Alpha-adrenergic Receptors Research Articles

The prostaglandin pathway is activated in patients who fail medical therapy for benign prostatic hyperplasia with lower urinary tract symptoms.

Little is known about how benign prostatic hyperplasia (BPH) develops and why patients respond differently to medical therapy designed to reduce lower urinary tract symptoms (LUTS). The Medical Therapy of Prostatic Symptoms (MTOPS) trial randomized men with symptoms of BPH and followed response to medical therapy for up to 6 years. Treatment with a 5α-reductase inhibitor (5ARI) or an alpha-adrenergic receptor antagonist (α-blocker) reduced the risk of clinical progression, while men treated with combination therapy showed a 66% decrease in risk of progressive disease. However, medical therapies for BPH/LUTS are not effective in many patients. The reasons for nonresponse or loss of therapeutic response in the remaining patients over time are unknown. A better understanding of why patients fail to respond to medical therapy may have a major impact on developing new approaches for the medical treatment of BPH/LUTS. Prostaglandins (PG) act on G-protein-coupled receptors (GPCRs), where PGE2 and PGF2 elicit smooth muscle contraction. Therefore, we measured PG levels in the prostate tissue of BPH/LUTS patients to assess the possibility that this signaling pathway might explain the failure of medical therapy in BPH/LUTS patients. Surgical BPH (S-BPH) was defined as benign prostatic tissue collected from the transition zone (TZ) of patients who failed medical therapy and underwent surgical intervention to relieve LUTS. Control tissue was termed Incidental BPH (I-BPH). I-BPH was TZ obtained from men undergoing radical prostatectomy for low-volume, low-grade prostatic adenocarcinoma (PCa, Gleason score ≤ 7) confined to the peripheral zone. All TZ tissue was confirmed to be cancer-free. S-BPH patients divided into four subgroups: patients on α-blockers alone, 5ARI alone, combination therapy (α-blockers plus 5ARI), or no medical therapy (none) before surgical resection. I-BPH tissue was subgrouped by prior therapy (either on α-blockers or without prior medical therapy before prostatectomy). We measured prostatic tissue levels of prostaglandins (PGF2α , PGI2 , PGE2 , PGD2 , and TxA2 ), quantitative polymerase chain reaction levels of mRNAs encoding enzymes within the PG synthesis pathway, cellular distribution of COX1 (PTGS1) and COX2 (PTGS2), and tested the ability of PGs to contract bladder smooth muscle in an in vitro assay. All PGs were significantly elevated in TZ tissues from S-BPH patients (n = 36) compared to I-BPH patients (n = 15), regardless of the treatment subgroups. In S-BPH versus I-BPH, mRNA for PG synthetic enzymes COX1 and COX2 were significantly elevated. In addition, mRNA for enzymes that convert the precursor PGH2 to metabolite PGs were variable: PTGIS (which generates PGI2 ) and PTGDS (PGD2 ) were significantly elevated; nonsignificant increases were observed for PTGES (PGE2 ), AKR1C3 (PGF2α ), and TBxAS1 (TxA2 ). Within the I-BPH group, men responding to α-blockers for symptoms of BPH but requiring prostatectomy for PCa did not show elevated levels of COX1, COX2, or PGs. By immunohistochemistry, COX1 was predominantly observed in the prostatic stroma while COX2 was present in scattered luminal cells of isolated prostatic glands in S-BPH. PGE2 and PGF2α induced contraction of bladder smooth muscle in an in vitro assay. Furthermore, using the smooth muscle assay, we demonstrated that α-blockers that inhibit alpha-adrenergic receptors do not appear to inhibit PG stimulation of GPCRs in bladder muscle. Only patients who required surgery to relieve BPH/LUTS symptoms showed significantly increased tissue levels of PGs and the PG synthetic enzymes. Treatment of BPH/LUTS by inhibition of alpha-adrenergic receptors with pharmaceutical α-blockers or inhibiting androgenesis with 5ARI may fail because of elevated paracrine signaling by prostatic PGs that can cause smooth muscle contraction. In contrast to patients who fail medical therapy for BPH/LUTS, control I-BPH patients do not show the same evidence of elevated PG pathway signaling. Elevation of the PG pathway may explain, in part, why the risk of clinical progression in the MTOPS study was only reduced by 34% with α-blocker treatment.

Involvement of Toll-Like Receptor 4 in Decreased Vasopressor Response Following Trauma/Hemorrhagic Shock.

OBJECTIVES:Refractory vascular failure due to the inability of vascular smooth muscle to respond to vasoconstrictors such as phenylephrine is a final common pathway for severe circulatory shock of any cause, including trauma/hemorrhagic shock. Increased inflammation, Toll-like receptor 4 activation, and decreased response of the alpha-1 adrenergic receptors which control vascular tone have been reported in trauma/hemorrhagic shock.HYPOTHESIS:In trauma/hemorrhagic shock, Toll-like receptor 4 activation contributes to vascular failure via decreased bioavailability of adrenergic receptors.DESIGN AND MEASUREMENTS:Trauma/hemorrhagic shock was induced in Wistar rats (laparotomy combined with mean arterial pressure at 40 mm Hg for 90 min followed by 2 hr resuscitation with Lactated Ringers solution). To inhibit Toll-like receptor 4, resatorvid (TAK-242) and resveratrol were used, and plasma was collected. Smooth muscle cells were incubated with lipopolysaccharide (10 ng/mL) or plasma. Inflammatory cytokines were screened using dot-blot. Toll-like receptor 4 and nuclear factor κB activation and cellular localization of the alpha-1 adrenergic receptor were measured by immunofluorescence imaging and Western blot analysis. Clustered regularly interspaced short palindromic repeats/Cas9 was used to knock out Toll-like receptor 4, and calcium influx following stimulation with phenylephrine was recorded.MAIN RESULTS:Trauma/hemorrhagic shock caused a decreased response to phenylephrine, whereas Toll-like receptor 4 inhibition improved blood pressure. Trauma/hemorrhagic shock plasma activated the Toll-like receptor 4/nuclear factor κB pathway in smooth muscle cells. Double labeling of Toll-like receptor 4 and the alpha-1 adrenergic receptor showed that these receptors are colocalized on the cell membrane. Activation of Toll-like receptor 4 caused cointernalization of both receptors. Calcium influx was impaired in cells incubated with trauma/hemorrhagic shock plasma but restored when Toll-like receptor 4 was knocked out or inhibited.CONCLUSIONS:Activation of the Toll-like receptor 4 desensitizes vascular smooth muscle cells to vasopressors in experimental trauma/hemorrhagic shock by reducing the levels of membrane alpha-1 adrenergic receptor.

Alpha and beta adrenergic receptors modulate keratinocyte migration.

Keratinocyte migration into skin wounds is the step of the healing process that correlates with the wound closure rate. Keratinocyte migration, and wound epithelialization are decreased when beta 2-adrenergic receptors (B2AR) are activated by 1 μM epinephrine/adrenaline, resulting in delayed wound healing in human and mouse skin. In the present study, we found paradoxically, that in a subset of keratinocyte strains exposure to low concentrations of epinephrine (0.1 nM) increased, rather than decreased, their migratory rate. We find that both the alpha- and the beta-adrenergic receptors are expressed in human keratinocytes, and expression of alpha-2 AR subtypes demonstrated for the first time. Therefore, we tested if the alpha-AR could be modulating the increased migratory response observed in these cell strains. By using specific inhibitors to alpha-AR, we demonstrated that blocking A2B-AR could reverse the rapid cell migration induced by the 0.1 nM epinephrine. Phosphorylation of ERK was elevated after 1–10 minutes of the low epinephrine treatment and the A2B-AR inhibitor blocked the ERK phosphorylation. The results suggest that both the A2B-AR and B2AR mediate keratinocyte migration, in which with a low level of epinephrine treatment, A2B-AR could alter the B2AR signals and regulate the migration rate.

Mediators of human ureteral smooth muscle contraction-a role for erythropoietin, tamsulosin and Gli effectors.

BackgroundUreteral contractility is a poorly understood process. Contractions have been demonstrated to occur in the smooth muscle layers of the ureter. Previous work suggests the involvement of Gli family proteins and erythropoietin (EPO) in regulating mammalian ureteral smooth muscle contraction. We sought to devise a method by which the effects of these proteins and tamsulosin on distal human ureteral tissue contractility could be investigated to better understand mechanisms regulating human ureteral function.MethodsIRB approval was obtained to procure portions of extraneous distal ureteral tissue from living donor renal transplants. Contractility was measured by placing the tissue in Krebs buffer and stimulating via a uniform electric current. Contractile force was recorded with each stimulation with and without the presence of a Gli inhibitor (GANT61) or EPO. Each ureteral specimen was subsequently fixed and tested by immunohistochemistry to determine Gli, EPO and alpha-adrenergic receptor activity.ResultsElectrical field stimulation successfully elicited contractions in the ureteral tissue. Administering tamsulosin decreased force and duration of ureteral contractions. Inhibiting Gli signaling decreased contractility and EPO decreased ureteral contractile forces within 5 minutes of administration versus untreated controls. Staining confirmed Gli1 protein and α-adrenergic receptor expression in ureteral smooth muscle and epithelial tissue with EPO receptor expression confined to the epithelial layer.ConclusionsDistal ureteral contractile forces are decreased by inhibition of Gli family proteins and the α-adrenergic receptor. EPO acts within five minutes, suggesting ion channel involvement instead of changes in gene expression. Continuing work will elucidate the role of these proteins in coordinating ureteral contractions. This has implications for the use of pharmacologic methods to address ureteral contractility and dysfunctional peristalsis during stone passage, ureteroscopy, in transplant patients and potentially to reduce symptoms from ureteral stents.

Dexmedetomidine maintains blood-brain barrier integrity by inhibiting Drp1-related endothelial mitochondrial dysfunction in ischemic stroke.

Stroke is the second leading cause of death and long-term disability worldwide, which lacks effective treatment. Perioperative stroke is associated with much higher rates of mortality and disability. The neuroprotective role of dexmedetomidine (Dex), a highly selective agonist of alpha2-adrenergic receptor, has been reported in a stroke rat model, and it was found that pretreatment of Dex before stroke could alleviate blood-brain barrier (BBB) breakdown. However, the underlying mechanisms are still unknown. As the brain endothelial cells are the main constituents of BBB and in high demand of energy, mitochondrial function of endothelial cells plays an important role in the maintenance of BBB. Given that dynamin-related protein 1 (Drp1) is a protein mediating mitochondrial fission, with mitochondrial fusion that balances mitochondrial morphology and ensures mitochondria function, the present study was designed to investigate the possible role of Drp1 in endothelial cells involved in the neuroprotective effects of Dex in ischemic stroke. Our results showed that preconditioning with Dex reduced infarction volume, alleviated brain water content and BBB damage, and improved neurological scores in middle cerebral artery occlusion rats. Meanwhile, Dex enhanced cell activity and decreased cell apoptosis in oxygen-glucose deprivation human brain microvascular endothelial cells in vitro. These protective effects of Dex were correlated with the mitochondrial morphology integrality of endothelial cells, mediated by increased phosphorylation of serine 637 in Drp1, and could be reversed by α2-adrenergic receptor antagonist Yohimbine and AMP-activated protein kinase inhibitor Compound C. These findings suggest new molecular pathways involved in the neuroprotective effects of Dex in ischemic stroke. As Dex is routinely used as a sedative drug clinically, our findings provide molecular evidence that it has perioperative neuroprotection from ischemic stroke.

Intraoperative floppy iris syndrome: an updated review of literature.

Intraoperative Floppy iris syndrome(IFIS) remains a challenge for surgeons during phacoemulsification. Initially, it was related to the use of tamsulosin, an alpha adrenergic receptor blocker used in benign prostatic hyperplasia. Subsequently, other alpha adrenergic receptor such as alfuzosin, terazosin and doxazosin alongwith different other class of medications and systemic risk factors were identified. Other class of medications includes 5-alpha reductase inhibitor, angiotensin receptor antagonist, benzodiazepines, antipsychotics and antidepressants. Other risk factors include increasing age, male gender, diabetes, hypertension and decreased preoperative pupil diameter. It is very important for surgeons to identify these risk factors preoperatively and take appropriate preoperative and intraoperative measures to tackle the dreaded complications of IFIS. Sometimes, it is important for an ophthalmologist to work in cooperation with physician and urologist to minimize the complications. In conclusion, awareness of the risk factors associated with IFIS, their detailed preoperative assessment and intraoperative measures and surgical intervention is crucial in addressing IFIS. Lack of awareness can turn a routine, uneventful surgery into one with significant visual morbidity.

Abstracts of the 30th Annual Pediatric Pharmacy Association (PPA) Meeting April 21 to 25, 2021

Abstracts of the 30th Annual Pediatric Pharmacy Association (PPA) Meeting April 21 to 25, 2021

The role of anterior insula-brainstem projections and alpha-1 noradrenergic receptors for compulsion-like and alcohol-only drinking.

Compulsion-like alcohol drinking (CLAD), where consumption continues despite negative consequences, is a major obstacle to treating alcohol use disorder. The locus coeruleus area in the brainstem and norepinephrine receptor (NER) signaling in forebrain cortical regions have been implicated in adaptive responding under stress, which is conceptually similar to compulsion-like responding (adaptive responding despite the presence of stress or conflict). Thus, we examined whether anterior insula (aINS)-to-brainstem connections and alpha-1 NERs regulated compulsion-like intake and alcohol-only drinking (AOD). Halorhodopsin inhibition of aINS-brainstem significantly reduced CLAD, with no effect on alcohol-only or saccharin intake, suggesting a specific aINS-brainstem role in aversion-resistant drinking. In contrast, prazosin inhibition of alpha-1 NERs systemically reduced both CLAD and AOD. Similar to systemic inhibition, intra-aINS alpha-1-NER antagonism reduced both CLAD and AOD. Global aINS inhibition with GABAR agonists also strongly reduced both CLAD and AOD, without impacting saccharin intake or locomotion, while aINS inhibition of calcium-permeable AMPARs (with NASPM) reduced CLAD without impacting AOD. Finally, prazosin inhibition of CLAD and AOD was not correlated with each other, systemically or within aINS, suggesting the possibility that different aINS pathways regulate CLAD versus AOD, which will require further study to definitively address. Together, our results provide important new information showing that some aINS pathways (aINS-brainstem and NASPM-sensitive) specifically regulate compulsion-like alcohol consumption, while aINS more generally may contain parallel pathways promoting CLAD versus AOD. These findings also support the importance of the adaptive stress response system for multiple forms of alcohol drinking.

Ligands of Adrenergic Receptors: A Structural Point of View.

Adrenergic receptors are G protein-coupled receptors for epinephrine and norepinephrine. They are targets of many drugs for various conditions, including treatment of hypertension, hypotension, and asthma. Adrenergic receptors are intensively studied in structural biology, displayed for binding poses of different types of ligands. Here, we summarized molecular mechanisms of ligand recognition and receptor activation exhibited by structure. We also reviewed recent advances in structure-based ligand discovery against adrenergic receptors.

Modulation of the Rat Intergeniculate Leaflet of the Thalamus Network by Norepinephrine

Circadian rhythms are regulated by a set of brain structures, one of which is the Intergeniculate Leaflet of the Thalamus (IGL). The most recognised role of the IGL is the integration of a variety of stimuli affecting rhythmicity, such as lighting conditions, received by the eye, or light-independent (non-photic) cues, the information about which is delivered via the activation of the non-specific projections. One of them is the norepinephrinergic system originating in the brainstem Locus Coeruleus (LC). In order to investigate the effect of norepinephrine (NE) on the IGL neurons we have performed ex vivo recordings using the extracellular multi-electrode array technique as well as the intracellular whole-cell patch clamp. Using both agonists and antagonists of specific NE receptor subtypes, we confirmed the presence of functional α1-, α2- and β-adrenergic receptors within the investigated structure, allowing NE to exert multiple types of effects on different IGL neurons, mainly depolarisation of the neurons projecting to the Suprachiasmatic Nuclei – the master circadian pacemaker, and various responses exhibited by the cells creating the connection with the contralateral IGL. Moreover, NE was shown to affect IGL cells both directly and via modulation of the synaptic network, in particular the miniature inhibitory postsynaptic currents. To the best of our knowledge, these are the first studies to confirm the effects of NE on the activity of the IGL network.

Alpha-1 adrenergic receptor antagonists to prevent hyperinflammation and death from lower respiratory tract infection.

In severe viral pneumonia, including Coronavirus disease 2019 (COVID-19), the viral replication phase is often followed by hyperinflammation, which can lead to acute respiratory distress syndrome, multi-organ failure, and death. We previously demonstrated that alpha-1 adrenergic receptor (⍺1-AR) antagonists can prevent hyperinflammation and death in mice. Here, we conducted retrospective analyses in two cohorts of patients with acute respiratory distress (ARD, n = 18,547) and three cohorts with pneumonia (n = 400,907). Federated across two ARD cohorts, we find that patients exposed to ⍺1-AR antagonists, as compared to unexposed patients, had a 34% relative risk reduction for mechanical ventilation and death (OR = 0.70, p = 0.021). We replicated these methods on three pneumonia cohorts, all with similar effects on both outcomes. All results were robust to sensitivity analyses. These results highlight the urgent need for prospective trials testing whether prophylactic use of ⍺1-AR antagonists ameliorates lower respiratory tract infection-associated hyperinflammation and death, as observed in COVID-19.

A role of noradrenergic receptors in anxiolytic-like effect of high CRF in the rat frontal cortex

Corticotropin releasing factor (CRF) is a neuropeptide widely distributed in the brain as a hormonal modulator and neurotransmitter. The best known behavioral function of CRF is activation of stress and anxiety via the hypothalamus and limbic structures but the role of CRF in the cortex is still poorly understood. Our previous studies have shown anxiolytic-like effects of high doses of CRF injected into the Fr2 frontal cortex and involvement of CRF1 receptors (R) in that effect. These results seemed to be controversial as most other studies suggested anxiogenic and not anxiolytic effects of CRF1R stimulation. Since stress is associated with adrenergic system, in the present study, we focused on participation of alpha1 and alpha2 or beta adrenergic receptors in the anxiolytic-like effect of CRF. Moreover, we verified whether these effects of CRF in the Fr2 were really connected with CRF1R.Male Wistar rats were bilaterally microinjected with CRF in a dose of 0.2 μg/1 μl/site or with the specific agonist of CRF1R, stressin 1 (0.2–0.0125 μg/1 μl/site) into the Fr2 area. The elevated plus maze (EPM) test was performed 30 min later to assess the anxiolysis. An involvement of noradrenergic receptors in the CRF induced anxiolytic-like effect in the Fr2 was studied by pretreatment with the alpha1 antagonist prazosin, alpha2 agonist clonidine, alpha2 antagonist RS 79948 or beta antagonist propranolol, 20–30 min before CRF. The influence on anxiety was assessed in the EPM test. The results show that anxiolytic behavior after CRF microinjection into the Fr2 area seems to be mainly connected with the CRF1R activation because a similar effect was observed after stressin 1 administration and it was blocked by CRF1R antagonist. The results observed after administration of noradrenergic ligands indicated that anxiolytic effects of CRF in the Fr2 engaged the alpha1 and alpha2 adrenergic receptors but not beta receptors.

Alpha adrenergic receptors have role in the inhibitory effect of electrical low frequency stimulation on epileptiform activity in rats

Aim: Low frequency stimulation (LFS) inhibits neuronal hyperexcitability following epileptic activity. However, knowledge about LFS’ inhibitory mechanisms is lacking. Here, α1 and α2 adrenergic receptors’ roles in mediating LFS inhibitory action on high-K+ induced epileptiform activity (EA) was examined in rat hippocampal slices. Materials and methods: LFS (1 Hz, 900 pulses) was applied to the Schaffer collaterals. Whole-cell, patch clamp recording was used to measure changes in CA1 pyramidal neurons’ excitability. By applying high-K+ on hippocampal slices, EA was induced, and neuronal excitability increased. Results: When administered at the beginning of EA, LFS reduced neuronal excitability. In the presence of prazosin (10 µM, an α1 adrenergic receptor antagonist) and yohimbine (5 µM, an α2 adrenergic receptor antagonist), LFS’ typically has a restorative impact on EA-induced membrane potential hyperpolarization and spike firing frequency, but this effect was reduced after high-K+ washout; These antagonists did not have a significant effect on LFS’ inhibitory action on spike firing during EA. Conclusion: These findings suggest that LFS’ anticonvulsant effect, on neuronal hyperexcitability following high-K+ EA, may be mediated partly through α adrenergic receptors in hippocampal slices.

Dexmedetomidine use and mortality in mechanically ventilated patients with severe burns: A cohort study using a national inpatient database in Japan

BackgroundDexmedetomidine is an alpha 2-adrenergic receptor agonist. Apart from its sedative effects, dexmedetomidine can potentially reduce mortality through its anti-inflammatory effect. However, the impact of dexmedetomidine on in-hospital outcomes of patients with severe burns remains unclear. Therefore, we aimed to elucidate the association between dexmedetomidine use and mortality in mechanically ventilated patients with severe burns, using a Japanese nationwide database of in-hospital patients. MethodsWe included adults with severe burns (burn index ≥ 10) who were registered in the Japanese Diagnosis Procedure Combination national inpatient database from 2010 to 2018, started mechanical ventilation within 3 days of admission, and received any sedative drug (dexmedetomidine, midazolam, or propofol). One-to-one propensity score matching was performed between patients who received dexmedetomidine on the day of mechanical ventilation initiation (dexmedetomidine group) and those who did not receive dexmedetomidine (control group). The primary outcome was all-cause 30-day in-hospital mortality. Secondary outcomes were length of hospital stay and duration of mechanical ventilation in patients and survivors. ResultsEligible patients (n = 1888) were classified into the dexmedetomidine group (n = 371) or the control group (n = 1517). After one-to-one propensity score matching, we compared 329 patients from both groups. No significant difference was observed in the 30-day mortality between patients in the dexmedetomidine and control groups (22.8% vs. 22.5%, respectively; odds ratio, 1.02; 95% confidence interval, 0.71–1.46). Moreover, there were no significant differences between patients in the dexmedetomidine and control groups in terms of the length of hospital stay or the duration of mechanical ventilation. ConclusionsWe found no significant association between dexmedetomidine use and in-hospital outcomes (mortality, length of hospital stay, and length of mechanical ventilation) in mechanically ventilated patients with severe burns. Dexmedetomidine use may not improve the aforementioned outcomes; therefore, its selection should be based on the patient’s general condition and the target level of sedation.

An emerging role for microglia in stress-effects on memory.

Stressful experiences evoke, among others, a rapid increase in brain (nor)epinephrine (NE) levels and a slower increase in glucocorticoid hormones (GCs) in the brain. Microglia are key regulators of neuronal function and contain receptors for NE and GCs. These brain cells may therefore potentially be involved in modulating stress effects on neuronal function and learning and memory. In this review, we discuss that stress induces (1) an increase in microglial numbers as well as (2) a shift toward a pro‐inflammatory profile. These microglia have (3) impaired crosstalk with neurons and (4) disrupted glutamate signaling. Moreover, microglial immune responses after stress (5) alter the kynurenine pathway through metabolites that impair glutamatergic transmission. All these effects could be involved in the impairments in memory and in synaptic plasticity caused by (prolonged) stress, implicating microglia as a potential novel target in stress‐related memory impairments.

Enhanced Vasoconstriction in Sickle Cell Disease is Mediated by ET A Receptor‐Dependent Induction of alpha 1A ‐Adrenergic Receptor Expression

Sickle cell disease (SCD) patients are at a significant risk for developing the potentially fatal vaso-occlusive crisis, which has been associated with stressful stimuli. We previously reported that SCD mice display enhanced alpha-1 adrenergic receptor mediated vasoconstriction along with increased alpha-1A receptor expression in the aorta. Also, we have demonstrated SCD mice have higher levels endothelin-1 (ET-1), a potent vasoconstrictor mediating actions via the ETA receptor as well as increased reactive oxygen species (ROS). The mechanisms underlying the enhanced vasoconstriction in SCD mice remains largely unknown. We hypothesized that the enhanced vasoconstrictor response to alpha-1 adrenergic receptor stimulation is meditated through an ET-1/ETA receptor and ROS axis. Male 16-week-old humanized SCD (HbSS) and genetic control (HbAA) mice were utilized for all experiments. Vascular reactivity of isolated thoracic aortic (100–150 μm diameter) rings to phenylephrine (PE) and potassium chloride (KCl) were examined using wire myography. Mice were pre-treated for two weeks with the specific ETA receptor antagonist, ambrisentan (AMB). Thoracic aortic rings were also pre-incubated with superoxide dismutase-polyethylene glycol (PEG-SOD) for 30 minutes ex vivo at 100 units/ml. HbSS mice treated with AMB demonstrated a significantly blunted maximum vasoconstrictive response to PE when compared to control HbSS mice (132.3 ± 3.69 vs. 113.5 ± 4.59 %KCl, p=0.003). HbSS mice treated with ex vivo PEG-SOD alone did not demonstrate a blunted maximum vasoconstrictive response to PE stimulation (132.3 ± 3.69 vs. 121.6 ± 5.36 %KCl, p>0.05). However, HbSS mice treated with a combination of AMB and ex vivo PEG-SOD demonstrated an overall significant decrease response to PE stimulation compared to HbSS control and HbSS treated with AMB alone (132.3 ± 3.69 vs. 113.5 ± 4.59 vs. 95.6 ± 11.3 %KCl, p=0.006, p=0.015, respectively). HbAA mice showed similar vasoconstrictive response across treatments (84.9 ± 5.38 vs. 78.2 ± 6.89 vs. 75.1 ± 9.92 %KCl, p>0.05). Graded concentration responses to KCl were similar between genotypes and pharmacologic treatment. To demonstrate the translational nature of this enhanced vasoconstriction response, flow mediated dilation was used in human subjects with SCD. A subset of the human subjects were treated with AMB or placebo for 3 months. SCD patients treated with AMB for 3 months did show a significant increase in baseline diameter compared to placebo (1.54 % vs -2.05 % change in diameter, p = 0.012). However, there was no increase in flow-mediated dilation supporting a role for the ET-1/ETA pathway and an enhanced vasoconstrictor response in SCD in humans. These findings suggest the enhancement of alpha-1 adrenergic mediated vasoconstriction is, in part, mediated through an ETA receptor dependent mechanism. These data suggest that reactive oxygen species contribute to this enhancement and may work synergistically with the ETA receptor pathway to produce the enhanced vasoconstrictive phenotype seen in SCD.

69-Year-Old Man With Nasal Congestion and Pre-Syncope

69-Year-Old Man With Nasal Congestion and Pre-Syncope

Fentanyl, but not Morphine, Increases Cortical Electroencephalographic (EEG) Delta Power during Wakefulness in C57BL/6J (B6) Mice

Cortical EEG signals comprise differing oscillations that represent lower-level traits characteristic of higher-level behavioral phenotypes such as sleep and wakefulness (Physiology 32:60, 2017). Opioid-induced changes in neurobehavioral function also are reflected in altered EEG frequency and amplitude. These EEG features have the potential to provide insights regarding the desired actions and undesired side effects of opioids. In rat, morphine (Anesthesiology 103:779, 2005) and fentanyl (Sci Rep 9:14122, 2019) increase EEG slow waves. Studies of mice provide the additional opportunity to use mutated or transgenic animals with features potentially relevant to respiratory depression or addiction. This study is testing the hypothesis that subcutaneous injection of fentanyl and morphine causes dose-dependent EEG changes in wild-type mice. Experiments were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. Adult male B6 mice (n = 4) were implanted with telemeters (DSI HD-X02) for wireless recording of cortical EEG and neck muscle electromyogram. The recordings were used to quantify the effects of opioids on sleep and wakefulness. These studies demonstrated dose-dependent sleep disruption by both opioids (see companion abstract by Zebadua Unzaga et al.). EEG data were digitized by DSI Ponemah software and analyzed using public domain software for creating multi-tapered EEG spectrograms (J Vis Exp 153:e60333, 2019). Analyses were based on EEG data from the first 30 min after opioid injection, when all mice were awake. EEG power was expressed as percent change relative to saline (vehicle control) for six frequency bands: delta (0.5 to 4 Hz), theta (4 to 8 Hz), alpha (8 to 13 Hz), sigma (12 to 15 Hz), beta (13 to 30 Hz), and gamma (30 to 60 Hz). Increasing half log doses (mg/kg) ranged from 0.001 to 3 for fentanyl and 0.01 to 30 for morphine. Repeated measures ANOVA with the Geisser-Greenhouse correction showed a significant dose main-effect of fentanyl on EEG delta power (F(1.857, 5.571) = 7.454, p = 0.0276) and alpha power (F(1.705, 5.116) = 6.316, p = 0.0439). For fentanyl (3 mg/kg) compared to saline, Cohen's d = 2.9 for delta power. By contrast, for morphine there was a significant main effect of dose only for EEG sigma power (F(2.162, 6.486) = 6.557, p = 0.0265). Comparing morphine (30 mg/kg) to saline, the Cohen's d = 1.1 for sigma power. Although both fentanyl and morphine are full mu-opioid receptor agonists, they significantly altered EEG power at different frequency bands. Fentanyl, but not morphine, blocks alpha-1 adrenergic receptor subtypes and vesicular reuptake of several monoamine transmitters (J Pharmacol Exp Ther 343:376, 2020). These results support the interpretation that the differential effects of fentanyl and morphine on EEG power may be due, in part, to non-mu-opioid receptor of fentanyl.

Activation of human skin mast cells by vancomycin via MrgX2: Comparison to the effects of brimonidine

Abstract Vancomycin (VCM) is a glycopeptide antibiotic that inhibits bacterial peptidoglycan biosynthesis and is used for the treatment of certain Gram-positive bacterial infections of the eyes. However, the use of VCM is associated with several potential adverse reactions. We examined the mechanism of activation of human mast cells by vancomycin (VCM) and compared to brimonidine, an alpha2-adrenergic receptor agonist used to treat in glaucoma. VCM induced degranulation and cytokine secretion by SMCs, which were suppressed by pertussis toxin (PTX) (similar to MrgX2 ligands). Brimonidine induced less degranulation than VCM that was partially inhibited by PTX. MrgX2 expression on the surface of SMCs was detected by FACS analysis. VCM enhanced FceRI-mediated degranulation and cytokine secretion in a dose-dependent manner, whereas brimonidine failed to induce cytokine production and suppressed FceRI-mediated cytokine secretion. VCM induced maximal ERK-phosphorylation by 5 min similar to compound 48/80, an MrgX2 ligand and inhibited by PTX. VCM- but not brimonidine-induced degranulation of MCs was significantly inhibited by QWF, an MrgX2 antagonist. This study indicates that VCM but not brimonidine activates human SMCs via MrgX2 and that MCs become more responsive to VCM when co-stimulated by aggregating FceRI, which may occur when sensitive patients exposed to aeroallergen and VCM.

Perivascular adipose tissue (PVAT)‐mediated buffering of vasoconstriction in placental ischemia‐induced hypertensive rats

Preeclampsia is a pregnancy-specific hypertensive disorder diagnosed along with systemic endothelial and vascular dysfunction. A novel regulator of vascular tone is the perivascular adipose tissue (PVAT) that surrounds most blood vessels and functions to blunt vasoconstriction. However, it is unknown whether PVAT becomes dysfunctional in preeclampsia. Preeclampsia arises from placental ischemia to elicit release of pro-vasoconstrictive factors into the maternal circulation. In this study, it was hypothesized that placental ischemic rats have attenuated PVAT-mediated buffering of vasoconstriction. Placental ischemia was induced on gestational day 14 in Sprague-Dawley rats by conducting the Reduced Uterine Perfusion Pressure (RUPP) protocol. Conscious blood pressure measurements (carotid catheter) and wire myography were conducted on day 19. Mean arterial blood pressure was significantly elevated (P<0.05) in RUPP (N=6, 117±3 mmHg) vs. Pregnant (N=6, 97±3 mmHg) rats. In aortas, the maximum response (as % increase in force from baseline tension) to KCl-induced depolarization was not affected (P>0.05) by RUPP or the presence of PVAT: Pregnant+PVAT (43±9%) vs. Pregnant-PVAT (42±5%) and RUPP+PVAT (42±%7) vs. RUPP-PVAT (48±7%). Furthermore, phenylephrine (Phe, alpha1-adrenergic receptor agonist)-induced vasoconstriction was similar (P>0.05) in Pregnant+PVAT (69±11%) vs. Pregnant-PVAT (70±12%) and RUPP+PVAT (38±12%) vs. RUPP-PVAT (34±9%), with the 2-way ANOVA detecting a main effect only for RUPP (P<0.05). It was then assessed whether the endothelium controls PVAT function whereby denuding the endothelium did not impact (P>0.05) any of the respective Phe responses in Pregnant+PVAT-Endo (60±12%), Pregnant-PVAT-Endo (72±11%), RUPP+PVAT-Endo (30±15%), or RUPP-PVAT-Endo (22±7%). In smaller, third-order mesenteric arteries, direct depolarization by KCl responses were similar (P>0.05) between Pregnant+PVAT (176±19%), Pregnant-PVAT (149±17%), RUPP+PVAT (148±30%), and RUPP-PVAT (167±27%). As for Phe, the 2-way ANOVA identified a main effect (P<0.05) only for PVAT to blunt Phe-induced vasoconstriction in Pregnant+PVAT (259±38%) vs. Pregnant-PVAT (337±26%) and RUPP+PVAT (176±39%) vs. RUPP-PVAT (379±47%), with no difference for the influence of PVAT between RUPP and Pregnant groups. Removal of the endothelium further attenuated Phe responses in the PVAT groups: Pregnant+PVAT-Endo (174±66%) and RUPP+PVAT-Endo (-3±3%) vs. their respective groups without PVAT: Pregnant-PVAT-Endo (379±39%) and RUPP-PVAT-Endo (339±167%). In fact, the degree of this attenuation to Phe-induced vasoconstriction was the greatest (P<0.05) in the RUPP+PVAT-Endo (-104±3%) group compared all other groups: RUPP-PVAT-Endo (19±59%), Pregnant+PVAT-Endo (10±11%), and Pregnant-PVAT-Endo (10±11%). In conclusion, as it was found that removal of the endothelium allows for enhanced PVAT blunting of vasoconstriction in RUPP rats, these data indicate that placental ischemia drives endothelial dysfunction to subsequently disrupt PVAT function in small arteries during pregnancy.