Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A-(1–11)-NH2) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure–activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide’s affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [35S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (Ki = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [35S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.

The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides’ opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2′-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.

. Low temperatures and high hydrostatic pressures are typical of the deep sea. The effects of these parameters on transmembrane signal transduction were determined through a study of the A, adenosine recep-tor-inhibitory guanine nucleotide binding protein-ade-nylyl cyclase system in brain membranes of the bathyal teleost fish. Antimora rostrata (Moridae). The components of this system were analyzed at 5C and 1 atm, and the role of the A, receptor in the modulation of adenylyl cyclase was determined. The A, selective radioligand N h - ['H]cyclohexyladenosine bound saturably, reversibly, and with high affinity. The K^ of N 6 -['H]cyclohexyladen-osine estimated from kinetic measurements was 1.11 nAf; the Kj determined from equilibrium binding was 4.86 nAf. [ 32 P]ADP-ribosylation of brain membranes by pertussis toxin labeled substrates with apparent molecular masses of 39,000 to 4 1 ,000 Da. Basal adenylyl cyclase activity was inhibited in a concentration-dependent manner by the A! adenosine receptor agonist N 6 -cyclo- pentyladenosine (IC 50 = 5.08 nM). The inhibition of ad-enylyl cyclase activity was dependent upon GTP. Basal adenylyl cyclase activity was unaffected by 272 atm of pressure. The efficacy of 100 /iA/ N 6 -cyclopentyladeno- sine as an inhibitor of adenylyl cyclase was the same at atmospheric pressure and at 272 atm. The inhibition of adenylyl cyclase by the agonist 5'-N-ethylcarboxami- doadenosine (100 pM) at 272 atm was twice that observed at atmospheric pressure. Although consideration

Voltage-gated sodium channel (VGSC) activators promote neurite outgrowth byaugmenting intracellular Na+concentration ([Na+]i) and upregulating N-methyl-D-aspartate receptor(NMDAR) function. NMDAR activation stimulates calcium (Ca2+)influx and increases brain-derivedneurotrophic factor (BDNF) release and activation of tropomyosin receptor kinase B (TrkB)signaling. The BDNF-TrkB pathway has been implicated in activity-dependent neuronaldevelopment. We have previously shown that antillatoxin (ATX), a novel lipopeptide isolatedfrom the cyanobacteriumMoorea producens, is a VGSC activator that produces an elevation of [Na+]i.Here we address the effect of ATX on the synthesis and release of BDNF and determine the signalingmechanisms by which ATX enhances neurite outgrowth in immature cerebrocortical neurons. ATXtreatment produced a concentration-dependent release of BDNF. Acute treatment with ATX alsoresulted in increased synthesis of BDNF. ATX stimulation of neurite outgrowth was prevented bypretreatment with a TrkB inhibitor or transfection with a dominant-negative Trk-B. The ATXactivation of TrkB and Akt was blocked by both a NMDAR antagonist (MK-801) and a VGSCblocker (tetrodotoxin). These results suggest that VGSC activators such as the structurally novelATX may represent a new pharmacological strategy to promote neuronal plasticity through a NMDAR-BDNF-TrkB-dependent mechanism

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na+]I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca2+) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca2+-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.

Kappa opioid receptor (KOR) antagonists have recently shown potential for treating drug addiction and mood disorders. The linear acetylated dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,d-Ala8]dynorphin A-(1-11)NH2), synthesized in our laboratory, demonstrated potent and selective KOR antagonism. Cyclization of arodyn could potentially stabilize the bioactive conformation and enhance its metabolic stability. The cyclization strategy employed involved ring closing metathesis between adjacent meta- or para-substituted Tyr(allyl) residues in the "message" sequence that were predicted in a docking study to yield analogs that would bind to the KOR with binding poses similar to arodyn. Consistent with the modeling, the resulting analogs retained KOR affinity similar to arodyn; the peptides involving cyclization between para O-allyl groups also retained high KOR selectivity, with one analog exhibiting KOR antagonist potency (KB = 15 nM) similar to arodyn. These promising cyclized analogs with constrained aromatic residues represent novel leads for further exploration of KOR pharmacology.

The dynorphin (Dyn) A analog arodyn (Ac[Phe1-3,Arg4,d-Ala8]Dyn A-(1–11)NH2) is a κ opioid receptor-selective antagonist, but as a linear peptide it is conformationally flexible and subject to metabolism by proteases. To restrict its conformational flexibility both short- and long-range cyclizations via ring-closing metathesis (RCM) involving residues in both the N-terminal “message” and C-terminal “address” sequences were explored. Cyclization between allyglycine (AllGly) residues in positions 5 and 8 yielded peptides with the highest κ opioid receptor affinity (Ki = 54 and 63 nM) and selectivity for κ over µ receptors (185- and 149-fold) comparable to arodyn (175-fold), with similar results for the peptides with the cis and trans double bond configurations; both isomers exhibited competitive antagonism of κ opioid receptors with potencies similar to arodyn. The minor cis isomer cyclized between AllGly residues in positions 2 and 8 exhibited similar κ receptor affinity to arodyn, but lacked selectivity for κ over µ opioid receptors. These results provide important structure-activity relationship information for cyclization of arodyn that we are using in the design of the next generation of cyclic arodyn analogs.

Brevetoxins and ciguatoxins are lipid-soluble polyether marine natural products that produce, respectively, neurotoxic shellfish poisoning and Ciguatera in humans. They bind to voltage-gated sodium channels at neurotoxin site 5 in a mutually exclusive manner. This binding modifies the gating properties of sodium channels leading to depolarization of neurons. While both ciguatoxins and brevetoxins produce membrane depolarization, Pacific ciguatoxin produces a prolongation of action potentials and afterhyperpolarization that is likely due to a blockade of voltage-gated K+ channels. Using primary cultures of cerebellar granule cells we have demonstrated that both brevetoxin and ciguatoxin produce excitotoxicity with Caribbean ciguatoxin displaying high potency. The ability of gating modifiers of sodium channels to trigger sodium influx can also be assessed using the Na+-sensitive fluorescent dye, sodium-binding benzofuran isophthalate. Murine neocortical neurons in primary culture were used to demonstrate that the brevetoxin PbTx-1 is a full agonist while PbTx-2 and -3 have somewhat lower efficacies. The increment in neuronal [Na+]i exceeded 40 mM at high concentrations of brevetoxins. Intracellular [Na+] increments have been demonstrated to increase NMDA receptor-mediated whole-cell currents and we have explored the ability of brevetoxin to augment NMDA receptor function. Brevetoxin treatment was found to increase both the mean open time and open probability of NMDA receptors. In addition brevetoxin enhanced cerebrocortical neuron neurite outgrowth, dendritic arbor complexity, dendritic filopodia density and synaptic density. Given these actions of brevetoxin in vitro, we used a photothrombotic stroke model in adult thy1-YFP mice to assess the effects of brevetoxin on structural plasticity and functional recovery following a unilateral stroke. Epicortical brevetoxin treatment enhanced dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. Brevetoxin also produced a robust improvement of motor recovery. These results suggest that brevetoxin may represent a novel pharmacologic approach to augment activity-dependent recovery from a stroke.

We explored the utility of the real-time FLIPR Membrane Potential (FMP) assay as a method to assess kappa opioid receptor (KOR)-induced hyperpolarization. The FMP Blue dye was used to measure fluorescent signals reflecting changes in membrane potential in KOR expressing CHO (CHO-KOR) cells. Treatment of CHO-KOR cells with kappa agonists U50,488 or dynorphin [Dyn (1−13)NH2] produced rapid and concentration-dependent decreases in FMP Blue fluorescence reflecting membrane hyperpolarization. Both the nonselective opioid antagonist naloxone and the κ-selective antagonists nor-binaltorphimine (nor-BNI) and zyklophin produced rightward shifts in the U50,488 concentration-response curves, consistent with competitive antagonism of the KOR mediated response. The decrease in fluorescent emission produced by U50,488 was blocked by overnight pertussis toxin pretreatment, indicating the requirement for PTX-sensitive G proteins in the KOR mediated response. We directly compared the potency of U50,488 and Dyn (1−13)NH2 in the FMP and [35S]GTPγS binding assays, and found that both were approximately 10 times more potent in the cellular fluorescence assay. The maximum responses of both U50,488 and Dyn (1−13)NH2 declined following repeated additions, reflecting receptor desensitization. We assessed the efficacy and potency of structurally distinct KOR small molecule and peptide ligands. The FMP assay reliably detected both partial agonists and stereoselectivity. Using KOR-selective peptides with varying efficacies, we found that the FMP assay allowed high throughput quantification of peptide efficacy. These data demonstrate that the FMP assay is a sensitive method for assessing κ-opioid receptor induced hyperpolarization, and represents a useful approach for quantification of potency, efficacy and desensitization of KOR ligands.

The oxytocin-arginine vasopressin (OT-AVP) ligand-receptor family influences a variety of physiological, behavioral, and social behavioral processes in the brain and periphery. The OT-AVP family is highly conserved in mammals, but recent discoveries have revealed remarkable diversity in OT ligands and receptors in New World Monkeys (NWMs) providing a unique opportunity to assess the effects of genetic variation on pharmacological signatures of peptide ligands. The consensus mammalian OT sequence has leucine in the 8th position (Leu8-OT), whereas a number of NWMs, including the marmoset, have proline in the 8th position (Pro8-OT) resulting in a more rigid tail structure. OT and AVP bind to OT's cognate G-protein coupled receptor (OTR), which couples to various G-proteins (Gi/o, Gq, Gs) to stimulate diverse signaling pathways. CHO cells expressing marmoset (mOTR), titi monkey (tOTR), macaque (qOTR), or human (hOTR) OT receptors were used to compare AVP and OT analog-induced signaling. Assessment of Gq-mediated increase in intracellular calcium (Ca2+) demonstrated that AVP was less potent than OT analogs at OTRs from species whose endogenous ligand is Leu8-OT (tOTR, qOTR, hOTR), relative to Pro8-OT. Likewise, AVP-induced membrane hyperpolarization was less potent at these same OTRs. Evaluation of (Ca2+)-activated potassium (K+) channels using the inhibitors apamin, paxilline, and TRAM-34 demonstrated that both intermediate and large conductance Ca2+-activated K+ channels contributed to membrane hyperpolarization, with different pharmacological profiles identified for distinct ligand-receptor combinations. Understanding more fully the contributions of structure activity relationships for these peptide ligands at vasopressin and OT receptors will help guide the development of OT-mediated therapeutics.

Substance abuse remains a serious public health crisis, affecting millions of people worldwide. Macrocyclic tetrapeptides like CJ-15,208 and [d-Trp]CJ-15,208 demonstrate opioid activity shown to attenuate the rewarding effects of cocaine in conditioned place preference assays in mice, making them promising lead compounds for treating substance abuse. In the present study, we report the rational design, synthesis, conformational analysis, and continued pharmacological evaluation of the novel macrocyclic tetrapeptide cyclo[Pro-Sar-Phe-d-Phe] to further explore this unique molecular scaffold. This peptide was rationally designed based on X-ray and NMR structures of related macrocyclic tetrapeptides. Following synthesis, its solution-phase conformations were determined by NMR and molecular modeling. The peptide adopted multiple conformations in polar solvents, but a single conformation in chloroform that is stabilized by intramolecular hydrogen bonding. The peptide is orally bioavailable, producing antinociception and antagonism of kappa opioid receptor (KOR) stimulation following oral administration in a mouse 55 °C warm-water tail-withdrawal assay. Notably, cyclo[Pro-Sar-Phe-d-Phe] blocked both stress- and drug-induced reinstatement of cocaine and morphine conditioned place preference in mice following oral administration, and displayed a decreased side-effect profile compared to morphine. Thus, cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for the treatment of substance abuse.

A thiazole-containing cyclic depsipeptide with 11 amino acid residues, named pagoamide A (1), was isolated from laboratory cultures of a marine Chlorophyte, Derbesia sp. This green algal sample was collected from America Samoa, and pagoamide A was isolated using guidance by MS/MS-based molecular networking. Cultures were grown in a light- and temperature-controlled environment and harvested after several months of growth. The planar structure of pagoamide A (1) was characterized by detailed 1D and 2D NMR experiments along with MS and UV analysis. The absolute configurations of its amino acid residues were determined by advanced Marfey's analysis following chemical hydrolysis and hydrazinolysis reactions. Two of the residues in pagoamide A (1), phenylalanine and serine, each occurred twice in the molecule, once in the d- and once in the l-configuration. The biosynthetic origin of pagoamide A (1) was considered in light of other natural products investigations with coenocytic green algae.

Arginine vasopressin (AVP) and oxytocin (OT) are nonapeptides that bind to G-protein coupled receptors and influence social behaviors. Consensus mammalian AVP and OT (Leu8-OT) sequences are highly conserved. In marmosets, an amino acid change in the 8th position of the peptide (Pro8-OT) exhibits unique structural and functional properties. There is ∼85 % structural homology between the OT receptor (OTR) and vasopressin 1a receptor (V1aR) resulting in significant cross-reactivity between the ligands and receptors. Chinese hamster ovary (CHO) cells expressing marmoset (mV1aR), macaque (qV1aR), or human vasopressin receptor 1a (hV1aR) were used to assess AVP, Leu8-OT and Pro8-OT pharmacological profiles. To assess activation of Gq, functional assays were performed using Fluo-3 to measure ligand-induced Ca2+ mobilization. In all three V1aR-expressing cell lines, AVP was more potent than the OT ligands. To assess ligand-induced hyperpolarization, FLIPR Membrane Potential (FMP) assays were performed. In all three V1aR lines, AVP was more potent than the OT analogs. The distinctive U-shaped concentration-response curve displayed by AVP may reflect enhanced desensitization of the mV1aR and hV1aR, which is not observed with qV1aR. Evaluation of Ca2+-activated potassium (K+) channels using the inhibitors apamin, paxilline, and TRAM-34 demonstrated that both intermediate and large conductance Ca2+-activated K+ channels contributed to membrane hyperpolarization, with different pharmacological profiles identified for distinct ligand-receptor combinations. Taken together, these data suggest differences in ligand-receptor signaling that may underlie differences in social behavior. Integrative studies of behavior, genetics and ligand-receptor interaction will help elucidate the connection between receptor pharmacology and social behaviors.

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues’ stereochemistry on the peptides’ opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Emerging literature suggests that after a stroke, the peri-infarct region exhibits dynamic changes in excitability. In rodent stroke models, treatments that enhance excitability in the peri-infarct cerebral cortex promote motor recovery. This increase in cortical excitability and plasticity is opposed by increases in tonic GABAergic inhibition in the peri-infarct zone beginning three days after a stroke in a mouse model. Maintenance of a favorable excitatory–inhibitory balance promoting cerebrocortical excitability could potentially improve recovery. Brevetoxin-2 (PbTx-2) is a voltage-gated sodium channel (VGSC) gating modifier that increases intracellular sodium ([Na+]i), upregulates N-methyl-D-aspartate receptor (NMDAR) channel activity and engages downstream calcium (Ca2+) signaling pathways. In immature cerebrocortical neurons, PbTx-2 promoted neuronal structural plasticity by increasing neurite outgrowth, dendritogenesis and synaptogenesis. We hypothesized that PbTx-2 may promote excitability and structural remodeling in the peri-infarct region, leading to improved functional outcomes following a stroke. We tested this hypothesis using epicortical application of PbTx-2 after a photothrombotic stroke in mice. We show that PbTx-2 enhanced the dendritic arborization and synapse density of cortical layer V pyramidal neurons in the peri-infarct cortex. PbTx-2 also produced a robust improvement of motor recovery. These results suggest a novel pharmacologic approach to mimic activity-dependent recovery from stroke.

This chapter explores the use of drugs for veterinary mental health care and behavior modification. It focuses on amino acid neurotransmitters. In addition to their role in intermediary metabolism, certain amino acids function as small molecule neurotransmitters in the central and peripheral nervous systems. Application of excitatory amino acids such as glutamic acid and aspartic acid typically depolarize mammalian neurons, while inhibitory amino acids such as gamma aminobutyric acid (GABA) and glycine characteristically hyperpolarize neurons. Ketamine is an anesthetic agent that was first introduced in clinical trials in the 1960s. Upon activation, the GABAergic neurons release GABA from presynaptic terminals into the synaptic cleft. GABAergic systems serve important regulatory functions in the brain such as vigilance, anxiety, muscle tension, epileptogenic activity, and memory.

Identifying neurons that have functional opioid receptors is fundamental for the understanding of the cellular, synaptic and systems actions of opioids. Current techniques are limited to post hoc analyses of fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), to label opioid receptors based on a chemical approach termed ‘traceless affinity labeling’. In this approach, a high affinity antagonist naltrexamine is used as the guide molecule for a transferring reaction of acylimidazole at the receptor. This reaction generates a fluorescent dye covalently linked to the receptor while naltrexamine is liberated and leaves the binding site. The labeling induced by this reagent allowed visualization of opioid-sensitive neurons in rat and mouse brains without loss of function of the fluorescently labeled receptors. The ability to locate endogenous receptors in living tissues will aid considerably in establishing the distribution and physiological role of opioid receptors in the CNS of wild type animals.

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu8-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro8-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (Gs) or inhibition (Gi/o) of adenylyl cyclase, stimulation of potassium channel currents (Gi), and activation of phospholipase C (Gq). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro8-OT was more efficacious than Leu8-OT in measures of Gq activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro8-OT and Leu8-OT differed with respect to Gq signaling. In both mOTR- and hOTR-expressing cells, Leu8-OT was more potent and modestly more efficacious than Pro8-OT in inducing hyperpolarization. In mOTR cells, Leu8-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for Gi/o activation; however, the Pro8-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a Gq signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated K+ channels.