHz are usual components of the defensive response of rats. However, depending on the neural substrate that is activated, such as the dorsal periaqueductal gray (dPAG), USV emissions may be reduced. Activation of neurokinin-1 (NK-1)-mediated mechanisms of the dPAG causes analgesia, reduced 22 kHz USVs, and anxiogenic-like effects in rats exposed to the elevated plus maze (EPM). Involvement of other types of neurokinin receptors in this activation has not yet been evaluated. Objectives  The present study examined whether local injections of the selective NK-3 agonist senktide (1-100 pmol/0.2 μL) into the dPAG can (1) cause anxiogenic effects in the EPM, (2) influence novelty-induced 22 kHz USVs, or (3) change nociceptive reactivity in the tail-flick test. Results  Senktide elicited a significant increase in exploratory behavior, an effect accompanied by hyperalgesia and an increase in the number of 22 kHz USVs. The nociceptive effects, increased locomotor activity, and USV emissions elicited by local injections of senktide (50 pmol/0.2 μL) were reduced by prior injections of the selective NK-3 receptor antagonist SB222200 (50 pmol/0.2 μL) into the dPAG. Conclusions  These findings show that NK-3 receptors in the dPAG mediate nociceptive responses in this area, contrasting with the known fear-related processes mediated by NK-1 receptors in the dPAG. Both hyperalgesia and fear-related processes are accompanied by emissions of 22 kHz USVs.