Comparison of Preoperative Alpha-blockade for Resection of Paraganglioma and Pheochromocytoma.

OBJECTIVE: Phenoxybenzamine (nonselective, noncompetitive alpha-blocker) is the preferred drug for preoperative treatment of pheochromocytoma, but doxazosin (selective, competitive alpha-blocker) may be equally effective. We compared the efficacy of doxazosin vs phenoxybenzamine. METHODS: We conducted a prospective study of patients undergoing pheochromocytoma or paraganglioma resection by randomizing pretreatment with phenoxybenzamine or doxazosin at a single tertiary referral center. The high cost of phenoxybenzamine led to high crossover to doxazosin. Randomization was halted, and a consecutive historical cohort of phenoxybenzamine patients was included for a case-control study design. The efficacy of alpha-blockade was assessed with preinduction infusion of incremental doses of phenylephrine. The primary outcomes were mortality, cardiovascular complications, and intensive care unit admission. The secondary outcomes were hemodynamic instability index (proportion of operation outside of hemodynamic goals), adequacy of blockade by the phenylephrine titration test, and drug costs. RESULTS: Twenty-four patients were prospectively enrolled (doxazosin, n = 20; phenoxybenzamine, n = 4), and 15 historical patients treated with phenoxybenzamine were added (total phenoxybenzamine, n = 19). No major cardiovascular complications occurred in either group. The phenylephrine dose-response curves showed less blood pressure rise in the phenoxybenzamine than in the doxazosin group (linear regression coefficient = 0.008 vs 0.018, P = .01), suggesting better alpha-blockade in the phenoxybenzamine group. The median hemodynamic instability index was 14% vs 13% in the phenoxybenzamine and doxazosin groups, respectively (P = .56). The median highest daily cost of phenoxybenzamine was $442.20 compared to $5.06 for doxazosin. CONCLUSION: Phenoxybenzamine may blunt intraoperative hypertension better than doxazosin, but this difference did not translate to fewer cardiovascular complications and is offset by a considerably increased cost.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Identification of serotonin 2A receptor as a novel HCV entry factor by a chemical biology strategy.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Although several HCV protease/polymerase inhibitors were recently approved by U.S. FDA, the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistance. Viral entry is an essential target step for antiviral development, but FDA-approved HCV entry inhibitor remains exclusive. Here we identify serotonin 2A receptor (5-HT2AR) is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy. The silencing of 5-HT2AR and clinically available 5-HT2AR antagonist suppress cell culture-derived HCV (HCVcc) in different liver cells and primary human hepatocytes at late endocytosis process. The mechanism is related to regulate the correct plasma membrane localization of claudin 1 (CLDN1). Moreover, phenoxybenzamine (PBZ), an FDA-approved 5-HT2AR antagonist, inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs. The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice. Our results not only expand the understanding of HCV entry, but also present a promising target for the invention of HCV entry inhibitor.

Anti-tumor activity of phenoxybenzamine and its inhibition of histone deacetylases.

The principal finding from this study was the recognition that the α-adrenergic antagonist, phenoxybenzamine, possesses histone deacetylase inhibitory activity. Phenoxybenzamine is approved by the United States Food and Drug Administration for the treatment of hypertensive crises associated with tumors of the adrenal medulla, pheochromocytomas. It has several "off label" indications relative to its capacity to relax vascular smooth muscle and smooth muscle of the urogenital tract. The drug also has a long history of apparent efficacy in ameliorating, and perhaps reversing, the severe symptoms of neuropathic pain syndromes. Our interest in this feature of the drug relates to the fact that certain types of neuropathic pain, in particular complex regional pain syndrome, demonstrate a proliferative nature, with the capacity to spread from an injured limb, for example, to a non-injured limb and perhaps to essentially the entire body. Sensory neuronal sprouting in the spinal cord has been observed under conditions where there is a high sensory input from painful stimuli. Searches of gene expression signatures in the BroadBuild02 Molecular Signature Database using their connectivity map software suggested that phenoxybenzamine may have histone deacetylase inhibitory activity. Studies by others have reported inhibitory effects of phenoxybenzamine on growth, invasion and migration of human tumor cell cultures and, in one study, inhibition of tumor expansion in animal experiments. Inhibitory effects on human tumor cell cultures are also reported in the present study. Phenoxybenzamine was also found to have histone deacetylase inhibitory activity; histone deacetylase isoforms 5, 6, and 9 were the most sensitive to inhibition by phenoxybenzamine. The importance of elevated levels of these isoforms as biomarkers of poor prognosis in human malignant disease, and the recognized suppression of tumor growth that may accrue from their inhibition, opens consideration of possible translation of phenoxybenzamine to new clinical applications. This might be facilitated by the fact that phenoxybenzamine is already an approved drug entity. There appears to be no previous report of the activity of phenoxybenzamine as a histone deacetylase inhibitor.

Quantitative systems pharmacology analysis of drug combination and scaling to humans: the interaction between noradrenaline and vasopressin in vasoconstriction.

BACKGROUND AND PURPOSE: Development of combination therapies has received significant interest in recent years. Previously, a two-receptor one-transducer (2R-1T) model was proposed to characterize drug interactions with two receptors that lead to the same phenotypic response through a common transducer pathway. We applied, for the first time, the 2R-1T model to characterize the interaction of noradrenaline and arginine-vasopressin on vasoconstriction and performed inter-species scaling to humans using this mechanism-based model. EXPERIMENTAL APPROACH: Contractile data were obtained from in vitro rat small mesenteric arteries after exposure to single or combined challenges of noradrenaline and arginine-vasopressin with or without pretreatment with the irreversible α-adrenoceptor antagonist, phenoxybenzamine. Data were analysed using the 2R-1T model to characterize the observed exposure-response relationships and drug-drug interaction. The model was then scaled to humans by accounting for differences in receptor density. KEY RESULTS: With receptor affinities set to published values, the 2R-1T model satisfactorily characterized the interaction between noradrenaline and arginine-vasopressin in rat small mesenteric arteries (relative standard error ≤20%), as well as the effect of phenoxybenzamine. Furthermore, after scaling the model to human vascular tissue, the model also adequately predicted the interaction between both agents on human renal arteries. CONCLUSIONS AND IMPLICATIONS: The 2R-1T model can be of relevance to quantitatively characterize the interaction between two drugs that interact via different receptors and a common transducer pathway. Its mechanistic properties are valuable for scaling the model across species. This approach is therefore of significant value to rationally optimize novel combination treatments.

Noradrenergic antagonists mitigate amphetamine-induced recovery.

Brain injury, including that due to stroke, leaves individuals with cognitive deficits that can disrupt daily aspect of living. As of now there are few treatments that shown limited amounts of success in improving functional outcome. The use of stimulants such as amphetamine have shown some success in improving outcome following brain injury. While the pharmacological mechanisms for amphetamine are known; the specific processes responsible for improving behavioral outcome following injury remain unknown. Understanding these mechanisms can help to refine the use of amphetamine as a potential treatment or lead to the use of other methods that share the same pharmacological properties. One proposed mechanism is amphetamine's impact upon noradrenaline (NA). In the current, study noradrenergic antagonists were administered prior to amphetamine to pharmacologically block α- and ß-adrenergic receptors. The results demonstrated that the blockade of these receptors disrupted amphetamines ability to induce recovery from hemispatial neglect using an established aspiration lesion model. This suggests that amphetamine's ability to ameliorate neglect deficits may be due in part to noradrenaline. These results further support the role of noradrenaline in functional recovery. Finally, the development of polytherapies and combined therapeutics, while promising, may need to consider the possibility that drug interactions can negate the effectiveness of treatment.

Comparison of phenoxybenzamine and doxazosin in perioperative management of patients with pheochromocytoma.

BACKGROUND: Adrenalectomy with preoperative pharmacological preparation is strongly recommended in patients diagnosed with pheochromocytoma, in order to prevent perioperative complications. AIM: To compare phenoxybenzamine (PhB) and doxazosin (DOX) in terms of perioperative haemodynamic status in patients with pheochromocytoma, who have been prepared for adrenalectomy. METHODS: Retrospective analysis of 44 patients with pheochromocytoma (aged 16-80 years, 29 females) who underwent adrenalectomy. Patients were divided into two groups: 35 patients on DOX and nine patients on PhB. RESULTS: Mean time of preparation for surgery was 38.8 days in the DOX group and 18.3 days in the PhB group (p = 0.04). No statistically significant differences between the DOX and PhB groups in intraoperative blood pressure (BP) fluctuations were found: < 170/100 mm Hg (34% vs. 44%, respectively, p = 0.42), ≥ 200/110 mm Hg (40% vs. 22%, respectively, p = 0.28). Mean greatest intraoperative systolic BP (195 ± 53 vs. 166 ± 42 mm Hg, p = 0.21) and diastolic BP (98 ± 20 vs. 89 ± 46 mm Hg, p = 0.21), and mean lowest intraoperative systolic BP (87 ± 13 vs. 79 ± 17 mm Hg, p = 0.25) and diastolic BP (49 ± 8 vs. 46 ± 12 mm Hg, p = 0.60) were not different between the DOX and PhB groups, respectively. Sodium nitroprusside was administrated in 30% DOX vs. 11% PhB patients (p = 0.25). Laparoscopic surgery was conducted in 97% DOX vs. 89% PhB patients (p = 0.64). Postoperative BP drop below 90/60 mm Hg was noted in 48% of the DOX vs. 43% of the PhB group (p = 0.56). Negative correlation was found between the length of DOX administration with maximal intraoperative systolic BP (r = -0.45, p = 0.006) and diastolic BP (r = -0.39, p = 0.019). CONCLUSIONS: There are no clinically relevant differences between patients with pheochromocytoma, who have been prepared for adrenalectomy with DOX or PhB.

Dexmedetomidine Inhibits Phenylephrine-induced Contractions via Alpha-1 Adrenoceptor Blockade and Nitric Oxide Release in Isolated Rat Aortae.

The goal of this in vitro study was to examine the effect of the alpha-2 adrenoceptor agonist dexmedetomidine on phenylephrine (alpha-1 adrenoceptor agonist)-induced contraction in isolated rat aortae and to elucidate the associated cellular mechanisms, with a particular focus on alpha-1 adrenoceptor antagonism. Dexmedetomidine dose-response curves were generated in isolated endothelium-intact and endothelium-denuded rat aortae precontracted with phenylephrine or 5-hydroxytryptamine. Endothelium-denuded aortic rings were pretreated with either dexmedetomidine or the reversible alpha-1 adrenoceptor antagonist phentolamine, followed by post-treatment with the irreversible alpha-1 adrenoceptor blocker phenoxybenzamine. Control rings were treated with phenoxybenzamine alone. All rings were repeatedly washed with Krebs solution to remove all pretreatment drugs, including phenoxybenzamine, phentolamine and dexmedetomidine. Phenylephrine dose-response curves were then generated. The effect of rauwolscine on the dexmedetomidine-mediated change in phenylephrine-induced endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined using western blotting. The magnitude of the dexmedetomidine-mediated inhibition of phenylephrine-induced contraction was higher in endothelium-intact aortae than in endothelium-denuded aortae or endothelium-intact aortae treated with Nω-nitro-L-arginine methyl ester. However, dexmedetomidine did not significantly alter 5-hydroxytryptamine-induced contraction. In further experiments, prazosin attenuated dexmedetomidine-induced contraction. Additionally, pretreatment with either dexmedetomidine plus phenoxybenzamine or phentolamine plus phenoxybenzamine produced greater phenylephrine-induced contraction than phenoxybenzamine alone, suggesting that dexmedetomidine protects aortae from the alpha-1 adrenoceptor blockade induced by phenoxybenzamine. Rauwolscine attenuated the dexmedetomidine-mediated enhancement of phenylephrine-induced eNOS phosphorylation. Taken together, these results suggest that dexmedetomidine attenuates phenylephrine-induced contractions via alpha-1 adrenoceptor blockade and endothelial nitric oxide release in the isolated rat aorta.

Molecular Mechanisms of Action of M5 Muscarinic Acetylcholine Receptor Allosteric Modulators.

Recently, the first subtype-selective allosteric modulators of the M5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca(2+) mobilization in a recombinant cell line stably expressing the human M5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhancements in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the pathway investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity cooperativity estimates were consistent with values derived from functional assays in some instances but not others, suggesting additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenoxybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modulators of the M5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs.

Anxiolytic effect of the GPR103 receptor agonist peptide P550 (homolog of neuropeptide 26RFa) in mice. Involvement of neurotransmitters.

The GPR103 receptor is a G protein-coupled receptor, which plays a role in several physiological functions. However, the role of the GPR103 receptor in anxiety has not been clarified. The first aim of our study was to elucidate the involvement of the GPR103 receptor in anxious behavior. Mice were treated with peptide P550, which is the mouse homolog of neuropeptide 26RFa and has similar activity for the GPR103 receptor as neuropeptide 26RFa. The anxious behavior was investigated using an elevated plus-maze paradigm. The second aim of our study was to investigate the underlying neurotransmissions. Accordingly, mice were pretreated with a nonselective muscarinic acetylcholine receptor antagonist, atropine, a γ-aminobutyric acid subunit A (GABAA) receptor antagonist, bicuculline, a non-selective 5-HT2 serotonergic receptor antagonist, cyproheptadine, a mixed 5-HT1/5-HT2 serotonergic receptor antagonist, methysergide, a D2, D3, D4 dopamine receptor antagonist, haloperidol, a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a nonselective ß-adrenergic receptor antagonist, propranolol. Our results demonstrated that peptide P550 reduces anxious behavior in elevated plus maze test in mice. Our study shows also that GABAA-ergic, α- and ß-adrenergic transmissions are all involved in this action, whereas 5-HT1 and 5-HT2 serotonergic, muscarinic cholinergic and D2, D3, D4 dopaminergic mechanisms may not be implicated.

Anti-tumor activity of phenoxybenzamine hydrochloride on malignant glioma cells.

Phenoxybenzamine hydrochloride (PHEN) is a selective antagonist of both α-adrenoceptor and calmodulin that exhibits anticancer properties. The aim of this study was to explore the anti-tumor function of PHEN in glioma. Cell proliferation assay was used to assess glioma cell growth. Migration and invasion capacity of glioma cells was monitored by wound-healing and transwell assay, respectively. Neurosphere formation test was adopted for the tumorigenesis of glioma cells, which was also confirmed by soft agar cloning formation test in vitro and a nude mouse model in vivo. Finally, we explored the potential pathway utilized by PHEN using Western blot and immunofluoresce staining. PHEN exhibited a significant inhibitory effect on the proliferation of both U251 and U87MG glioma cell lines in a positive dose-dependent manner. PHEN apparently attenuated the malignancy of glioma in terms of migration and invasion and also suppressed the tumorigenic capacity both in vitro and in vivo. Mechanism study showed that PHEN promoted tumor suppression by inhibiting the TrkB-Akt pathway. The results of the present study demonstrated that PHEN suppressed the proliferation, migration, invasion, and tumorigenesis of glioma cells, induced LINGO-1 expression, and inhibited the TrkB-Akt pathway, which may prove to be the mechanisms underlying the anti-tumor effect of PHEN on glioma cells.

Targeting the cis-dimerization of LINGO-1 with low MW compounds affects its downstream signalling.

BACKGROUND AND PURPOSE: The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. EXPERIMENTAL APPROACH: LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein-protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. KEY RESULTS: LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. CONCLUSIONS AND IMPLICATIONS: Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.

Neurotransmitter-mediated anxiogenic action of PACAP-38 in rats.

The action of PACAP-38 was studied by measuring the anxiogenic-anxiolytic behavior of rats in an elevated plus maze. PACAP-38 was administered into the lateral brain ventricle and the behavior of the animals was measured 3h later. The possible involvement of transmitters was measured by pretreating the animals with receptor blockers which alone did not influence the task, but in the doses used were effective with other neuropeptides. The receptor antagonist PACAP 6-38 (a PAC 1/VPAC2 receptor antagonist of PACAP-38 receptor), haloperidol (a non-selective dopamine receptor antagonist), phenoxybenzamine (an α1/α2ß-adrenergic receptor antagonist), propranolol(a ß-adrenergic receptor antagonist), bicuculline (a gamma-aminobutyric acid subunit A receptor antagonist), methysergide (a nonselective 5-HT2 serotonergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), naloxone (a nonselective opioid receptor antagonist) and nitro-l-arginine which acts by blocking the enzyme nitric oxide synthase, thereby blocking the nitric oxide synthesis, were tested. The following parameters were measured: the time spent in open arms/the time spent in total entries. PACAP-38 decreased the ratio of time spent in open arms to the time spent in total entries, indicating anxiogenic action. The total number of entries was not altered significantly either by PACAP-38 or by the receptor blockers. The following receptor blockers diminished the action of PACAP-38: PACAP 6-38,haloperidol, methysergide, naloxone and nitro-l-arginine. Pretreatment with atropine, phenoxybenzamine, propranolol and bicuculline did not influence the action of PACAP-38 on the time spent in open arms. The results demonstrate that PACAP-38 administered into the lateral brain ventricle exerted anxiogenic action at 3 h following treatment. Pretreatment of the animals with various receptor blockers indicated that a nonselective dopaminergic receptor antagonist, 5HT2 serotonergic and opioid receptors, nitric oxide and PAC1 receptors are involved in the anxiogenic action induced by PACAP-38.

Neuropeptide AF induces anxiety-like and antidepressant-like behavior in mice.

Little is known about the action of neuropeptide AF (NPAF) on anxiety and depression. Only our previous study provides evidence that NPAF induces anxiety-like behavior in rats. Therefore, the aim of the present study was to investigate the action of NPAF on depression-like behavior and the underlying neurotransmissions in mice. In order to determine whether there are species differences between rats and mice, we have investigated the action of NPAF on anxiety-like behavior in mice as well. A modified forced swimming test (mFST) and an elevated plus maze test (EPMT) were used to investigate the depression and anxiety-related behaviors, respectively. Mice were treated with NPAF 30min prior to the tests. In the mFST, the animals were pretreated with a non-selective muscarinic acetylcholine receptor antagonist, atropine, a non-selective 5-HT2 serotonergic receptor antagonist, cyproheptadine, a mixed 5-HT1/5-HT2 serotonergic receptor antagonist, methysergide, a D2/D3/D4 dopamine receptor antagonist, haloperidol, a α1/α2ß-adrenergic receptor antagonist, prazosin or a non-selective ß-adrenergic receptor antagonist, propranolol 30min before the NPAF administration. In the mFST, NPAF decreased the immobility time and increased the climbing and swimming times. This action was reversed completely by methysergide and partially by atropine, whereas cyproheptadine, haloperidol, prazosin and propranolol were ineffective. In the EPMT, NPAF decreased the time spent in the arms (open/open+closed). Our results demonstrate that NPAF induces anti-depressant-like behavior in mice, which is mediated, at least in part, through 5HT2-serotonergic and muscarinic cholinergic neurotransmissions. In addition, the NPAF-induced anxiety is species-independent, since it develops also in mice.

Predictors of hemodynamic instability during surgery for pheochromocytoma.

BACKGROUND: Resection of pheochromocytoma is often associated with hemodynamic instability (HDI). We examined patient and tumor factors that may influence HDI. The effect of pretreatment with nonselective α blockade phenoxybenzamine (PXB) versus selective α blockade on HDI and outcomes was also evaluated. METHODS: The records of 91 patients who underwent adrenalectomy between 2002 and 2013 were retrospectively reviewed. HDI was determined by number of intraoperative episodes of systolic blood pressure (SBP) > 200 mmHg, those greater than or less than 30 % of baseline, heart rate > 110 bpm, and the need for postoperative vasopressors. Fishers exact, t test and regressions were performed. RESULTS: Among 91 patients, 78 % received PXB, 18 % selective α blockade and 4 % no adrenergic blockade. Patient demographics, tumor factors and surgical approach were similar among the blockade groups. On multivariate analysis, increasing tumor size was associated with a significant rise in the number of episodes of SBP > 30 % [rate ratio (RR) 1.40] and an increased postoperative vasopressor requirement [odds ratio (OR) 1.23]. Open adrenalectomy and use of selective blockade were associated with an increased number of episodes of SBP > 200 mmHg (RR 27.8 and RR 20.9, respectively). Open adrenalectomy was also associated with increased readmissions (OR 12.3), complications (OR 5.6), use of postoperative vasopressors (OR 4.4) and hospital stay (4.6 days longer). There were no differences in other HDI measurements or postoperative outcomes among the blockade groups. CONCLUSIONS: Tumor size, open adrenalectomy, and type of α blockade were associated with intraoperative HDI during pheochromocytoma resection. Selective blockade was associated with significantly more episodes of intraoperative hypertension but no perioperative adverse outcomes.

Urocortin 3 administration impairs fear motivated learning in mice is mediated by transmitters.

Urocortin 3 (Ucn 3) was found to impair passive avoidance learning in male and female mice. The possible involvement of transmitters in the action, the animals were pretreated with the following receptor antagonists in doses which alone could not influence the measurement. Haloperidol (a D2, dopamine receptor antagonist), phenoxybenzamine (a nonselective α1-adrenergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), bicuculline (a γ-aminobutyric acid subunit A receptor antagonist), nitro-L-arginine (a nitric oxide synthase inhibitor), antalarmin (a CRF1 receptor antagonist) and astressin 2B (a CRF2 receptor antagonist). Haloperidol, phenoxybenzamine, bicuculline, atropine, nitro-L-arginine and astressin 2B prevented the action of Ucn 3, in both sexes, whereas antalarmin exerted no action in either male or female animals.

Transmitter mediation of the anxiolytic action of apelin-13 in male mice.

The widespread distribution of apelin-13 and apelin receptors in the brain and periphery suggests an important function of this neuropeptide in regulatory processes in the organism. In previous work we found that apelin-13 facilitates the consolidation of passive avoidance learning in rats. In the present work we demonstrate that apelin-13 exerts anxiolytic action in an elevated plus maze in mice. In order to assess the possible involvement of transmitters in this action, the animals were pretreated with the following receptor blockers in doses which themselves did not influence the behavioral paradigm: atropine (a nonselective muscarinic acetylcholine receptor antagonist), haloperidol (a D2, D3, D4 dopamine receptor antagonist), phenoxybenzamine (a nonselective α1-adrenergic receptor antagonist), methysergide (a nonselective 5-HT2 serotonergic receptor antagonist), propranolol (a ß-adrenergic receptor antagonist), naloxone (a nonselective opioid receptor antagonist) and bicuculline (a γ-aminobutyric acid subunit A receptor antagonist. Phenoxybenzamine, haloperidol, propranolol and methysergide prevented the action of apelin-13, whereas atropine, naloxone and bicuculline were ineffective. The data suggest that apelin-13 elicits its anxiolytic action via α-adrenergic, dopaminergic, ß-adrenergic and 5-HT2 serotonergic mediation.

Phenoxybenzamine is neuroprotective in a rat model of severe traumatic brain injury.

Phenoxybenzamine (PBZ) is an FDA approved α-1 adrenergic receptor antagonist that is currently used to treat symptoms of pheochromocytoma. However, it has not been studied as a neuroprotective agent for traumatic brain injury (TBI). While screening neuroprotective candidates, we found that phenoxybenzamine reduced neuronal death in rat hippocampal slice cultures following exposure to oxygen glucose deprivation (OGD). Using this system, we found that phenoxybenzamine reduced neuronal death over a broad dose range (0.1 µM-1 mM) and provided efficacy when delivered up to 16 h post-OGD. We further tested phenoxybenzamine in the rat lateral fluid percussion model of TBI. When administered 8 h after TBI, phenoxybenzamine improved neurological severity scoring and foot fault assessments. At 25 days post injury, phenoxybenzamine treated TBI animals also showed a significant improvement in both learning and memory compared to saline treated controls. We further examined gene expression changes within the cortex following TBI. At 32 h post-TBI phenoxybenzamine treated animals had significantly lower expression of pro-inflammatory signaling proteins CCL2, IL1ß, and MyD88, suggesting that phenoxybenzamine may exert a neuroprotective effect by reducing neuroinflammation after TBI. These data suggest that phenonxybenzamine may have application in the treatment of TBI.

Prospective study to compare peri-operative hemodynamic alterations following preparation for pheochromocytoma surgery by phenoxybenzamine or prazosin.

BACKGROUND: Prospective studies comparing the efficacy of selective versus nonselective alpha blockers for preoperative preparation of pheochromocytoma (PCC) are lacking. In this prospective nonrandomized study, we compared the outcome of preoperative preparation with phenoxybenzamine (PBZ) and prazosin (PRZ) in terms of perioperative hemodynamic alterations. METHODS: The study was conducted at a tertiary referral center from July 2010 to December 2012. Thirty-two patients with PCC underwent operation after adequate preparation with PBZ (n = 15) or PRZ (n = 17). Five pediatric and adolescent patients were excluded because of different hemodynamics in this population. Perioperative monitoring was done for pulse rate (PR) and blood pressure(BP) alterations, occurrence of arrhythmias, and time taken to achieve hemodynamic stability. Groups were compared with the Mann-Whitney test, Student's t test, and the χ2 test as applicable. RESULTS: Patients in the two groups were similar in age,gender, 24 h urinary metanephrine and normetanephrine levels, and type of procedure. Patients prepared with PRZ had significantly more intraoperative episodes of transient hypertension (systolic BP ≥ 160 mmHg) and hypertensive urgency (BP >180/110 mmHg) (p 0.02, 0.03, respectively). More patients receiving PRZ suffered from transient severe hypertension (SBP ≥ 220 mmHg) (p 0.03). The PRZ group also had more median maximum SBP (233 mmHg vs PBZ 181.5 mmHg) (p = 0.01) and lesser median minimum SBP (71 mmHg vs PBZ 78 mmHg) (p 0.03). No significant differences were found between the study groups for changes in PR, postoperative BP alterations,occurrence of arrhythmias, and time taken to achieve hemodynamic stability. CONCLUSIONS: PBZ was found superior to PRZ in having fewer intraoperative hemodynamic fluctuations.

Kinetics of 5-HT2B receptor signaling: profound agonist-dependent effects on signaling onset and duration.

The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptor-mediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine2B (5-HT2B) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Gαq-mediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signal-regulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptor-dissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.