Papers by Christian Felder
Journal of Biological Chemistry, 2016
Establishing the in vivo activation status of G protein-coupled receptors (GPCRs) would not only ... more Establishing the in vivo activation status of G protein-coupled receptors (GPCRs) would not only indicate physiological roles of GPCRs but would also aid drug-discovery by establishing drug:receptor engagement. Here we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (S228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR positive allosteric modulator, BQCA, enhanced acetylcholine-mediated phosphorylation at S228. These data supported the hypothesis that phosphorylation at S228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated S228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and BQCA. Finally, S228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at S228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampal based memory and learning.
Cerebral cortex (New York, N.Y. : 1991), Jan 15, 2015
Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their i... more Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires ...
Journal of steroid biochemistry, 1983
Experimental conditions for the optimum detection and measurement of the cytosolic progestogen re... more Experimental conditions for the optimum detection and measurement of the cytosolic progestogen receptor in human prostatic hyperplasia and neoplasia are described. In presence of 20 mM molybdate ion and a reaction time of 0.5-2 h at 0-2 degrees C, we are able to detect the appearance of a [6.7-3H]-17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione ([3H]-R5020) and [3H]-progesterone binding moiety in human prostatic cytosol. The [3H]-R5020 binding protein sediments at approximately 8-11S in a glycerol density gradient centrifuged in a Sorvall TV865 rotor (vertical rotor). Maximal binding of [3H]-R5020 occurs at 30 min at 25 degrees C and 1 h at 0 degree C. Considerable overall improvement in receptor detection and measurement was made when gradient centrifugation was carried out in a vertical rotor instead of swing bucket rotor. The specifically bound [3H]-R5020 is displaced by progesterone, triamcinolone acetonide and R5020, but not by cortisol, dihydrotestosterone, 17 beta-estradiol,...
The Journal of Neuroscience, Jul 15, 1997
culture, both the CB1 receptor agonist [3-(1,1-dimethylheptyl)-11-hydroxy-⌬ 8 tetrahydrocannabino... more culture, both the CB1 receptor agonist [3-(1,1-dimethylheptyl)-11-hydroxy-⌬ 8 tetrahydrocannabinol] (HU210) and the D2 receptor agonist quinpirole inhibited forskolin-stimulated cAMP accumulation when applied separately. In contrast, HU210 and quinpirole in combination augmented cAMP accumulation. This augmentation was blocked by the CB1 receptor antagonist SR141716A or the D2 antagonist sulpride. Pertussis toxin treatment of striatal neurons prevented the inhibition of cAMP accumulation by D2 receptors but unmasked a cannabinoid receptor-mediated stimulatory effect on cAMP accumulation. The cannabinoid receptor-stimulated accumulation of cAMP was blocked in a concentration-dependent manner by SR141716A, suggesting that the response was regulated through the CB1 receptor. Similar augmentation of cAMP accumulation after pertussis toxin treatment was observed in Chinese hamster ovary (CHO) cells transfected with, and stably expressing, the CB1 receptor. This stimulation of cAMP was not Ca 2ϩ -sensitive and was unaffected by a range of protein kinase inhibitors. Treatment of the pertussis toxin-treated cells with cholera toxin before CB1 receptor activation amplified the stimulatory pathway, suggesting that this response was mediated through a G s -type G-protein. Stimulation of cAMP accumulation was not observed after pertussis toxin treatment of CHO cells expressing the human CB2 receptor, suggesting that this novel signaling pathway is unique to the cannabinoid CB1 receptor.
Pharmacology Therapeutics, Apr 30, 2001
The active principle in marijuana, Delta(9)-tetrahydrocannabinol (THC), has been shown to have wi... more The active principle in marijuana, Delta(9)-tetrahydrocannabinol (THC), has been shown to have wide therapeutic application for a number of important medical conditions, including pain, anxiety, glaucoma, nausea, emesis, muscle spasms, and wasting diseases. Delta(9)-THC binds to and activates two known cannabinoid receptors found in mammalian tissue, CB1 and CB2. The development of cannabinoid-based therapeutics has focused predominantly on the CB1 receptor, based on its predominant and abundant localization in the CNS. Like most of the known cannabinoid agonists, Delta(9)-THC is lipophilic and relatively nonselective for both receptor subtypes. Clinical studies show that nonselective cannabinoid agonists are relatively safe and provide therapeutic efficacy, but that they also induce psychotropic side effects. Recent studies of the biosynthesis, release, transport, and disposition of anandamide are beginning to provide an understanding of the role of lipid transmitters in the CNS. This review attempts to link current understanding of the basic biology of the endocannabinoid nervous system to novel opportunities for therapeutic intervention. This new knowledge may facilitate the development of cannabinoid receptor-targeted therapeutics with improved safety and efficacy profiles.
Molecular Pharmacology
The recently discovered endogenous agonist for the cannabinoid receptor, anandamide (arachidonyle... more The recently discovered endogenous agonist for the cannabinoid receptor, anandamide (arachidonylethanolamide), can be formed enzymatically by the condensation of arachidonic acid with ethanolamine. 5Z,8Z,11Z-Eicosatrienoic acid (mead acid) has been found to substitute for arachidonic acid in the sn-2 position of phospholipids and accumulate during periods of dietary fatty acid deprivation in rats. In the present study, the chemically synthesized ethanolamide of mead acid was evaluated as a potential agonist at the two known subtypes of cannabinoid receptor: CB1 (central) and CB2 (peripheral). This compound was equipotent to anandamide in competing with [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the human CB1 receptor and from ATt-20 cells expressing the human CB2 receptor. Mead ethanolamide was also equipotent to anandamide in inhibiting forskolin-stimulated cAMP accumulation in cells expressing the CB1 receptor. It inhibited N-type calcium currents with a lower potency than anandamide. Mead and arachidonic acid were equally efficacious as substrates for the enzymatic synthesis of their respective ethanolamides in rat and adult human hippocampal P2 membranes. Palmitic acid was not an effective substrate for the enzymatic synthesis of palmitoyl ethanolamide. Mead ethanolamide exhibits several characteristics of a novel agonist to CB1 and CB2 receptors and may represent another candidate endogenous ligand for the CB1 receptor. Due to the anticonvulsant properties of GABA and the positional similarity of L-serine to ethanolamine in membrane phospholipids, these compounds were synthetically coupled to arachidonic acid, and their resulting arachidonamides were tested as potential cannabinoid agonists. The arachidonamides of GABA and L-serine were inactive in both binding and functional assays at the CB1 receptor.
Pediatric Research
Studies were designed to develop an animal depletion is responsible for the development of metabo... more Studies were designed to develop an animal depletion is responsible for the development of metabolic alkamodel for the syndrome of hypochloremic, hypokalemic losis and resultant electrolyte imbalances (9-1 1). Either citrate metabolic alkalosis (HMA), and failure to thrive in infants alone or in combination with other counter anions used to due to intake of chloride-deficient formula. Littermate replace chloride in soy formulas has also been implicated in the canine puppies, 2 wk old, were fed soy formula containing development of HMA (12, 14). normal chloride, 20 mEq/liter (NC, n = S), or low chloride, Essential to the further study of CDS in human infants is a
Pediatric Research
ABSTRACT
Neuropeptides
Phospholipase D belongs to a group of membrane associated phospholipases which have been shown to... more Phospholipase D belongs to a group of membrane associated phospholipases which have been shown to be activated by G-protein coupled neurotransmitter receptors. Phosphatidylcholine is the primary substrate for phospholipase D generating phosphatidic acid (PA) and choline. In the presence of 1% ethanol, phospholipase D catalyzes a transphosphatidylation reaction generating phosphatidylethanol (PEt) which is an indicator of phospholipase D activation. In the present study, we utilized Chinese hamster ovary (CHO) cells stably transfected with and expressing a rat V1a vasopressin receptor to study the regulation of phospholipase D by protein kinase C and calcium. Arginine-vasopressin (AVP) stimulated the release of 3H-PEt and 3H-PA in cells pre-labelled overnight with 3H-palmitic acid. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the release of PEt and PA that was additive with AVP over 15 min. However, long-term stimulation with PMA, which desensitizes protein kinase C, decreased PEt production while simultaneously increasing PA production. Differential regulation of PEt and PA production by PMA suggests the existence of more than one phospholipase D isoenzyme. Though differentially regulated by protein kinase C, both AVP-stimulated PEt and PA production required extracellular and not intracellular calcium.
Journal of Biological Chemistry
In Chinese hamster ovary cells transfected with m5 muscarinic receptors, carbachol stimulates bot... more In Chinese hamster ovary cells transfected with m5 muscarinic receptors, carbachol stimulates both calcium influx and calcium release from intracellular stores. The marine toxin maitotoxin (MTX) elicits a similar response on calcium influx. Carbachol-and MTX-induced calcium influx can be inhibited by the proposed blockers of receptor-operated calcium channels (ROCC), CAI and SK&F 96365. Both carbachol and MTX induce a significant increase in total protein tyrosine phosphorylation, which is dependent on extracellular calcium and can be inhibited by CAI and SK&F 96365. Phospholipase C--y was identified as one of the substrates subject to calcium-dependent tyrosine phosphorylation following carbachol or MTX stimulation.
PloS one, 2015
Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic r... more Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic receptors have been suggested as possible future targets for the treatment of schizophrenia and drug abuse. The muscarinic ligand (5R,6R)-6-(3-butylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane (BuTAC) exhibits high affinity for muscarinic receptors with no or substantially less affinity for a large number of other receptors and binding sites, including the dopamine receptors and the dopamine transporter. In the present study, we wanted to examine the possible antipsychotic-like effects of BuTAC in primates. To this end, we investigated the effects of BuTAC on d-amphetamine-induced behaviour in antipsychotic-naive Cebus paella monkeys. Possible adverse events of BuTAC, were evaluated in the same monkeys as well as in monkeys sensitized to antipsychotic-induced extrapyramidal side effects. The present data suggests that, the muscarinic receptor ligand BuTAC exhibits antipsychotic-l...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 1997
Cannabinoids act at the CB1 receptor to inhibit adenylate cyclase activity via a pertussis toxin-... more Cannabinoids act at the CB1 receptor to inhibit adenylate cyclase activity via a pertussis toxin-sensitive G-protein. Within the striatum, CB1 receptors have been shown to be localized on the same neurons as Gi-coupled dopamine D2 receptors. In this study we have examined the interactions of CB1 and D2 receptors on adenylate cyclase. In striatal neurons in primary culture, both the CB1 receptor agonist [3-(1, 1-dimethylheptyl)-11-hydroxy-Delta8tetrahydrocannabinol] (HU210) and the D2 receptor agonist quinpirole inhibited forskolin-stimulated cAMP accumulation when applied separately. In contrast, HU210 and quinpirole in combination augmented cAMP accumulation. This augmentation was blocked by the CB1 receptor antagonist SR141716A or the D2 antagonist sulpride. Pertussis toxin treatment of striatal neurons prevented the inhibition of cAMP accumulation by D2 receptors but unmasked a cannabinoid receptor-mediated stimulatory effect on cAMP accumulation. The cannabinoid receptor-stimula...
Drug Discovery, Development, and Manufacturing, 2010
... TWO POTENCY ASSAYS Brian J. Eastwood, Amy K. Chesterfield, Mary C. Wolff, and Christian C. Fe... more ... TWO POTENCY ASSAYS Brian J. Eastwood, Amy K. Chesterfield, Mary C. Wolff, and Christian C. Felder Eli Lilly and Company Indianapolis, Indiana 15.1 INTRODUCTION 668 ... See Carrasco and Jover [14], for a summary and classification of these methods. ...
Current Protocols in Neuroscience, 2001
Scintillation proximity assay technologies provide a rapid non-separation method to measure commo... more Scintillation proximity assay technologies provide a rapid non-separation method to measure common biological interactions using radioactively tagged molecules. This unit identifies potential uses of the technology for the measurement of receptor-ligand binding, cAMP accumulation, GTP binding to heterotrimeric G proteins, protease activity and cellular uptake.
Pediatric Research, 1985
N e g a t i v e NaB a n d h y p o n a t r e m i a h a v e b e e n r e p o r t e d f o r g r o w i... more N e g a t i v e NaB a n d h y p o n a t r e m i a h a v e b e e n r e p o r t e d f o r g r o w i n g Althouah olasma CL levels I" NS have heen found t o he IOW or normal. t h e i r . . , p r e t e r m i n f a n t s . I t h a s b e e n s u g g e s t e d t h a t l e s s Na i s i n t e r~r e t a t l o n remains amhlguous.
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Papers by Christian Felder