Alpha-2a Research Articles

Safety and cardiovascular effects of multiple-dose administration of aripiprazole and olanzapine in a randomised clinical trial.

To assess adverse events (AEs) and safety of aripiprazole (ARI) and olanzapine (OLA) treatment. Twenty-four healthy volunteers receiving five daily oral doses of 10 mg ARI and 5 mg OLA in a crossover clinical trial were genotyped for 46 polymorphisms in 14 genes by qPCR. Drug plasma concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Blood pressure (BP) and 12-lead electrocardiogram were measured in supine position. AEs were also recorded. ARI decreased diastolic BP on the first day and decreased QTc on the third and fifth day. OLA had a systolic and diastolic BP, heart rate and QTc lowering effect on the first day. Polymorphisms in ADRA2A, COMT, DRD3 and HTR2A genes were significantly associated to these changes. The most frequent adverse drug reactions (ADRs) to ARI were somnolence, headache, insomnia, dizziness, restlessness, palpitations, akathisia and nausea while were somnolence, dizziness, asthenia, constipation, dry mouth, headache and nausea to OLA. Additionally, HTR2A, HTR2C, DRD2, DRD3, OPRM1, UGT1A1 and CYP1A2 polymorphisms had a role in the development of ADRs. OLA induced more cardiovascular changes; however, more ADRs were registered to ARI. In addition, some polymorphisms may explain the difference in the incidence of these effects among subjects.

α1-Adrenergic Receptors in Neurotransmission, Synaptic Plasticity, and Cognition.

α1-adrenergic receptors are G-Protein Coupled Receptors that are involved in neurotransmission and regulate the sympathetic nervous system through binding and activating the neurotransmitter, norepinephrine, and the neurohormone, epinephrine. There are three α1-adrenergic receptor subtypes (α1A, α1B, α1D) that are known to play various roles in neurotransmission and cognition. They are related to two other adrenergic receptor families that also bind norepinephrine and epinephrine, the β- and α2-, each with three subtypes (β1, β2, β3, α2A, α2B, α2C). Previous studies assessing the roles of α1-adrenergic receptors in neurotransmission and cognition have been inconsistent. This was due to the use of poorly-selective ligands and many of these studies were published before the characterization of the cloned receptor subtypes and the subsequent development of animal models. With the availability of more-selective ligands and the development of animal models, a clearer picture of their role in cognition and neurotransmission can be assessed. In this review, we highlight the significant role that the α1-adrenergic receptor plays in regulating synaptic efficacy, both short and long-term synaptic plasticity, and its regulation of different types of memory. We will also present evidence that the α1-adrenergic receptors, and particularly the α1A-adrenergic receptor subtype, are a potentially good target to treat a wide variety of neurological conditions with diminished cognition.

Vascular α1A Adrenergic Receptors as a Potential Therapeutic Target for IPAD in Alzheimer's Disease.

Drainage of interstitial fluid from the brain occurs via the intramural periarterial drainage (IPAD) pathways along the basement membranes of cerebral capillaries and arteries against the direction of blood flow into the brain. The cerebrovascular smooth muscle cells (SMCs) provide the motive force for driving IPAD, and their decrease in function may explain the deposition of amyloid-beta as cerebral amyloid angiopathy (CAA), a key feature of Alzheimer’s disease. The α-adrenoceptor subtype α1A is abundant in the brain, but its distribution in the cerebral vessels is unclear. We analysed cultured human cerebrovascular SMCs and young, old and CAA human brains for (a) the presence of α1A receptor and (b) the distribution of the α1A receptor within the cerebral vessels. The α1A receptor was present on the wall of cerebrovascular SMCs. No significant changes were observed in the vascular expression of the α1A-adrenergic receptor in young, old and CAA cases. The pattern of vascular staining appeared less punctate and more diffuse with ageing and CAA. Our results show that the α1A-adrenergic receptor is preserved in cerebral vessels with ageing and in CAA and is expressed on cerebrovascular smooth muscle cells, suggesting that vascular adrenergic receptors may hold potential for therapeutic targeting of IPAD.

Treatment of Coronavirus Disease 2019 Patients With Prolonged Postsymptomatic Viral Shedding With Leflunomide: A Single-center Randomized Controlled Clinical Trial.

ObjectiveTo evaluate the efficacy and safety of leflunomide, an approved dihydroorotate dehydrogenase inhibitor, to treat COVID-19 patients with prolonged post-symptomatic viral shedding.MethodsWe conducted a prospective, randomized, controlled, open-label trial involving hospitalized adult COVID-19 patients with prolonged PCR positivity. Patients were randomly assigned to receive either leflunomide (50 mg, q12h, three consecutive times, orally; then 20 mg, once daily for 8 days), in addition to nebulized interferon alpha 2a (IFN α-2a, 3 million IU each time, twice daily for 10 days), or nebulized IFN α-2a alone for 10 days. The primary end point was the duration of viral shedding.ResultsA total of 50 COVID-19 patients with prolonged PCR positivity were randomized into 2 groups; 26 were assigned to the leflunomide group, and 24 were assigned to the interferon alone group. Treatment with leflunomide was not associated with a difference from the interferon alone group in the duration of viral shedding (hazard ratio for negative RT-PCR, 0.70; 95% confidence interval, 0.391-1.256; P=0.186). In addition, the patients given leflunomide did not have a substantially shorter length of hospital stay than patients treated with interferon alone, with median (IQRs) durations of 29.0 (19.3-47.3) days and 33.0 (29.3-42.8) days, respectively, P=0.170. Two leflunomide recipients were unable to complete the full 10-day course of administration due to adverse events.ConclusionsIn COVID-19 patients with prolonged PCR positivity, no benefit in terms of the duration of viral shedding was observed with the combined treatment of leflunomide and IFN α-2a beyond IFN α-2a alone.

New insights on the arylpiperazinylalkyl pyridazinone ET1 as potent antinociceptive and anti-inflammatory agent

Pyridazine derivatives, such as arylpiperazinylalkyl pyridazinones, display antinociceptive effects to thermal and chemical stimuli. Here, we extended our previous knowledge on the pharmacological profile of 4-amino-6-methyl-2-(3-(4-(4-methylcyclohexa-1,3-dien-1-yl)piperazin-1-yl)propyl)-5-vinylpyridazin-3(2H)-one, here referred as ET1, paving the way for the comprehension of its complete mechanism of action. To this aim, we have evaluated the mouse behavioural responses in several animal models of pain, the effect of ET1 in the murine model of zymosan-induced paw oedema and air-pouch, assessing the cytokines and the cellular phenotype and finally, an in vitro radioligand binding study was performed on a panel of 30 different receptors. In the formalin test, ET1 reduced both neurogenic and inflammatory phase of nociception induced by the aldehyde. Similarly, ET1 strongly reduced paw licking response in the capsaicin test, the abdominal stretching in the writhing test and the carrageenan-induced thermal hyperalgesia. ET1 also evoked a long-lasting reduction of thermal hyperalgesia. Furthermore, ET1 produced a long-lasting anti-inflammatory effect in the zymosan-induced mouse paw oedema and air-pouch through the selective inhibition of inflammatory monocytes recruitment and the modulation of IL-1β, IL-6, TNF-α and MCP-1. Binding experiments confirmed an inhibitory effect on adrenergic α1A, α1B and α2A receptors subtypes and, for the first time, a moderate affinity was observed for the following receptors: histamine H1, imidazoline I2, sigma non-opioid intracellular receptor 1 and σ2. These results prompt ET1 as a potent analgesic and anti-inflammatory agent, and support the possibility that it may be suitable for clinical applications in a wide-range of inflammatory-based diseases.

Comparative Analysis of Cardiac Effects of α1A-Adrenoreceptor Stimulation In Vivo and Ex Vivo in Newborn Rats.

The study examined the effects of α1A-adrenoceptor stimulation on chronotropic function of Langendorff-perfused isolated heart ex vivo and on cardiac chronotropy in vivo in 7-day-old rats. α1A-Adrenergic receptor agonist A-61603 reduced heart chronotropy only in the whole organism. No chronotropic effects of selective stimulation of α1A-adrenergic receptors on isolated hearts were observed in ex vivo experiments. These findings suggest that α1A-adrenergic receptors are not implicated in HR regulation in newborn rats. Bradycardia induced by activation of these receptors in vivo is most likely associated with reflex influences on the heart and changes in the vascular tone in the whole organism.

Abstract 408: An Essential Protective Role for Cardiomyocyte Alpha1a-adrenergic Receptors in a Mouse Model of Myocardial Infarction

Our previous work reveals that dabuzalgron, an oral agonist of alpha-1A adrenergic receptors (α1A-ARs) protects rodents from pathological cardiac stress, a finding in agreement with genetic studies of global α1A-AR overexpression/inactivation. However, it remains largely obscure whether cardiomyocyte-specific actions of α1A-ARs alone determines these cardioprotective effects. To test this essential gap in knowledge, we generated a new mouse line lacking α1A-AR only on cardiomyocytes by crossing αMHC-cre mice with floxed α1A-KO mice (CMKO= cre+ fl/fl, CMWT= cre-fl/fl). After validating specific deletion, baseline features were measured by conscious echo. No overt structural or functional anomaly was identified in either group. Fractional Shortening (FS, %) was virtually identical in 2 groups (51.7±0.7 vs. 52.4±5.3, p=NS). Next, both groups were subjected to permanent left anterior descending (LAD) ligation. By day 14, CMKO had significantly higher mortality (9/15 vs 2/13, p=0.025). To determine early changes that might account for this difference, hearts were harvested at day 3 post-ligation from another cohort. HW did not differ between two groups (7.0±1.1 vs. 7.2±1.1 mg/g·BW, p=NS), neither did LW (8.0±2.1 vs. 7.2±1.0 mg/g·BW, p=NS). Serum troponin level was numerically higher in CMKO mice (8.4±12.4 vs 4.8±6.2 ng/mL,p=0.38). CMKO mice had 51% larger injury (infarcted and leukocyte-infiltrating area) compared to CMWT (31.0±10.5 vs. 20.5±4.9% of LV area, p=0.05). Our preliminary postmortem examination suggested a higher trend of cardiac rupture in injured CMKO (3/5 vs 0/5). To test myocardial biomechanical properties between 2 groups, ventricles from mice 3d post ligation were then subjected to biaxial mechanical stress test. Myocardium from infracted CMKO mice exhibited 70% more stiffness (dt Stress/dt Strain) CMWT (190±34.29 vs.111±42 kPa, p=0.004). Collectively, our results demonstrate that mice deficient of α1A-AR on cardiomyocytes had lower late survival, more profound early pathological injury & adverse mechanical properties after LAD ligation, suggesting that activation of cardiomyocyte α1A-ARs plays an essential role in protecting experimental myocadiac infarction, holds the promise of future clinical implications.

Mirtazapine, an α2 Antagonist-Type Antidepressant, Reverses Pain and Lack of Morphine Analgesia in Fibromyalgia-Like Mouse Models.

Treatment of fibromyalgia is an unmet medical need; however, its pathogenesis is still poorly understood. In a series of studies, we have demonstrated that some pharmacological treatments reverse generalized chronic pain but do not affect the lack of morphine analgesia in the intermittent cold stress (ICS)-induced fibromyalgia-like pain model in mice. Here we report that repeated intraperitoneal treatments with mirtazapine, which is presumed to disinhibit 5-hydroxytriptamine (5-HT) release and activate 5-HT1 receptor through mechanisms of blocking presynaptic adrenergic α2 and postsynaptic 5-HT2 and 5-HT3 receptors, completely reversed the chronic pain for more than 4 to 5 days after the cessation of treatments. The repeated mirtazapine treatments also recovered the morphine analgesia after the return of nociceptive threshold to the normal level. The microinjection of small interfering RNA (siRNA) adrenergic α2a receptor (ADRA2A) into the habenula, which showed a selective upregulation of α2 receptor gene expression after ICS, reversed the hyperalgesia but did not recover the morphine analgesia. However, both reversal of hyperalgesia and recovery of morphine analgesia were observed when siRNA ADRA2A was administered intracerebroventricularly. As the habenular is reported to be involved in the emotion/reward-related pain and hypoalgesia, these results suggest that mirtazapine could attenuate pain and/or augment hypoalgesia by blocking the habenular α2 receptor after ICS. The recovery of morphine analgesia in the ICS model, on the other hand, seems to be mediated through a blockade of α2 receptor in unidentified brain regions. SIGNIFICANCE STATEMENT: This study reports possible mechanisms underlying the complete reversal of hyperalgesia and recovery of morphine analgesia by mirtazapine, a unique antidepressant with adrenergic α2 and serotonergic receptor antagonist properties, in a type of intermittently repeated stress (ICS)-induced fibromyalgia-like pain model. Habenula, a brain region which is related to the control of emotional pain, was found to play key roles in the antihyperalgesia, whereas other brain regions appeared to be involved in the recovery of morphine analgesia in the ICS model.

Pilot study of tenofovir disoproxil fumarate and pegylated interferon-alpha 2a add-on therapy in Japanese patients with chronic hepatitis B.

A prospective pilot study of tenofovir disoproxil fumarate (TDF) and pegylated interferon alpha 2a (P-IFN) add-on therapy was conducted to evaluate its efficacy in reducing viral antigen levels in Japanese patients with chronic hepatitis B (UMIN 000020179). Patients with chronic hepatitis B receiving maintenance TDF therapy and exhibiting hepatitis B surface antigen (HBsAg) level > 800IU/ml were divided into two arms. P-IFN was added for 48weeks in the add-on arm (n = 32), while TDF monotherapy was maintained in the control arm (n = 51). Both groups were followed for 96weeks after baseline measurements. Almost all patients in the control arm displayed a slow and constant reduction in HBsAg during follow-up. In contrast, roughly half of the add-on arm exhibited a sharp decline in HBsAg during P-IFN administration, which disappeared after halting P-IFN. At 96weeks after baseline, 41% (13/32) of patients in the add-on arm had shown a rapid decrease in HBsAg, versus 2% (1/51) in the control arm (p < 0.001). Add-on therapy and increased cytotoxic T-cell response were significant factors associated with a rapid decrease in HBsAg according to multivariate analysis. In addition, higher HB core-related antigen (HBcrAg) level at baseline (p = 0.001) and add-on therapy (p = 0.036) were significant factors associated with a rapid reduction in HBcrAg. TDF and P-IFN add-on therapy in Japanese patients with chronic hepatitis B facilitated rapid decreases in HBsAg and HBcrAg. Further studies are needed to improve early HBsAg clearance rate.

Effect of biphasic temperature regime on therapeutic recombinant protein production in the green alga Chlamydomonas reinhardtii

Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-α2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-α2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 °C to 15 °C in mid-exponential growth phase increased the accumulation of Cr.IFN-α2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 °C to 35 °C accelerated cell growth, while negatively impacting the production of Cr.IFN-α2a. After a two-step chromatography purification, the Cr.IFN-α2a produced was estimated to be 53% pure with a yield of 90 μg/L of culture. The amino acid sequence of Cr.IFN-α2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-α2a was found to be 10 times lower than the human IFN-α2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-α2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-α2a in C. reinhardtii.

From the Literature

From the Literature

Interaction between Mesocortical and Mesothalamic Catecholaminergic Transmissions Associated with NMDA Receptor in the Locus Coeruleus

Noncompetitive N-methyl-D-aspartate/glutamate receptor (NMDAR) antagonists contribute to the pathophysiology of schizophrenia and mood disorders but improve monoaminergic antidepressant-resistant mood disorder and suicidal ideation. The mechanisms of the double-edged sword clinical action of NMDAR antagonists remained to be clarified. The present study determined the interaction between the NMDAR antagonist (MK801), α1 adrenoceptor antagonist (prazosin), and α2A adrenoceptor agonist (guanfacine) on mesocortical and mesothalamic catecholaminergic transmission, and thalamocortical glutamatergic transmission using multiprobe microdialysis. The inhibition of NMDAR in the locus coeruleus (LC) by local MK801 administration enhanced both the mesocortical noradrenergic and catecholaminergic coreleasing (norepinephrine and dopamine) transmissions. The mesothalamic noradrenergic transmission was also enhanced by local MK801 administration in the LC. These mesocortical and mesothalamic transmissions were activated by intra-LC disinhibition of transmission of γ-aminobutyric acid (GABA) via NMDAR inhibition. Contrastingly, activated mesothalamic noradrenergic transmission by MK801 enhanced intrathalamic GABAergic inhibition via the α1 adrenoceptor, resulting in the suppression of thalamocortical glutamatergic transmission. The thalamocortical glutamatergic terminal stimulated the presynaptically mesocortical catecholaminergic coreleasing terminal in the superficial cortical layers, but did not have contact with the mesocortical selective noradrenergic terminal (which projected terminals to deeper cortical layers). Furthermore, the α2A adrenoceptor suppressed the mesocortical and mesothalamic noradrenergic transmissions somatodendritically in the LC and presynaptically/somatodendritically in the reticular thalamic nucleus (RTN). These discrepancies between the noradrenergic and catecholaminergic transmissions in the mesocortical and mesothalamic pathways probably constitute the double-edged sword clinical action of noncompetitive NMDAR antagonists.

Disseminated Pleural Malignant Melanoma

A 68-year-old man was admitted to our hospital with a dyspnea. On physical examination the patient was not distressed. Chest X-ray demonstrated an extensive left pleural effusion (A). Pleural aspiration showed an exsudate with elevated LDH of 464 U/L (0-100 U/l). The diagnostic thoracoscopy revealed disseminated black nodules on the visceral and parietal pleura (B) and a pleurodesis was performed after obtaining multiple biopsies. The melanoma cells showed a strong cytoplasmatic HMB-45 reaction (C). Immunohistochemical staining revealed a malignant melanoma with expression of PD-L1 in 3% of the tumor cells, the moleculargenetic examination proofed a BRAF V600 mutation. The patient was treated with the kinase inhibitor Trametinib and the tyrosinkinase inhibitor Tafiniar. He had initially a good response but died 10 months later after presenting to our clinic. 3 years prior the patient had a removal of a malign melanoma on the right upper thorax wall and the surgical exploration showed micrometastases in the sentinel lymphnode of the right axilla. (SSM Clark Level IV). At that time a immunotherapy with interferon alpha 2a was initiated.

Single Nucleotide Polymorphism Analysis in HIV and Kaposi's Sarcoma Disease by Microarray Technique.

Emergence of Kaposi's Sarcoma in the cases other than HIV, following the use of immunosuppressant drugs, demonstrates that it is related to weak immunity. The fact that this malignancy does not occur in every HIV-positive patient suggests that genetic predisposition may also be effective. Replacement of one of the base pairs of adenine, guanine, cytosine, and thymine that constitute the DNA sequence in the human genome with another base pair can affect susceptibility to disease, response to treatment, and immunity. The purpose of this study is to analyze the Single Nucleotide Polymorphism that could predispose to Kaposi's sarcoma of an HIV-infected patient and to identify which nucleotides such SNPs correspond to, using the microarray technology. The blood samples of individuals, one of whom was diagnosed with Kaposi's Sarcoma HIV (+) visiting the outpatient clinic of infectious diseases polyclinic of Harran University Research and Practice Hospital and of a healthy individual with no Kaposi's Sarcoma, were used in the study. Following the DNA isolation of the blood samples taken from the respective individuals, a SNP analysis was conducted on the microarray device. 204,000 SNPs obtained were scanned later on in the databases in an attempt to identify the SNPs related to Kaposi's Sarcoma. In the 204,000 SNP screenings, we scrutinized the SNPs that differ in the case of Kaposi's Sarcoma [KS (+) and HIV (+)] on the basis of Control [KS(-) and HIV(-)] and HIV+ [KS(-)], and two SNPs of the ENDRA gene, three SNPs of the ADRA1A gene, six SNPs of the STIM1 gene, four SNPs of the EFNB2 gene, and one SNP of the CD209 gene were found to be different. However, when it comes to all SNPs (all the 204.000 SNPs) screened in terms of allele, it was observed that the AA and BB alleles were lower in the patient with Kaposi's Sarcoma [KS (+) and HIV (+)] compared to other groups and AB alleles were found to be higher than others in the patient with Kaposi's sarcoma [KS] (+) and HIV (+)]. In the microarray study we have conducted, 204,000 SNPs were screened for Control (HIV-) HIV (+) and HIV (+) patient with Kaposi's Sarcoma. It was found that 32,362 of those SNPs had different alleles in the Kaposi's Sarcoma [KS + HIV (+)] patient, while they had the same ones in the control [KS (-) and HIV (-)] and HIV + [KS (-)] group. 16 of the 32,362 SNPs took place among the genes related to Kaposi's Sarcoma. In the cases of Kaposi's Sarcoma with suspected diagnosis, it can be used as a beneficial laboratory test.

2090-P: Noradrenergic Stimulus as a Potential Inducer of Human ß-Cell Dedifferentiation

β dedifferentiation is recognized as one of the main mechanisms responsible for the disappearance of β cells from pancreatic islets of diabetic subjects, but the underlying process is still unclear. Noradrenergic innervation is known to inhibit insulin secretion, and in pancreas of diabetic subjects a 3-fold increase of noradrenergic fibers was observed compared to nondiabetic subjects, directly related to the % of dedifferentiated cells within the islets. The increase in fibers has been associated with worsening β cell function, suggesting a role in the pathogenesis of the disease and perhaps in the process of dedifferentiation. The aim of our study was to use in vitro human β cell models to explore a direct role of innervation in the process of noradrenergic dedifferentiation. The human β cell line EndoC-βH1 was exposed to different concentrations of noradrenaline (NA) (0.4 μM, 4 μM and 40 μM) for 24 and 72 h. The expression of β cell dedifferentiation markers was assessed before and after stimulation of the α2A adrenergic receptor, by morphology (immunofluorescence (IF)), qPCR and ELISA. FGF2 (100 ng/mL), a known inducer of β dedifferentiation in vitro, was used as a positive control. NA treatment did not have any impact on β cell viability, while, as expected, it slightly reduced insulin secretion after 24 h. The expression of β cell dedifferentiation markers (ALDH1a3) was increased by approximately 30% after stimulation with NA, while mature β cell marker were reduced (NKX6.1) up to 60% after 72 h (by IF detection). Moreover, the mRNA expression of FOXO1, the main transcription factor involved in the dedifferentiation process, was reduced up to 35.4% after 72 h stimulation with NA. We observed a dose and time dependent pattern in all the experiments. In conclusion, the data suggest a direct role of noradrenergic fibers in the dedifferentiation process, potentially identifying a new target for the prevention and therapy of type 2 diabetes. Disclosure F. Cinti: None. F. Fantuzzi: None. A. Carfí: None. T. Mezza: None. C. Cefalo: None. S. Moffa: None. G. Sorice: None. A. Giaccari: Speaker’s Bureau; Self; Amgen, AstraZeneca, Eli Lilly and Company, Merck Sharp &amp; Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi-Aventis. M. Cnop: None. Funding European Foundation for the Study of Diabetes

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

New Serotoninergic Ligands Containing Indolic and Methyl Indolic Nuclei: Synthesis and In Vitro Pharmacological Evaluation.

Serotonin is an important biogenic amine and is implicated in wideranging physiological and physiopathological processes. Pharmacological manipulation of the serotoninergic system is believed to have a great therapeutic potential. In order to identify selective ligands for 5-HT1A, 5-HT2A and 5-HT2C receptors two series of 4-substituted piperazine derivatives, bearing indolic or methyl indolic nuclei, were synthesized. All the compounds, synthesized by standard solution methods, were evaluated for 5- HT1A, 5-HT2A and 5-HT2C receptors. The highest affine and selective compounds have been evaluated also on dopaminergic (D1 and D2) and adrenergic (α1A and α2A) receptors. Several of the newly synthesized molecules showed affinity in the nanomolar range for 5- HT1A, 5-HT2A and 5-HT2C receptors and moderate to no affinity for other relevant receptors (D1, D2, α1A and α2A). Compounds 7f and 10a showed a nanomolar affinity towards 5-HT1A with an in vitro pharmacologic profile compatible with antipsychotic drugs.

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

GPCR Kinase 2 (GRK2) Phosphosphorylation of the Alpha 2A Adrenergic Receptor in Intact Cells

The α2A‐adrenergic receptor (α2A‐AR) is activated by the catecholamine norepinephrine and plays a variety of roles including neuromodulation, vasoconstriction in the cardiovascular system, regulation of insulin secretion and platelet aggregation. Platelet α2A‐AR serves as an excellent substrate for agonist‐dependent phosphorylation by G protein‐coupled receptor (GPCR) kinase 2 (GRK2) in vitro. α2A‐AR is structurally distinct from the well‐characterized β2‐adrenergic receptor (β2AR) in possessing a large (~100 amino acid) third intracellular loop containing four successive serine residues that serve as phospho‐acceptors. When overexpressed with GRK2, the receptor is phosphorylated at all 4 phospho‐sites. Our long‐term goal is to understand how GRK2 interacts with and is activated by GPCRs. We have mapped residues on GRK2 that are required for phosphorylation of β2AR and recruitment to α2A‐AR in intact cells. We wish to assay phosphorylation of α2A‐AR in intact cells and because no commercially available α2A‐AR phosphorylation site antibodies currently exist, we set out to develop such a reagent. An antibody that recognizes α2A‐AR phosphorylation at serines 296–299 was generated and characterized. This antibody detects an epinephrine‐stimulated signal that is increased two‐fold by transfection of GRK2 in COS‐7 cells. We also found that the α2A‐AR was phosphorylated relatively well in the absence of epinephrine and transfected GRK2 suggesting that α2A‐AR is a good substrate for an endogenous COS‐7 cell kinase. Mutagenesis of the receptor has allowed us to measure agonist‐ and GRK‐dependent phosphorylation of α2A‐AR. We are now in a position to characterize GRK2 phosphorylation of this receptor.Support or Funding InformationThis work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).

Zerumbone Modulates α2A-Adrenergic, TRPV1, and NMDA NR2B Receptors Plasticity in CCI-Induced Neuropathic Pain In Vivo and LPS-Induced SH-SY5Y Neuroblastoma In Vitro Models.

Zerumbone has shown great potential in various pathophysiological models of diseases, particularly in neuropathic pain conditions. Further understanding the mechanisms of action is important to develop zerumbone as a potential anti-nociceptive agent. Numerous receptors and pathways function to inhibit and modulate transmission of pain signals. Previously, we demonstrated involvement of the serotonergic system in zerumbone’s anti-neuropathic effects. The present study was conducted to determine zerumbone’s modulatory potential involving noradrenergic, transient receptor potential vanilloid type 1 (TRPV1) and N-methyl-D-aspartate (NMDA) receptors in chronic constriction injury (CCI)-induced in vitro and lipopolysaccharide (LPS)-induced SH-SY5Y in vitro neuroinflammatory models. von Frey filament and Hargreaves plantar tests were used to assess allodynia and hyperalgesia in the chronic constriction injury-induced neuropathic pain mouse model. Involvement of specific adrenoceptors were investigated using antagonists— prazosin (α1-adrenoceptor antagonist), idazoxan (α2-adrenoceptor antagonist), metoprolol (β1-adrenoceptor antagonist), ICI 118,551 (β2-adrenoceptor antagonist), and SR 59230 A (β3-adrenoceptor antagonist), co-administered with zerumbone (10 mg/kg). Involvement of excitatory receptors; TRPV and NMDA were conducted using antagonists capsazepine (TRPV1 antagonist) and memantine (NMDA antagonist). Western blot was conducted to investigate the effect of zerumbone on the expression of α2A-adrenoceptor, TRPV1 and NMDA NR2B receptors in CCI-induced whole brain samples of mice as well as in LPS-induced SH-SY5Y neuroblastoma cells. Pre-treatment with α1- and α2-adrenoceptor antagonists significantly attenuated both anti-allodynic and anti-hyperalgesic effects of zerumbone. For β-adrenoceptors, only β2-adrenoceptor antagonist significantly reversed the anti-allodynic and anti-hyperalgesic effects of zerumbone. β1-adrenoceptor antagonist only reversed the anti-allodynic effect of zerumbone. The anti-allodynic and anti-hyperalgesic effects of zerumbone were both absent when TRPV1 and NMDA receptors were antagonized in both nociceptive assays. Zerumbone treatment markedly decreased the expression of α2A-adrenoceptor, while an up-regulation was observed of NMDA NR2B receptors. Expression of TRPV1 receptors however did not significantly change. The in vitro study, representing a peripheral model, demonstrated the reduction of both NMDA NR2B and TRPV1 receptors while significantly increasing α2A-adrenoceptor expression in contrast to the brain samples. Our current findings suggest that the α1-, α2-, β1- and β2-adrenoceptors, TRPV1 and NMDA NR2B are essential for the anti-allodynic and antihyperalgesic effects of zerumbone. Alternatively, we demonstrated the plasticity of these receptors through their response to zerumbone’s administration.