Biphasic Time Course Research Articles

Half-sarcomeres relax with a biphasic time-course in spatially-explicit simulations with series compliance

Half-sarcomeres relax with a biphasic time-course in spatially-explicit simulations with series compliance

Synaptotagmin 7 Sculpts Short-Term Plasticity at a High Probability Synapse.

Synapses with high release probability (Pr ) tend to exhibit short-term synaptic depression. According to the prevailing model, this reflects the temporary depletion of release-ready vesicles after an initial action potential (AP). At the high-Pr layer 4 to layer 2/3 (L4-L2/3) synapse in rodent somatosensory cortex, short-term plasticity appears to contradict the depletion model: depression is absent at interstimulus intervals (ISIs) <50 ms and develops to a maximum at ∼200 ms. To understand the mechanism(s) underlying the biphasic time course of short-term plasticity at this synapse, we used whole-cell electrophysiology and two-photon calcium imaging in acute slices from male and female juvenile mice. We tested several candidate mechanisms including neuromodulation, postsynaptic receptor desensitization, and use-dependent changes in presynaptic AP-evoked calcium. We found that, at single L4-L2/3 synapses, Pr varies as a function of ISI, giving rise to the distinctive short-term plasticity time course. Furthermore, the higher-than-expected Pr at short ISIs depends on expression of synaptotagmin 7 (Syt7). Our results show that two distinct vesicle release processes summate to give rise to short-term plasticity at this synapse: (1) a basal, high-Pr release mechanism that undergoes rapid depression and recovers slowly (τ = ∼3 s) and (2) a Syt7-dependent mechanism that leads to a transient increase in Pr (τ = ∼100 ms) after the initial AP. We thus reveal how these synapses can maintain a very high probability of neurotransmission for multiple APs within a short time frame. Key words : depression; facilitation; short-term plasticity; synaptotagmin 7.

Quercetin improves and protects Calu-3 airway epithelial barrier function

Introduction: In light of the impact of airway barrier leaks in COVID-19 and the significance of vitamin D in COVID-19 outcomes, including airway barrier protection, we investigated whether the very common dietary flavonoid quercetin could also be efficacious in supporting airway barrier function.Methods: To address this question, we utilized the widely used airway epithelial cell culture model, Calu-3.Results: We observed that treating Calu-3 cell layers with quercetin increased transepithelial electrical resistance while simultaneously reducing transepithelial leaks of 14C-D-mannitol (Jm) and 14C-inulin. The effects of quercetin were concentration-dependent and exhibited a biphasic time course. These effects of quercetin occurred with changes in tight junctional protein composition as well as a partial inhibition of cell replication that resulted in decreased linear junctional density. Both of these effects potentially contribute to improved barrier function. Quercetin was equally effective in reducing the barrier compromise caused by the pro-inflammatory cytokine TNF-α, an action that seemed to derive, in part, from reducing the elevation of ERK 1/2 caused by TNF-α.Discussion: Quercetin improved Calu-3 barrier function and reduced TNF-α-induced barrier compromise, mediated in part by changes in the tight junctional complex.

Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated gene expression was mostly located on chromosomes 16-22. Protein-coding transcripts formed the majority with more than 90% of all differentially expressed genes and followed a continuous trend of downregulation, whereas retained introns demonstrated a biphasic time-course. The gene expression pattern of hypergravity response was not comparable with other stress factors such as oxidative stress, heat shock or inflammation. Furthermore, we tested a routine centrifugation protocol that is widely used to harvest cells for subsequent RNA analysis and detected a huge impact on the transcriptome compared to non-centrifuged samples, which did not return to baseline within 15 min. Thus, we recommend carefully studying the response of any cell types used for any experiments regarding the hypergravity time and levels applied during cell culture procedures and analysis.

Behaviors associated with formalin-induced pain in the Japanese quail (Coturnix coturnix japonica)

The present study aimed to investigate behaviors associated with formalin-induced nociception in the Japanese quail (Coturnix japonica). The tarsometatarsus region of the male quail's foot (shank) was injected with 40µl of formalin (0.3, 0.6, and 0.9%) solutions or normal saline. Two distinct nociceptive behaviors that is injected foot lifting and flinching/shaking behaviors were recorded over 45 minutes. Also, unstimulated preening, eye closing, and chamber wall pecking behaviors were recorded during the observation period. Subcutaneous injection of formalin 0.6 and 0.9% significantly increased the foot lifting activity in the 0-5 minutes (acute phase) and 15-45 minutes (inflammatory phase) after administration. But the injection of formalin 0.3% significantly increased foot lifting activity only in the acute phase. In the other recorded behaviors, we did not find this biphasic time course pattern. All formalin concentrations induced significant foot flinching/shaking activity only in the acute phase. Subcutaneous injection of formalin 0.9% but not 0.6 and 0.3% significantly increased the duration of eye closure behavior between 20-45 minutes after administration. Injection of all formalin concentrations caused a significant decrease in the unstimulated preening activity between the period of 10 to 45 minutes after administration. Injection of all formalin concentrations caused a significant decrease in the observation chamber wall pecking activity between the period 30 to 45 minutes after administration. These results suggest that the quail formalin test can be considered a relevant, sensitive, and specific technique that helps researchers to use it in the determination of the neurobiology and pharmacology of nociception in the avian species.

Chronic facial inflammatory pain-induced anxiety is associated with bilateral deactivation of the rostral anterior cingulate cortex

Patients with chronic pain, especially orofacial pain, often suffer from affective disorders, including anxiety. Previous studies largely focused on the role of the caudal anterior cingulate cortex (cACC) in affective responses to pain, long-term potentiation (LTP) in cACC being thought to mediate the interaction between anxiety and chronic pain. But recent evidence indicates that the rostral ACC (rACC), too, is implicated in processing affective pain. However, whether such processing is associated with neuronal and/or synaptic plasticity is still unknown. We addressed this issue in a chronic facial inflammatory pain model (complete Freund's adjuvant model) in rats, by combining behavior, Fos protein immunochemistry and ex vivo intracellular recordings in rACC slices prepared from these animals. Facial mechanical allodynia occurs immediately after CFA injection, peaks at post-injection day 3 and progressively recovers until post-injection days 10–11, whereas anxiety is delayed, being present at post-injection day 10, when sensory hypersensitivity is relieved, but, notably, not at post-injection day 3. Fos expression reveals that neuronal activity follows a bi-phasic time course in bilateral rACC: first enhanced at post-injection day 3, it gets strongly depressed at post-injection day 10. Ex vivo recordings from lamina V pyramidal neurons, the rACC projecting neurons, show that both their intrinsic excitability and excitatory synaptic inputs have undergone long-term depression (LTD) at post-injection day 10. Thus chronic pain processing is associated with dynamic changes in rACC activity: first enhanced and subsequently decreased, at the time of anxiety-like behavior. Chronic pain-induced anxiety might thus result from a rACC deactivation-cACC hyperactivation interplay.

Sensory adaptation at ribbon synapses in the zebrafish lateral line.

Adaptation is used by sensory systems to adjust continuously their sensitivity to match changes in environmental stimuli. In the auditory and vestibular systems, the release properties of glutamate-containing vesicles at the hair-cell ribbon synapses play a crucial role in sensory adaptation, thus shaping the neural response to sustained stimulation. How ribbon synapses regulate the release of glutamate and how they modulate afferent responses in vivo is still largely unknown. Here, we have used two-photon imaging and electrophysiology to investigate the synaptic transfer characteristics of the hair cells in the context of sensory adaptation in live zebrafish. Prolonged and repeated water-jet stimulation of the hair-cell stereociliary bundles caused adaptation of the action potential firing rate elicited in the afferent neurons. By monitoring glutamate at ribbon synapses using time-lapse imaging, we identified two kinetically distinct release components: a rapid response that was exhausted within 50-100 ms and a slower and sustained response lasting the entire stimulation. After repeated stimulations, the recovery of the fast component followed a biphasic time course. Depression of glutamate release was largely responsible for the rapid firing rate adaptation recorded in the afferent neurons. However, postsynaptic Ca2+ responses had a slower recovery time course than that of glutamate release, indicating that they are also likely to contribute to the afferent firing adaptation. Hair cells also exhibited a form of adaptation during inhibitory bundle stimulations. We conclude that hair cells have optimised their synaptic machinery to encode prolonged stimuli and to maintain their sensitivity to new incoming stimuli.

A Journey in Diabetes: From Clinical Physiology to Novel Therapeutics: The 2020 Banting Medal for Scientific Achievement Lecture.

Insulin resistance and β-cell dysfunction are the core pathophysiological mechanisms of all hyperglycemic syndromes. Advances in in vivo investigative techniques have made it possible to quantify insulin resistance in multiple sites (skeletal and myocardial muscle, subcutaneous and visceral fat depots, liver, kidney, vascular tissues, brain and intestine), to clarify its consequences for tissue substrate selection, and to establish its relation to tissue perfusion. Physiological modeling of β-cell function has provided a uniform tool to measure β-cell glucose sensitivity and potentiation in response to a variety of secretory stimuli, thereby allowing us to establish feedbacks with insulin resistance, to delineate the biphasic time course of conversion to diabetes, to gauge incretin effects, and to identify primary insulin hypersecretion. As insulin resistance also characterizes several of the comorbidities of diabetes (e.g., obesity, hypertension, dyslipidemia), with shared genetic and acquired influences, the concept is put forward that diabetes is a systemic disease from the outset, actually from the prediabetic stage. In fact, early multifactorial therapy, particularly with newer antihyperglycemic agents, has shown that the burden of micro- and macrovascular complications can be favorably modified despite the rising pressure imposed by protracted obesity.

A Proteolytic Site-Directed Affinity Label to Inhibit the Human ATP-Dependent Protease Caseinolytic Complex XP.

Human caseinolytic protease component X and P (hClpXP) is a heterooligomeric ATP-dependent protease. The hClpX subunit catalyzes ATP hydrolysis whereas the hClpP subunit catalyzes peptide bond cleavage. In this study, we generated a peptidyl chloromethyl ketone (dansyl-FAPAL-CMK) that inhibited the hClpP subunit through alkylation of the catalytic His122, which was detected by LC-MS. This inhibitor is composed of a peptide sequence derived from a hydrolyzed peptide product of a substrate cleaved by hClpXP. Binding of FAPAL positions the electrophilic chloromethyl ketone moiety near His122 where alkylation occurs. Dansyl FAPAL-CMK exhibits selectivity for hClpXP over other ATP-dependent proteases such as hLon and the 26S proteasome and abolishes hClpXP activity in HeLa cell lysate. Using the fluorogenic peptide substrate FR-Cleptide as reporter, we detected biphasic inhibition time courses; this supports a slow-binding, time-dependent, covalent inhibition mechanism that is often found in active-site directed affinity labels. Because this inhibitor reacts only with hClpXP but not hLon or the proteasome, it has the potential to serve as a chemical tool to help validate endogenous protein substrates of hClpXP in cell lysate, thereby benefiting investigation of the physiological functions of hClpXP in different cell types or tissue samples.

Oscillatory brain dynamics of pronoun processing in native Spanish speakers and in late second language learners of Spanish

AbstractA longstanding question in the second language acquisition literature is whether late second language (L2) learners process grammatical structures in a native-like manner. Here, we use Time Frequency Representation (TFR) analysis to test morpho-syntactic processing of clitic pronouns in native and late L2 learners of Spanish. The TFR results show overall similar power decreases in theta, alpha, and beta frequencies in both groups. Critically, the observed oscillatory effects persisted in time for native Spanish speakers, but declined earlier for L2 learners. We discuss the results using cell-assembly theory models for language processing (e.g., Pulvermüller, 1999) that posit a biphasic time-course for neural assemblies consisting of an early ignition (recognition) and a later reverberation (re-processing) phase. We propose a working hypothesis for L2 processing in tune with a cell-assembly theory suggesting that the length of the reverberation phase could be a distinguishing feature between native and L2 processing.

Drought stress induces a biphasic NO accumulation in Arabidopsis thaliana

ABSTRACTWe have recently reported the proteomic signature of the early (≤30 min) drought stress responses in Arabidopsis thaliana suspension cells challenged with PEG. We found an over-representation in the gene ontology categories “Ribosome” and “Oxidative stress along with an increased abundance of late embryogenesis abundant (LEA) and early response to dehydration (ERD) proteins. Since nitric oxide (NO) plays a pivotal role in plant responses to drought stress and induces LEA and DREB proteins, here we monitored the levels of NO in Arabidopsis cell suspensions and leaf disks challenged with PEG, and performed comparative analyses of the proteomics and transcriptomics data in public domain to search for a common set of early drought and NO responsive proteins.We show that under drought-stress, NO shows a biphasic time course, much like in response to ozone stress and that among the early drought and NO responsive proteins, the categories “DNA binding”, “Nucleotide binding” and “Transcription regulator activity” are enriched. Taken together, present study suggests that in Arabidopsis the changing NO levels may play a critical role in early drought responsive processes and notably in the transcriptional and translational reprograming observed under drought stress.

Apoptotic cell death in the mammalian central nervous system during development and in pathological conditions

Mammalian central nervous system (CNS) development involves genetically controlled, opposing processes, such as neuronal proliferation, migration, differentiation and death. The natural, developmental cell death is a ubiquitous phenomenon and is referred to as «programmed cell death» (PCD). Apoptosis, a type of PCD, is a central event in normal development of the CNS, playing an important role in the control of cell numbers and the establishment of neuronal circuitry. During embryogenesis, apoptosis takes place in proliferating cell populations and is involved in CNS morphogenesis. At later stages of development, apoptosis occurs in postmitotic neurons, because of the competition for limited supply of neurotrophic factors, that originally suppress the endogenous genetic death programme. Data concerning apoptotic cell death during normal CNS development of domestic mammals is lacking, therefore information about the developmental pattern of this phenomenon is restricted to rodents and rabbits. In these animals it has been suggested that apoptosis follows a mono- or biphasic time course and is completed duringan early, critical period of CNS development, that is characterized by morphological and functional neuronal maturation and synapse formation. Apart from its role in CNS development, apoptosis has also been implicated in neuronal loss accompanying neurodegenerative diseases and traumatic brain injuries in humans and animals. In the domestic canine brain, it has been shown that neurons die via apoptosis in Alzheimer's-like dementia, cerebellar abiotrophy, global and focal ischemia and virus-induced encephalopathies. In addition, cell death in ruminants with transmissible spongiform encephalopathy has been reported to be apoptotic in nature. A plethora of studies using animal models have been employed to elucidate the mechanisms than govern cell loss in neurological disorders. These studies provided strong evidence that experimental lesions of the connections between CNS areas and withdrawal of neurotrophic factors result in an increase of apoptosis, that is age-dependent. Specifically, developing neurons are more dependent on the integrity of their connections than mature ones. In addition, the response of neurons to apoptotic stimuli shows regional specificity. According to epidemiologic studies, CNS disorders are of major concern for animal and human public health, with a high socioeconomic impact. A major goal of neuroscientists is the development of therapeutic approaches for CNS repair. Contemporary strategies that are under trial include neurotrophic factor substitution and transplantation of stem cells. Investigation of the principles and mechanisms controlling cell loss in neurodegenerative diseases and traumatic brain injuries are universally considered of high priority and hopefully will lead to novel therapeutic approaches, with encouraging outcome. The present review summarises recent data on the molecular mechanisms and factors controlling neuronal apoptosis during development and in pathological conditions, describes popular animal models used in lesion studies and discusses therapeutic approaches aiming at preventing or restricting apoptotic cell death.

Factors Governing Autooxidation of Human Hemoglobin

Determining mechanisms for the autooxidation of hemoglobin is required for understanding and treating unstable hemoglobinopathies and for developing more stable hemoglobin based O2 carriers. Previous studies suggested significant differences in autooxidation rates of α and β subunits. We used an azide reaction assay to measure the concentrations of ferric α and β chains at different time points during autooxidation. Our results showed no differences between the subunits. To obtain more accurate time courses for autooxidation, we deconvoluted observed spectra into the decay of HbO2, metHb appearance, hemichrome generation, and increases in turbidity due to hemin loss and apoprotein precipitation. The time courses for HbO2 decay at high concentrations (≥ 100µM heme) accelerate implying cooperative autooxidation, where as at low concentrations (≤ 10uM) the time courses are biphasic. These results suggest that the biphasic time courses at low hemoglobin are due to differences between tetramers and dimers. We have also measured autooxidation rates for a recombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-α subunit. This hemoglobin shows a monophasic time course for autooxidation at both high and low protein concentrations, and the azide binding assay showed equal amounts of ferric α and β subunits. We have also examined recombinant mutant hemoglobins to examine the structural factors that govern autooxidation. Increased rates of autooxidation were found for rHb Providence, rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmed that the rate of autooxidation shows a bell-shaped dependence on oxygen concentration and increases markedly as the pH is decreased.Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the Robert A. Welch Foundation.

Application of Physiologically Based Absorption Modeling to Characterize the Pharmacokinetic Profiles of Oral Extended Release Methylphenidate Products in Adults.

A previously presented physiologically-based pharmacokinetic model for immediate release (IR) methylphenidate (MPH) was extended to characterize the pharmacokinetic behaviors of oral extended release (ER) MPH formulations in adults for the first time. Information on the anatomy and physiology of the gastrointestinal (GI) tract, together with the biopharmaceutical properties of MPH, was integrated into the original model, with model parameters representing hepatic metabolism and intestinal non-specific loss recalibrated against in vitro and in vivo kinetic data sets with IR MPH. A Weibull function was implemented to describe the dissolution of different ER formulations. A variety of mathematical functions can be utilized to account for the engineered release/dissolution technologies to achieve better model performance. The physiological absorption model tracked well the plasma concentration profiles in adults receiving a multilayer-release MPH formulation or Metadate CD, while some degree of discrepancy was observed between predicted and observed plasma concentration profiles for Ritalin LA and Medikinet Retard. A local sensitivity analysis demonstrated that model parameters associated with the GI tract significantly influenced model predicted plasma MPH concentrations, albeit to varying degrees, suggesting the importance of better understanding the GI tract physiology, along with the intestinal non-specific loss of MPH. The model provides a quantitative tool to predict the biphasic plasma time course data for ER MPH, helping elucidate factors responsible for the diverse plasma MPH concentration profiles following oral dosing of different ER formulations.

Rhombencephalitis: pictorial essay.

The term rhombencephalitis refers to inflammatory diseases affecting the hindbrain (brainstem and cerebellum). Rhombencephalitis has a wide variety of etiologies, including infections, autoimmune diseases, and paraneoplastic syndromes. Infection with bacteria of the genus Listeria is the most common cause of rhombencephalitis. Primary rhombencephalitis caused by infection with Listeria spp. occurs in healthy young adults. It usually has a biphasic time course with a flu-like syndrome, followed by brainstem dysfunction; 75% of patients have cerebrospinal fluid pleocytosis, and nearly 100% have an abnormal brain magnetic resonance imaging scan. However, other possible causes of rhombencephalitis must be borne in mind. In addition to the clinical aspects, the patterns seen in magnetic resonance imaging can be helpful in defining the possible cause. Some of the reported causes of rhombencephalitis are potentially severe and life threatening; therefore, an accurate initial diagnostic approach is important to establishing a proper early treatment regimen. This pictorial essay reviews the various causes of rhombencephalitis and the corresponding magnetic resonance imaging findings, by describing illustrative confirmed cases.

Disulfiram inhibits chocolate self-administration and reinstatement to chocolate seeking in rats

We recently found that the dopamine β-hydroxylase (DBH) inhibitor, nepicastat, reduced chocolate self-administration and reinstatement of chocolate-seeking behavior in free-fed rats and self-administration of regular food pellets in food-restricted rats. Here we show that disulfiram reproduced all these effects but exhibited a considerably different time-course. Wistar rats were trained to lever-respond for a chocolate solution (free-fed rats) or regular food pellets (food-restricted rats) under the Fixed Ratio (FR) 10 (FR10) schedule of reinforcement. Once lever-responding stabilized, rats were exposed to sessions under FR10 and Progressive Ratio (PR) schedule and reinstatement sessions. Acutely administered disulfiram (0, 25, 50, and 100mg/kg, i.p.) inhibited, with similar potency and effectiveness, lever-responding for chocolate solution and regular food pellets under the FR10 schedule. Disulfiram-induced inhibition of lever-responding for chocolate solution and regular food pellets showed a biphasic time-course: an early inhibition at 2h, which subsided within 24h, and a second long-lasting inhibition from 48 to 96h. Administered 48h beforehand, disulfiram reduced lever-responding for chocolate solution under the PR schedule and prevented cue-induced reinstatement of chocolate-seeking behavior. Spontaneous locomotor activity was reduced at 2 but not 48h after disulfiram administration. These results indicate that disulfiram reduced food seeking and consumption elicited by high palatability or appetite. It is suggested that the delayed inhibitory effect of disulfiram is likely mediated by a novel mechanism distinct from blockade of DBH or aldehyde dehydrogenase.

Peri-infarct depolarizations during focal ischemia in the awake Spontaneously Hypertensive Rat. Minimizing anesthesia confounds in experimental stroke

Anesthesia profoundly impacts peri-infarct depolarizations (PIDs), but only one prior report has described their monitoring during experimental stroke in awake animals. Since temporal patterns of PID occurrence are model specific, the current study examined PID incidence during focal ischemia in the awake Spontaneously Hypertensive Rat (SHR), and documented the impact of both prior and concurrent isoflurane anesthesia. For awake recordings, electrodes were implanted under isoflurane anesthesia 1day to 5weeks prior to occlusion surgery. Rats were then subjected to permanent or transient (2h) tandem occlusion of the middle cerebral and ipsilateral common carotid arteries, followed by PID monitoring for up to 3days. Comparison perfusion imaging studies evaluated PID-associated hyperemic transients during permanent ischemia under anesthesia at varied intervals following prior isoflurane exposure. Prior anesthesia attenuated PID number at intervals up to 1week, establishing 2weeks as a practical recovery duration following surgical preparation to avoid isoflurane preconditioning effects. PIDs in awake SHR were limited to the first 4h after permanent occlusions. Maintaining anesthesia during this interval reduced PID number, and prolonged their occurrence through several hours following anesthesia termination. Although PID number otherwise correlated with infarct size, PID suppression by anesthesia was not protective in the absence of reperfusion. PIDs persisted up to 36h after transient occlusions. These results differ markedly from the one previous report of such monitoring in awake Sprague–Dawley rats, which found an extended biphasic PID time course during 24h after both permanent and transient filament occlusions. PID occurrence closely reflects the time course of infarct progression in the respective models, and may be more useful than absolute PID number as an index of ongoing pathology.

Generation of cortical neurons from human induced-pluripotent stem cells by biodegradable polymeric microspheres loaded with priming factors

Ischemic stroke is often associated with loss of cortical neurons leading to various neurological deficits. A cell replacement based on stem cell transplantation to repair the damaged brain requires the generation of specific neuronal subtypes. Recently, induced pluripotent stem cells have been used to generate various subtypes of neurons in vitro for transplantation in stroke-damaged brains. However, whether these cells can be primed as neuronal precursors to become cortical projection neurons by means of biomaterials releasing differentiation factors is not known. Here, we report that microspheres of biodegradable poly(ester-amide) composed of adipic acid, L-phenyl-alanine and 1,4-butanediol, loaded with differentiation factors, can be used to fate human induced pluripotent stem cell-derived long-term expandable neuroepithelial-like stem cells to cortical projection neurons. The three factors, Wnt3A, BMP4 and cyclopamine, were released from loaded microspheres over at least one month following biphasic dynamic time course, promoting cortical differentiation of the cells in vitro. Microspheres did not evoke significant inflammatory response after transplantation into intact rodent brain. Our study shows the potential of biodegradable polymer microspheres to promote neuronal differentiation by continuous release of factors, thereby creating the appropriate microenvironment. This new strategy may improve the efficacy of stem cell-based therapeutic approaches.

Native CB1 receptor affinity, intrinsic activity and accumbens shell dopamine stimulant properties of third generation SPICE/K2 cannabinoids: BB-22, 5F-PB-22, 5F-AKB-48 and STS-135

In order to investigate the in vivo dopamine (DA) stimulant properties of selected 3rd generation Spice/K2 cannabinoids, BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, their in vitro affinity and agonist potency at native rat and mice CB1 receptors was studied. The compounds bind with high affinity to CB1 receptors in rat cerebral cortex homogenates and stimulate CB1-induced [35S]GTPγS binding with high potency and efficacy. BB-22 and 5F-PB-22 showed the lowest Ki of binding to CB1 receptors (0.11 and 0.13 nM), i.e., 30 and 26 times lower respectively than that of JWH-018 (3.38 nM), and a potency (EC50, 2.9 and 3.7 nM, respectively) and efficacy (Emax, 217% and 203%, respectively) as CB1 agonists higher than JWH-018 (EC50, 20.2 nM; Emax, 163%). 5F-AKB-48 and STS-135 had higher Ki for CB1 binding, higher EC50 and lower Emax as CB1 agonists than BB-22 and 5F-PB-22 but still comparatively more favourable than JWH-018.The agonist properties of all the compounds were abolished or drastically reduced by the CB1 antagonist/inverse agonist AM251 (0.1 μM). No activation of G-protein was observed in CB1-KO mice. BB-22 (0.003–0.01 mg/kg i.v.) increased dialysate DA in the accumbens shell but not in the core or in the medial prefrontal cortex, with a bell shaped dose–response curve and an effect at 0.01 mg/kg and a biphasic time-course. Systemic AM251 (1.0 mg/kg i.p.) completely prevented the stimulant effect of BB-22 on dialysate DA in the NAc shell. All the other compounds increased dialysate DA in the NAc shell at doses consistent with their in vitro affinity for CB1 receptors (5F-PB-22, 0.01 mg/kg; 5F-AKB-48, 0.1 mg/kg; STS-135, 0.15 mg/kg i.v.). 3rd generation cannabinoids can be even more potent and super-high CB1 receptor agonists compared to JWH-018. Future research will try to establish if these properties can explain the high toxicity and lethality associated with these compounds.

Characterization of V0162, a new long-acting antagonist at human M3 muscarinic acetylcholine receptors

The anticholinergic properties of the mequitazine enantiomer V0162 make it a drug candidate for the treatment of chronic obstructive airway diseases. Here, we compared V0162’s in vitro pharmacological activity at recombinant human M3 muscarinic acetylcholine receptors (hM3Rs) with that of other anticholinergics, using (i) a radioligand binding assay, (ii) a functional reporter gene assay and (iii) a bronchoconstriction inhibition assay on human bronchial preparations. V0162 had high affinity for hM3Rs, with a pKi varying from 9.01 after a 2h incubation to 9.21 after 23h. The other mequitazine enantiomer (V0114) was less potent. V0162 displayed rapid off-kinetics and a biphasic time course of binding. V0162 was found to be an antagonist behaving as an inverse agonist for hM3R-mediated reporter gene activation, with much the same efficacy as atropine, ipratropium and tiotropium. However, in contrast to ipratropium and atropine, V0162’s inhibitory potency was only slightly affected by compound washout. V0162 antagonized acetylcholine-mediated contractions in a human bronchial preparation; the pA2 values increased with the incubation time (up to 2h). Moreover, there was a progressive increase in V0162’s ability to inhibit electrically-induced contractions, which persisted after compound washout. In conclusion, V0162 is the most active mequitazine enantiomer at hM3Rs and shows a complex pattern of binding to the membrane compartment. These particular features may be of therapeutic value when persistent antagonism at hM3Rs is required.