Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.

Corticolimbic analysis of microRNAs and protein expressions in scopolamine-induced memory loss under stress.

The aim of the present study was to assess thealterations of corticolimbic microRNAs and protein expressions in the effect of scopolamine with or without stress on passive-avoidance memory in male Wistar rats. The expressions of miR-1, miR-10 and miR-26 and also the levels of p-CREB, CREB, C-FOS and BDNF in the prefrontal cortex (PFC), the hippocampus and the amygdala were evaluated using RT-qPCR and Western blotting techniques. The data showed that the administration of a muscarinic receptor antagonist, scopolamine or the exposure to 30 min stress significantly induced memory loss. Interestingly, the injection of an ineffective dose of scopolamine (0.5 mg/kg) alongside with exposure to an ineffective time of stress (10 min) impaired memory formation, suggesting a potentiative effect of stress on scopolamine response. Our results showed that memory formation was associated with the down-regulated expression of miR-1, miR-10 and miR-26 in the PFC and the hippocampus, but not the amygdala. The relative expression increase of miR-1 and miR-10 in the PFC and the hippocampus was shown in memory loss induced by scopolamine administration or 30-min stress. The PFC level of miR-10 and also hippocampal level of miR-1 and miR-10 were significantly up-regulated, while amygdala miR-1 and miR-26 were down-regulated in scopolamine-induced memory loss under stress. Memory formation increased BDNF, C-FOS and p-CREB/CREB in the PFC, the hippocampus and the amygdala. In contrast, the PFC, hippocampal and amygdala protein expressions were significantly decreased in memory loss induced by scopolamine administration (2 mg/kg), stress exposure (for 30 min) or scopolamine (0.5 mg/kg) plus stress (10 min). One of the most significant findings to emerge from this study is that the stress exposure potentiated the amnesic effect of scopolamine may via affecting the expressions of miRs and proteins in the PFC, the hippocampus and the amygdala. It is possible to hypothesis that corticolimbic signaling pathways play a critical role in relationship between stress and Alzheimer's disease.

NSC23766, a widely used inhibitor of Rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors.

Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M2 muscarinic acetylcholine receptor (M2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly Gißγ-mediated, adenosine-induced activation of Gi protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K(+) current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca(2+) concentrations in human embryonic kidney 293 (HEK-293) cells expressing M1, M2, or M3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M2 and M3 mAChR crystal structures confirmed this interpretation.

Autoantibodies in Sjögren's syndrome patients acutely inhibit muscarinic receptor function.

OBJECTIVES: Autoantibodies from the sera of Sjögren's syndrome patients (SS IgG) have been suggested to inhibit muscarinic receptor function. However, the acute nature of such an inhibitory effect remains controversial. In this study, we investigated the acute effects of SS IgG on muscarinic receptor function in human submandibular gland (HSG) cells. METHODS: The effects of autoantibodies on muscarinic receptor function were studied using microspectrofluorimetry, whole-cell patch clamp, immunofluorescence confocal microscopy, and a co-immunoprecipitation assay. RESULTS: Carbachol (CCh) was found to consistently increase intracellular calcium concentration ([Ca(2+) ](i) ) and activate K(+) current in HSG cells. However, pretreatment of the cells with SS IgG for 5 or 30 min significantly attenuated these responses, with a substantially more prominent effect after 30 min of treatment. Like CCh, adenosine 5'-triphosphate (ATP) also increased [Ca(2+) ](i) and activated K(+) currents in HSG cells, although pretreatment with SS IgG did not affect the cellular response to ATP. CCh was found to reorganize α-fodrin in HSG cells in a Ca(2+) -dependent manner. However, pretreatment with SS IgG prevented the cytoskeletal reorganization of α-fodrin induced by CCh. CONCLUSIONS: SS IgG acutely and reversibly inhibited muscarinic receptor function, thereby inhibiting the Ca(2+) mobilization necessary for the activation of K(+) currents and α-fodrin reorganization in HSG cells.

Evaluation of drug efflux transporter liabilities of darifenacin in cell culture models of the blood-brain and blood-ocular barriers.

AIMS: The objective of the present study was to evaluate drug efflux transporter interactions of darifenacin and examine the impact of such transporter interactions on darifenacin permeability in an in vitro model of the blood-brain barrier (BBB) and blood-ocular barrier (BOB). METHODS: Cell membranes expressing human P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) were examined for ATPase activity following darifenacin exposure (0-10 µM). Primary cultured bovine brain microvessel endothelial cells (BBMEC) and P-gp transfected Manin-Darby canine kidney epithelial cells (MDCKMDR1) were used to examine darifenacin permeability and drug efflux transporter responses. RESULTS: Concentration-dependent increases in ATPase activity was observed in P-gp membranes following darifenacin exposure. Both MRP and BCRP membrane preparations were unresponsive to darifenacin. Studies in both BBMEC and MDCKMDR1 monolayers confirmed a P-gp interaction for darifenacin and significantly greater efflux (basolateral to apical) permeability for darifenacin that was reduced by the P-gp inhibitor, elacridar. CONCLUSIONS: Darifenacin is a substrate for the P-gp drug efflux transporter present in both BBB and BOB. The P-gp drug efflux transporter liabilities of darifenacin may limit its penetration into brain and ocular tissue thereby reducing side effect potential.

Promising multifunctional anti-Alzheimer's dimer bis(7)-Cognitin acting as an activator of protein kinase C regulates activities of alpha-secretase and BACE-1 concurrently.

We have recently demonstrated that bis(7)-Cognitin, a promising multifunctional anti-Alzheimer's dimer, can remarkably reduce the generation of amyloid beta peptide (Abeta) by inhibiting beta-secretase (BACE-1) and activating alpha-secretase activity. In this study, the mechanism(s) underlying bis(7)-Cognitin's regulation of the activity of these two proteases was further investigated. In N2a cells stably expressing human amyloid precursor protein with the Swedish mutation (APPswe), the reduction in Abeta production induced by 1microM bis(7)-Cognitin was not altered by the co-pretreatment of muscarinic and nicotinic cholinergic receptor antagonists, indicating that the regulation of APP processing by this dimer is independent of cholinergic transmission. Furthermore, bis(7)-Cognitin (0.1-3microM) significantly increased protein kinase C (PKC) activity in cells and in vitro in a concentration-dependent manner. Administration of a PKC activator, phorbol 12-myristate 13-acetate (PMA), concentration-dependently increased the alpha-secretase cleavage products, and reduced the BACE-1 cleavage products. In addition, the inhibition of PKC prevented PMA- or bis(7)-Cognitin-induced alterations in alpha-secretase and BACE-1 activities, eliminating reductions in Abeta production seen with PMA or the dimer. These results strongly suggest that bis(7)-Cognitin may reduce the biosynthesis of Abeta by inhibiting BACE-1 and activating alpha-secretase concurrently through the direct activation of PKC. Combined with previous findings of direct inhibition of AChE and BACE-1 by this dimer, this work indicates that strategy may have potential to provide new insights into designing novel drugs that target multiple steps of aberrant APP processing to treat Alzheimer's disease.

[Characterization of muscarinic receptors in undifferentiated thyroid cells in Fisher rats]. / Caracterización de receptores muscarínicos en células indiferenciadas tiroideas de ratas Fisher.

INTRODUCTION: The parasympathetic autonomous nervous system exerts control over thyroid function by activation of the muscarinic receptors in follicular cells. Various pharmacological and molecular subtypes of muscarinic receptors (M(1), M(2), M(3), M(4), M(5)) have been identified in central nervous system and peripheral tissues. Controversy surrounds receptor characterization in thyroid cells. MATERIALS AND METHODS: Undifferentiated Fisher rat thyroid epithelial cells (FRT) were cultured. Association and dissociation kinetics assays and antagonist competition studies of the binding of (3)H-N-methylscopolamine ((3)H-NMS) to muscarinic receptors were performed to demonstrate the presence of muscarinic receptors. RESULTS: Specific muscarinic receptors in the plasma membrane of FRT cells were observed with an equilibrium dissociation constant (K(d)) of 0.44 nmol. The order of affinities obtained fitting the data to one binding site model in competition experiments with the muscarinic receptor antagonist was: dicyclomine > hexahydrosiladifenidol (HHSD) = 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine > himbacine = 11-[[2-[(diethylamino)methyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido (414)benzodiazepine (AF-DX 116). CONCLUSIONS: The results obtained indicate the existence of specific (3)H-NMS muscarinic binding sites located in the plasma membrane of FRT cells. The results obtained in competition experiments suggest that the receptors present in FRT cells belong to the M(3) subtype.

[Adjunctive therapies to glycaemic control of type 1 diabetes mellitus]. / Adjuvantes no tratamento da hiperglicemia do diabetes melito tipo 1.

Since Diabetes Control and Complications Trial (DCCT), intensive therapy has been directed at achieving glucose and glycosylated hemoglobin (HbA1c) values as close to normal as possible regarding safety issues. However, hyperglycemia (especially postprandial hyperglycemia) and hypoglicemia continue to be problematic in the management of type 1 diabetes. The objective of associating other drugs to insulin therapy is to achieve better metabolic control lowering postprandial blood glucose levels. Adjunctive therapies can be divided in four categories based on their mechanism of action: enhancement of insulin action (e.g. the biguanides and thiazolidinediones), alteration of gastrointestinal nutrient delivery (e.g. acarbose and amylin) and other targets of action (e.g. pirenzepine, insulin-like growth factor I and glucagon-like peptide-1). Many of these agents have been found to be effective in short-term studies with decreases in HbA1c of 0.5-1%, lowering postprandial blood glucose levels and decreasing daily insulin doses.

Atropine treatment modifies LPS-induced inflammatory response and increases survival.

OBJECTIVE: To explore the effect that Atropine, a competitive antagonist for the muscarinic acetylcholine receptor (mAChR), has on the response to LPS. SUBJECTS: Eight-week-old, male, B6 mice. TREATMENT: Mice were treated with Atropine prior to, or after LPS challenge. METHODS: Survival was monitored and analyzed via Kaplan-Meier analysis using the log-rank test. The effects of atropine on the inflammatory response (TNF-alpha, IL-6 and IL-10) were monitored at various time intervals following LPS injection in mice that were treated and not treated with atropine. RESULTS: Atropine administration prior to LPS induction of the inflammatory response resulted in reduced TNF-alpha and elevated IL-10 plasma levels without affecting the production of IL-6. This reduction in TNF-alpha levels was independent of the increase in IL-10 production. Atropine pretreatment improved the rate of survival from endotoxic shock in mice. The improved survival of mice after endotoxic shock could still be observed when atropine was administered several hours after LPS injection. CONCLUSION: The administration of atropine after injury may have a beneficial clinical effect.

Adjuvantes no tratamento da hiperglicemia do diabetes melito tipo 1: [revisão] / Adjunctive therapies to glycaemic control of type 1 diabetes mellitus: [review]

Desde o Diabetes Control and Complications Trial (DCCT), a terapia insulínica intensiva tem sido direcionada para alcançar valores de glicemia e hemoglobina glicada (HbA1c) tão próximos do normal quanto a segurança permita. Entretanto, a hiperglicemia (especialmente a hiperglicemia pós-prandial) e a hipoglicemia continuam a ser um problema no manejo do diabetes tipo 1. O objetivo de associar outras drogas à terapia insulínica é diminuir a glicemia pós-prandial. A terapia adjunta pode ser dividida em três grupos, conforme seu mecanismo de ação: 1. Aumento da ação da insulina (metformina e tiazolidinedionas); 2. Alteração da liberação de nutrientes no trato gastrintestinal (acarbose e amilina); 3. Outros modos de ação [pirenzepina, fator de crescimento insulina-símile (IGF-1) e peptídeo semelhante ao glucagon 1 (GLP-1). Muitos desses agentes mostraram, em estudos de curto prazo, diminuição de 0,5 por cento a 1 por cento na HbA1c, diminuir a hiperglicemia pós-prandial e as doses diárias de insulina.

Since Diabetes Control and Complications Trial (DCCT), intensive therapy has been directed at achieving glucose and glycosylated hemoglobin (HbA1c) values as close to normal as possible regarding safety issues. However, hyperglycemia (especially postprandial hyperglycemia) and hypoglicemia continue to be problematic in the management of type 1 diabetes. The objective of associating other drugs to insulin therapy is to achieve better metabolic control lowering postprandial blood glucose levels. Adjunctive therapies can be divided in four categories based on their mechanism of action: enhancement of insulin action (e.g. the biguanides and thiazolidinediones), alteration of gastrointestinal nutrient delivery (e.g. acarbose and amylin) and other targets of action (e.g. pirenzepine, insulin-like growth factor I and glucagon-like peptide-1). Many of these agents have been found to be effective in short-term studies with decreases in HbA1c of 0.5-1 percent, lowering postprandial blood glucose levels and decreasing daily insulin doses.

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function.

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.

Disparate cholinergic currents in rat principal trigeminal sensory nucleus neurons mediated by M1 and M2 receptors: a possible mechanism for selective gating of afferent sensory neurotransmission.

Neurons situated in the principal sensory trigeminal nucleus (PSTN) convey orofacial sensory inputs to thalamic relay regions and higher brain centres, and the excitability of these ascending tract cells is modulated across sleep/wakefulness states and during pain conditions. Moreover, acetylcholine release changes profoundly across sleep/wakefulness states and ascending sensory neurotransmission is altered by cholinergic agonists. An intriguing possibility is, therefore, that cholinergic mechanisms mediate such state-dependent modulation of PSTN tract neurons. We tested the hypotheses that cholinergic agonists can modulate PSTN cell excitability and that such effects are mediated by muscarinic receptor subtypes, using patch-clamp methods in rat and mouse. In all examined cells, carbachol elicited an electrophysiological response that was independent of action potential generation as it persisted in the presence of tetrodotoxin. Responses were of three types: depolarization, hyperpolarization or a biphasic response consisting of hyperpolarization followed by depolarization. In voltage-clamp mode, carbachol evoked corresponding inward, outward or biphasic currents. Moreover, immunostaining for the vesicle-associated choline transporter showed cholinergic innervation of the PSTN. Using muscarinic receptor antagonists, we found that carbachol-elicited PSTN neuron hyperpolarization was mediated by M2 receptors and depolarization, in large part, by M1 receptors. These data suggest that acetylcholine acting on M1 and M2 receptors may contribute to selective excitability enhancement or depression in individual, rostrally projecting sensory neurons. Such selective gating effects via cholinergic input may play a functional role in modulation of ascending sensory transmission, including across behavioral states typified by distinct cholinergic tone, e.g. sleep/wakefulness arousal levels or neuropathic pain conditions.

M1 muscarinic receptors contribute to, whereas M4 receptors inhibit, dopamine D1 receptor-induced [3H]-cyclic AMP accumulation in rat striatal slices.

In rat striatal slices labelled with [(3)H]-adenine and in the presence of 1 mM 3-isobutyl-1-methylxantine (IBMX), cyclic [(3)H]-AMP ([(3)H]-cAMP) accumulation induced by the dopamine D(1) receptor agonist SKF-81297 (1 microM; 177 +/- 13% of basal) was inhibited by the general muscarinic agonist carbachol (maximum inhibition 72 +/- 3%, IC(50) 0.30 +/- 0.06 microM). The muscarinic toxin 7 (MT-7), a selective antagonist at muscarinic M(1) receptors, reduced the effect of SKF-81297 by 40+/-7% (IC(50) 251+/- 57 pM) and enhanced the inhibitory action of a submaximal (1 microM) concentration of carbachol (69 +/- 4% vs. 40 +/- 7% inhibition, IC(50) 386 +/- 105 pM). The toxin MT-1, agonist at M(1) receptors, stimulated [(3)H]-cAMP accumulation in a modest but significant manner (137 +/- 11% of basal at 400 nM), an action additive to that of D(1) receptor activation and blocked by MT-7 (10 nM). The effects of MT-7 on D(1) receptor-induced [(3)H]-cAMP accumulation and the carbachol inhibition were mimicked by the PKC inhibitors Ro-318220 (200 nM) and Gö-6976 (200 nM). Taken together our results indicate that in addition to the inhibitory role of M(4) receptors, in rat striatum acetylcholine stimulates cAMP formation through the activation of M(1 )receptors and PKC stimulation.

Role of the urothelium in urinary bladder dysfunction following spinal cord injury.

A consequence of spinal cord injury is a change in bladder reflex pathways resulting in the emergence of detrusor hyperreflexia and increased activity of the urethral sphincter. A basis for some of these alterations could be changes in the environment of bladder sensory nerve endings at the target organ. Recent evidence suggests that the urothelium (the lining of the urinary bladder) plays a prominent role in modulating bladder sensory nerve ending excitability. It is conceivable that factors and processes affecting the plasticity of bladder neurons after spinal cord injury may be partly due to changes occurring in the urothelium. Although the urothelium has classically been thought of as a passive barrier to ions/solutes, a number of novel properties have been recently attributed to these cells. Our work and that of others clearly demonstrates that the urothelium exhibits both "sensor" (expression of sensor molecules or response to thermal, mechanical and chemical stimuli) as well as "transducer" (release of factors/transmitters) properties. Taken together, these and other findings discussed in this chapter suggest a sensory function for the urothelium and that alterations in urothelial properties may contribute to afferent abnormalities following spinal cord injury.

In vivo co-ordinated interactions between inhibitory systems to control glutamate-mediated hippocampal excitability.

We present an overview of the long-term adaptation of hippocampal neurotransmission to cholinergic and GABAergic deafferentation caused by excitotoxic lesion of the medial septum. Two months after septal microinjection of 2.7 nmol alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), a 220% increase of GABA(A) receptor labelling in the hippocampal CA3 and the hilus was shown, and also changes in hippocampal neurotransmission characterised by in vivo microdialysis and HPLC. Basal amino acid and purine extracellular levels were studied in control and lesioned rats. In vivo effects of 100 mm KCl perfusion and adenosine A(1) receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) on their release were also investigated. In lesioned animals GABA, glutamate and glutamine basal levels were decreased and taurine, adenosine and uric acid levels increased. A similar response to KCl infusion occurred in both groups except for GABA and glutamate, which release decreased in lesioned rats. Only in lesioned rats, DPCPX increased GABA basal level and KCl-induced glutamate release, and decreased glutamate turnover. Our results evidence that an excitotoxic septal lesion leads to increased hippocampal GABA(A) receptors and decreased glutamate neurotransmission. In this situation, a co-ordinated response of hippocampal retaliatory systems takes place to control neuron excitability.

Spinal gabapentin and antinociception: mechanisms of action.

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.

Altered expression of autonomic neurotransmitter receptors and proliferative responses in lymphocytes from a chronic mild stress model of depression: effects of fluoxetine.

We studied beta-adrenergic and muscarinic cholinergic receptor (MR) expression and proliferative response in lymphocytes from animals under chronic mild stress (CMS) model of depression (CMS animals). Animals were subjected to CMS (periods of food or water deprivation, changes in lighting conditions, tilted cage, etc.) for 12 weeks. CMS lymphocytes showed an altered mitogen-induced proliferation. CMS-B and -T lymphocytes showed an increment on beta-adrenoceptor number and on intracellular responses to a beta-agonist. CMS-T cells showed higher MR expression and lower cGMP responses than normal lymphocytes. MR were not detectable in normal B cells while CMS-B cells showed both MR expression and cGMP response. Beta and muscarinic stimulation influenced lymphocyte proliferative responses, in accordance with cAMP and cGMP responses. After 12 weeks of the CMS procedure, animals were treated with fluoxetine while the CMS procedure continued. Fluoxetine treatment reverted the alterations induced by CMS. These findings suggest a possible mechanism for the immune alterations found in depressive disorders and for the effect of fluoxetine treatment on immune response.

Assessment of the role of the inositol 1,4,5-trisphosphate receptor in the activation of transient receptor potential channels and store-operated Ca2+ entry channels.

The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.

The effect of tolterodine on the pharmacokinetics and pharmacodynamics of a combination oral contraceptive containing ethinyl estradiol and levonorgestrel.

BACKGROUND: Tolterodine is an antimuscarinic agent for the treatment of overactive bladder, a chronic condition that is particularly common in women. Given the prevalence pattern of overactive bladder and the widespread use of oral contraception, circumstances are likely to arise in which physicians may wish to prescribe tolterodine for patients already taking oral contraceptives. Based on a search of MEDLINE from 1990 to 2001, there have been no studies of whether concomitant use of these agents entails a risk of drug-drug interaction or conception. OBJECTIVE: This study investigated the effects of tolterodine on the pharmacokinetics and pharmacodynamics of a low-dose combination oral contraceptive (ethinyl estradiol 30 microg/levonorgestrel 150 microg). METHODS: This was an open-label, randomized, 2-period crossover study in healthy women. Oral contraception was given for 21 days either alone or in combination with oral tolterodine 2 mg BID (on days 1-14) over two 28-day contraceptive cycles. Pharmacokinetic assessments were performed on day 14 based on plasma levels of ethinyl estradiol and levonorgestrel up to 24 hours after dosing and serum tolterodine levels at 1 to 3 hours after dosing. The potential for pharmacodynamic interaction was assessed in terms of the risk of failure of suppression of ovulation based on serum levels of estradiol and progesterone measured throughout each cycle. RESULTS: Twenty-four healthy women (age, 23-41 years [mean, 30 years]; height, 155-178 cm [mean, 167 cm]; body weight, 51-75 kg [mean, 64 kg]) participated in the study. There was no evidence of a pharmacokinetic interaction between tolterodine and the steroid hormones in the oral contraceptive used, nor did the oral contraceptive show any relevant pharmacokinetic interaction with tolterodine. Serum levels of estradiol and progesterone indicated suppression of ovulation in both treatment periods. CONCLUSION: In this selected population. coadministration of tolterodine did not affect the contraceptive efficacy of a low-dose combination oral contraceptive containing ethinyl estradiol and levonorgestrel.