Timothy Angelotti MD, PhD

All Publications

• Expression and trafficking of functional G protein-coupled receptors are related, yet distinct, concepts. Naunyn-Schmiedeberg's archives of pharmacology Hurt, C. M., Angelotti, T. 2014; 387 (10): 1009-1012

  View details for DOI 10.1007/s00210-014-1028-7

  View details for PubMedID 25103411

• Common a2A and a2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking. Naunyn-Schmiedeberg's archives of pharmacology Hurt, C. M., Sorensen, M. W., Angelotti, T. 2014; 387 (6): 569-579

  Abstract

  Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.

  View details for DOI 10.1007/s00210-014-0972-6

  View details for PubMedID 24643471

  View details for PubMedCentralID PMC4035122

• REEP1 and REEP2 proteins are preferentially expressed in neuronal and neuronal-like exocytotic tissues. Brain research Hurt, C. M., Björk, S., Ho, V. K., Gilsbach, R., Hein, L., Angelotti, T. 2014; 1545: 12-22

  Abstract

  The six members of the Receptor Expression Enhancing Protein (REEP) family were originally identified based on their ability to enhance heterologous expression of olfactory receptors and other difficult to express G protein-coupled receptors. Interestingly, REEP1 mutations have been linked to neurodegenerative disorders of upper and lower motor neurons, hereditary spastic paraplegia (HSP) and distal hereditary motor neuropathy type V (dHMN-V). The closely related REEP2 isoform has not demonstrated any such disease linkage. Previous research has suggested that REEP1 mRNA is ubiquitously expressed in brain, muscle, endocrine, and multiple other organs, inconsistent with the neurodegenerative phenotype observed in HSP and dHMN-V. To more fully examine REEP1 expression, we developed and characterized a new REEP1 monoclonal antibody for both immunoblotting and immunofluorescent microscopic analysis. Unlike previous RT-PCR studies, immunoblotting demonstrated that REEP1 protein was not ubiquitous; its expression was restricted to neuronal tissues (brain, spinal cord) and testes. Gene expression microarray analysis demonstrated REEP1 and REEP2 mRNA expression in superior cervical and stellate sympathetic ganglia tissue. Furthermore, expression of endogenous REEP1 was confirmed in cultured murine sympathetic ganglion neurons by RT-PCR and immunofluorescent staining, with expression occurring between Day 4 and Day 8 of culture. Lastly, we demonstrated that REEP2 protein expression was also restricted to neuronal tissues (brain and spinal cord) and tissues that exhibit neuronal-like exocytosis (testes, pituitary, and adrenal gland). In addition to sensory tissues, expression of the REEP1/REEP2 subfamily appears to be restricted to neuronal and neuronal-like exocytotic tissues, consistent with neuronally restricted symptoms of REEP1 genetic disorders.

  View details for DOI 10.1016/j.brainres.2013.12.008

  View details for PubMedID 24355597

  View details for PubMedCentralID PMC3919455

• REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity PLOS ONE Bjork, S., Hurt, C. M., Ho, V. K., Angelotti, T. 2013; 8 (10)

  Abstract

  Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins.

  View details for DOI 10.1371/journal.pone.0076366

  View details for Web of Science ID 000325434500064

  View details for PubMedID 24098485

  View details for PubMedCentralID PMC3788743

• Systematic and Quantitative Analysis of G Protein-Coupled Receptor Trafficking Motifs G PROTEIN COUPLED RECEPTORS: TRAFFICKING AND OLIGOMERIZATION Hurt, C. M., Ho, V. K., Angelotti, T. 2013; 521: 171-187

  Abstract

  Plasma membrane expression of G protein-coupled receptors (GPCRs) is a dynamic process balancing anterograde and retrograde trafficking. Multiple interrelated cellular processes determine the final level of cell surface expression, including endoplasmic reticulum (ER) export/retention, receptor internalization, recycling, and degradation. These processes are highly regulated to achieve specific localization to subcellular domains (e.g., dendrites or basolateral membranes) and to affect receptor signaling. Analysis of potential ER trafficking motifs within GPCRs requires careful consideration of intracellular dynamics, such as protein folding, ER export and retention, and glycosylation. This chapter presents an approach and methods for qualitative and quantitative assessment of these processes to aid in accurate identification of GPCR trafficking motifs, utilizing the analysis of a hydrophobic extracellular trafficking motif in α2C adrenergic receptors as a model system.

  View details for DOI 10.1016/B978-0-12-391862-8.00009-0

  View details for Web of Science ID 000317147200009

  View details for PubMedID 23351739

• Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal TRAFFIC Angelotti, T., Daunt, D., Shcherbakova, O. G., Kobilka, B., Hurt, C. M. 2010; 11 (4): 560-578

  Abstract

  Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  View details for DOI 10.1111/j.1600-0854.2010.01033.x

  View details for Web of Science ID 000275530700012

  View details for PubMedID 20059747

  View details for PubMedCentralID PMC2919199

• Pharmacology and mechanism of action of pregabalin: The calcium channel alpha(2)-delta (alpha(2)-delta) subunit as a target for antiepileptic drug discovery EPILEPSY RESEARCH Taylor, C. P., Angelotti, T., Fauman, E. 2007; 73 (2): 137-150

  Abstract

  Pregabalin (Lyrica) is a new antiepileptic drug that is active in animal seizure models. Pregabalin is approved in US and Europe for adjunctive therapy of partial seizures in adults, and also has been approved for the treatment of pain from diabetic neuropathy or post-herpetic neuralgia in adults. Recently, it has been approved for treatment of anxiety disorders in Europe. Pregabalin is structurally related to the antiepileptic drug gabapentin and the site of action of both drugs is similar, the alpha2-delta (alpha2-delta) protein, an auxiliary subunit of voltage-gated calcium channels. Pregabalin subtly reduces the synaptic release of several neurotransmitters, apparently by binding to alpha2-delta subunits, and possibly accounting for its actions in vivo to reduce neuronal excitability and seizures. Several studies indicate that the pharmacology of pregabalin requires binding to alpha2-delta subunits, including structure-activity analyses of compounds binding to alpha2-delta subunits and pharmacology in mice deficient in binding at the alpha2-delta Type 1 protein. The preclinical findings to date are consistent with a mechanism that may entail reduction of abnormal neuronal excitability through reduced neurotransmitter release. This review addresses the preclinical pharmacology of pregabalin, and also the biology of the high affinity binding site, and presumed site of action.

  View details for DOI 10.1016/j.eplepsyres.2006.09.008

  View details for Web of Science ID 000245135700001

  View details for PubMedID 17126531

• Anesthetic implications of a near-lethal sodium azide exposure ANESTHESIA AND ANALGESIA Angelotti, T., Mireles, S., McMahon, D. 2007; 104 (1): 229-230

  View details for DOI 10.1213/01.ane.0000249841

  View details for Web of Science ID 000243040100085

  View details for PubMedID 17179300

• Molecular insights into alpha2 adrenergic receptor function: Clinical implications Seminars in Anesthesia, Perioperative Medicine and Pain Hurt CM, Angelotti T 2007; 26 (1): 28-34
• Differential targeting and function of alpha(2A) and alpha(2C) adrenergic receptor subtypes in cultured sympathetic neurons NEUROPHARMACOLOGY Brum, P. C., Hurt, C. M., Shcherbakova, O. G., Kobilka, B., Angelotti, T. 2006; 51 (3): 397-413

  Abstract

  Previous research suggested that alpha2A and alpha2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed alpha2A and alpha2CARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, alpha2A and alpha2CARs could be readily detected in SGN from wild-type mice. By Day 8, alpha2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas alpha2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, alpha2C ARs had relocated to somatodendritic and axonal sites and, unlike alpha2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for alpha2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous alpha2A and alpha2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the co-localization at Day 16 of alpha2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between alpha2A and alpha2C ARs in SGN.

  View details for DOI 10.1016/j.neuropharm.2006.03.032

  View details for Web of Science ID 000240811900001

  View details for PubMedID 16750543

• Verification of endotracheal tube placement by prehospital providers: is a portable fiberoptic bronchoscope of value? Air medical journal Angelotti, T., Weiss, E. L., Lemmens, H. J., Brock-Utne, J. 2006; 25 (2): 74-78

  Abstract

  This study was designed to examine whether a handheld, battery-operated fiberoptic bronchoscope (FOB) used to verify endotracheal tube (ETT) placement would be as sensitive and specific as other modes and whether a combination of multiple modes would further enhance the sensitivity and specificity of ETT placement verification.An academic hospital-based air medical program.This was a prospective, randomized study examining surgical patients undergoing general endotracheal anesthesia. Eighteen critical care transport (CCT) nurses, previously unfamiliar with FOB, were asked to identify intratracheal and intraesophageal ETTs by using misting, end-tidal carbon dioxide concentration (ETCO(2)), and FOB alone or with a combination of all three modes. The sensitivity and specificity of single and multimode verification were calculated and compared.Comparison of ETT verification by single mode alone revealed a rank order of sensitivity with ETCO(2) (0.97) > FOB (0.87) > misting (0.84), whereas all three modes had similar specificities (0.93-0.94). However, the use of the three-mode combination revealed a sensitivity and specificity of 1.0.As a single mode for ETT verification, use of a handheld, battery-operated FOB device allowed for direct visualization and had an 87% sensitivity and 93% specificity. When combined with misting and ETCO(2), FOB allowed 100% successful verification of ETT placement.

  View details for PubMedID 16516118

• A Subarachnoid Hemorrhage Presenting as Massive Nasopharyngeal Bleeding: A Case Report A & A PRACTICE Zvara, J., Hafen, R. D., Angelotti, T. 2018; 11 (1): 11–13

  View details for DOI 10.1213/XAA.0000000000000720

  View details for Web of Science ID 000437313900004

• A Subarachnoid Hemorrhage Presenting as Massive Nasopharyngeal Bleeding: A Case Report. A&A practice Zvara, J., Hafen, R. D., Angelotti, T. 2018

  Abstract

  Patients who present with a subarachnoid hemorrhage may have more than 1 intracranial aneurysm at risk, which may not be appreciated until a subsequent aneurysmal bleeding event occurs. We describe a patient who underwent successful aneurysmal clipping, but later presented urgently with large-volume epistaxis 48 hours after the procedure. After successful intubation of the patient, subsequent angiographic imaging determined that the massive intranasal/oral hemorrhage was due to bleeding through the former operative site, from rupture of a previously unrecognized aneurysm. This series of events demonstrates the importance of selecting the most at-risk aneurysm for surgical intervention.

  View details for PubMedID 29634555

• A Gap, and Opportunity, in the ICU Admission, Discharge, and Triage Guidelines. Critical care medicine Frakes, M. A., Wilcox, S. R., Bigham, M. T., Angelotti, T., Marcolini, E. G., Cohen, J. 2017; 45 (3): e337-e338

  View details for DOI 10.1097/CCM.0000000000002202

  View details for PubMedID 28212241

• Editorial comment: esophageal perforation and pneumothorax after routine intraoperative orogastric tube placement. A & A case reports Angelotti, T. 2014; 2 (10): 125-?

  View details for DOI 10.1213/XAA.0000000000000018

  View details for PubMedID 25611993

• Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of alpha(2)-Adrenergic Responses Is because of Ligand-selective Internalization JOURNAL OF BIOLOGICAL CHEMISTRY Lu, R., Li, Y., Zhang, Y., Chen, Y., Shields, A. D., Winder, D. G., Angelotti, T., Jiao, K., Limbird, L. E., Zhou, Y., Wang, Q. 2009; 284 (19): 13233-13243

  Abstract

  Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

  View details for DOI 10.1074/jbc.M807535200

  View details for Web of Science ID 000265688300070

  View details for PubMedID 19276088

  View details for PubMedCentralID PMC2676055

• Sex-specific modulation of spinal nociception by alpha(2)-adrenoceptors: Differential regulation by estrogen and testosterone NEUROSCIENCE Thompson, A. D., Angelotti, T., Nag, S., Mokha, S. S. 2008; 153 (4): 1268-1277

  Abstract

  Sex-related differences in antinociception produced by the activation of alpha(2)-adrenoceptors (alpha(2)-ARs) have been reported, however, the precise role of gonadal steroids is still unknown. Hence, we hypothesized that estrogen and testosterone modulate antinociceptive effects of clonidine (an alpha(2)-AR agonist) on N-methyl-D-aspartate- (NMDA) and heat-induced spinal nociception. We also investigated whether estrogen or testosterone alters the expression of alpha(2A)-adrenoceptors in the spinal cord. Sprague-Dawley (SD) rats were implanted with PE10 cannulae in the intrathecal space of the lumbosacral spinal cord and divided into male, proestrous and diestrous female, ovariectomized (OVX), estradiol-treated OVX (OVX+E), castrated male (GDX), testosterone (GDX+T) and estradiol-treated castrated male (GDX+E) groups. Clonidine dose-dependently inhibited NMDA-induced scratching behavior in the male and OVX groups but to a significantly lesser extent in the OVX+E group. It also increased the tail withdrawal latency in the male, OVX, diestrous and GDX+T groups but not in the OVX+E, proestrous, GDX and GDX+E groups. Levels of alpha(2A)-AR mRNA were significantly higher in the OVX, estradiol-treated OVX, GDX and GDX+E animals. In contrast, alpha(2A)-AR protein levels were higher in estradiol-treated OVX, GDX, GDX+T and GDX+E animals as compared with the male. Indeed, no correlations were observed between changes in the mRNA or protein levels of alpha(2A)-AR and behavioral observations. These results support our hypothesis that sex-related differences in alpha(2)-AR-mediated modulation of spinal nociception are gonadal hormone-dependent: estrogen attenuates antinociceptive effects in females whereas testosterone is required for the expression of antinociception in males. In addition, results also revealed that the mechanism of action of gonadal hormones may not involve a global alternation in expression of alpha(2A)-AR in the spinal cord. Estrogen-induced attenuation of alpha(2)-AR-mediated inhibition of nociception could contribute to the higher prevalence of pain syndromes in women.

  View details for DOI 10.1016/j.neuroscience.2008.03.008

  View details for Web of Science ID 000256742200035

  View details for PubMedID 18434028

• Evolutionarily conserved trafficking signal within the alpha2c adrenergic receptor restricts plasma membrane expression Angelotti, T., Daunt, D., Kobilka, B., Hurt, C. FEDERATION AMER SOC EXP BIOL. 2008

  View details for Web of Science ID 000208467809375

• Regulation of alpha 2C adrenergic receptor cell surface expression by its amino terminal domain. Angelotti, T., Kobilka, B., Hurt, C. LIPPINCOTT WILLIAMS & WILKINS. 2007: A32

  View details for Web of Science ID 000251398900119

• New methods for direct verification of correct endotracheal tube placement ANESTHESIA AND ANALGESIA Angelotti, T., Brock-Utne, J. 2007; 105 (4): 1168-1168

  View details for DOI 10.1213/01.ane.0000278153.05254.48

  View details for Web of Science ID 000249678500047

  View details for PubMedID 17898410

• Anesthesia for older patients with hypertrophic cardiomyopathy: is there cause for concern? JOURNAL OF CLINICAL ANESTHESIA Angelotti, T., Fuller, A., Rivera, L., Schmiesing, C. 2005; 17 (6): 478-481

  Abstract

  Hypertrophic cardiomyopathy (HCM) may remain clinically silent and undiagnosed until patients reach advanced age. We describe 2 older patients with previously undetected and probable late-onset HCM whose preoperative cardiac examination revealed only the presence of a systolic murmur. Both patients were diagnosed with HCM by perioperative echocardiography. We provide an algorithm for the evaluation of murmurs detected during the preoperative anesthesia evaluation, with emphasis on the clinical characteristics of HCM, and we discuss the perioperative management of these patients. In addition, recent findings concerning the natural progression of HCM are discussed, revealing differences between HCM in younger patient populations and that in older patient populations and its implications for anesthetic management.

  View details for DOI 10.1016/j.jclinane.2004.09.009

  View details for Web of Science ID 000232551100013

  View details for PubMedID 16171671

• Diagnosis of an unsuspected maternal hemorrhage via fetal heart rate tracing JOURNAL OF CLINICAL ANESTHESIA Doyle, C., Angelotti, T. 2004; 16 (6): 465-468

  Abstract

  Early detection of placental abruption often relies on the observation of vaginal bleeding; however, overt bleeding does not always occur. We report the case of an unsuspected placental abruption diagnosis that was prompted by an internal fetal scalp electrode tracing. The presence of a "normal" fetal heart rate (FHR) of approximately 150 beats per minute with poor variability, which matched the maternal heart rate (MHR), suggested that the tracing was not of fetal origin. An urgent ultrasound examination revealed a fetal demise with a possible concealed abruption, proving that the scalp electrode tracing was actually maternal in origin. Though reports of transmission of MHR via a deceased fetus are not new, it is uncommon for MHR to mimic a normal, reassuring FHR. This report reinforces the need for anesthesiologists to be adept at interpreting and integrating FHR monitors with maternal monitors prior to initiation of epidural analgesia.

  View details for DOI 10.1016/j.jclinane.2003.11.005

  View details for Web of Science ID 000225752400015

  View details for PubMedID 15567655

• Right subclavian artery injury ANESTHESIA AND ANALGESIA Angelotti, T., Amador, E. R. 2003; 96 (4): 1237-1237

  View details for DOI 10.1213/01.ANE.0000048363.63828.C4

  View details for Web of Science ID 000181808300076

  View details for PubMedID 12651702

• Nodular invasive tracheobronchitis due to Aspergillus in a patient with systemic lupus erythematosus LUPUS Angelotti, T., Krishna, G., Scott, J., Berry, G., Weinacker, A. 2002; 11 (5): 325-328

  Abstract

  Nodular or pseudomembranous tracheobronchitis due to infection by Aspergillus species is an uncommon presentation of invasive aspergillosis. Most cases have been described in severely immunocompromised hosts. We describe the case of a 23-year-old woman, with recently diagnosed systemic lupus erythematosus, who developed worsening respiratory function. Bronchoscopy revealed rapid development and progression of multiple nodular plaques in her trachea and bronchi. Endobronchial biopsy demonstrated invasive fungal infection with tissue necrosis and the presence of hyphal elements consistent with aspergillosis. To the best of our knowledge, this is only the second report of fulminant invasive tracheobronchitis due to Aspergillus in a patient with an autoimmune disease.

  View details for DOI 10.1191/0961203302lu206cr

  View details for Web of Science ID 000176537200011

  View details for PubMedID 12090570

• Activation of phosphatidylinositol 3-kinase is necessary for differentiation of FDC-P1 cells following stimulation of type III receptor tyrosine kinases CELL GROWTH & DIFFERENTIATION Kubota, Y., Angelotti, T., Niederfellner, G., Herbst, R., Ullrich, A. 1998; 9 (3): 247-256

  Abstract

  Signaling molecules that are responsible for proliferation and differentiation of hematopoietic cells following ectopic expression of receptor tyrosine kinases (RTKs) were investigated in the interleukin 3 (IL-3)-dependent hematopoietic cell line, FDC-P1. Cells were transfected with human platelet-derived growth factor receptor (PDGF-R), macrophage colony stimulating factor-1 receptor (CSF-1R), epidermal growth factor receptor (EGF-R), and chimeras consisting of the extracellular domain of EGF-R and the transmembrane and cytoplasmic domains of either HER2 (HER1-2) or c-kit (EK-R). All FDC-P1 transfectants proliferated in response to the corresponding growth factor in the absence of IL-3. However, only cells expressing PDGF-R, CSF-1R, and EK-R (type III RTKs) differentiated along the monocyte-macrophage lineage after treatment with their activating ligands. Analysis of proteins from these RTK-expressing cells revealed that a Mr 85,000 protein showed in vitro phosphorylation, and V8 protease peptide mapping showed that this protein was p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Accordingly, activation of PDGF-R-, CSF-1R-, and EK-R-expressing cells led to an increase in PI3-kinase activity. Expression of EK-R mutant Y721F, which lacked the known p85 binding site, blocked differentiation and activation of PI3-kinase, without affecting proliferation. Last, addition of wortmannin to cells expressing PDGF-R, CSF-1R, and EK-R blocked ligand-induced differentiation in a concentration-dependent manner, and this effect correlated with wortmannin's ability to inhibit PI3-kinase. Thus, ectopic expression of both type I and III RTKs could stimulate FDC-P1 proliferation in the absence of IL-3; however, only activation of type III RTKs led to differentiation via selective coupling to p85 and PI3-kinase activation.

  View details for Web of Science ID 000072452700006

  View details for PubMedID 9543391

• The "VH1-like" dual-specificity protein tyrosine phosphatases MOLECULES AND CELLS Martell, K. J., Angelotti, T., Ullrich, A. 1998; 8 (1): 2-11

  Abstract

  The number of dual-specificity protein tyrosine phosphatases has grown considerably in the last few years, and thus it would be helpful to organize these novel enzymes. The simple term "VH1-like" or "dual-specificity" phosphatase does not adequately reflect the different subclasses within this new and important phosphatase subfamily. In this article, we review the salient features of dual-specificity phosphatases and propose a novel classification scheme of these enzymes based on their structural motifs. Classification of dual-specificity phosphatases based on their motifs should serve as a useful organizational framework for bringing together this now large subgroup of phosphatases. Moreover, this classification scheme may hold predictive value, since some of these motifs may hold the key to new, undiscovered functional properties.

  View details for Web of Science ID 000072500600001

  View details for PubMedID 9571625

• Tissue-specific expression of splice variants of the mouse voltage-gated calcium channel alpha 2/delta subunit FEBS LETTERS Angelotti, T., Hofmann, F. 1996; 397 (2-3): 331-337

  Abstract

  Five different splice variants of mouse alpha2/delta subunit isoforms (alpha2a-e), which arose from various combinations of three alternatively spliced regions, were cloned with a combination of cDNA library screening and RT-PCR. Expression patterns and relative abundance of the various isoforms in mouse tissues were determined with an RNAse protection assay. Skeletal muscle and brain expressed single isoforms, alpha2a and alpha2b, respectively; however, the cardiovascular system expressed all five isoforms. Heart expressed mainly isoforms alpha2c and alpha2d while, in contrast to other species, aorta expressed predominantly alpha2a, the 'skeletal muscle' isoform. Smooth muscle-containing tissues expressed alpha2d and alpha2e. Thus, alpha2/delta isoforms are restricted in their tissue expression, suggesting an important functional role for the differentially spliced variants.

  View details for Web of Science ID A1996VV17100043

  View details for PubMedID 8955374

• Enhancement of recombinant alpha 1 beta 1 gamma 2L gamma-aminobutyric acid(A) receptor whole-cell currents by protein kinase C is mediated through phosphorylation of both beta 1 and gamma 2L subunits MOLECULAR PHARMACOLOGY Lin, Y. F., ANGELOTTI, T. P., Dudek, E. M., Browning, M. D., Macdonald, R. L. 1996; 50 (1): 185-195

  Abstract

  The gamma-aminobutyric acidA (GABA)A receptor (GABAR) beta 1 and gamma 2L subtypes have been shown to be phosphorylated in vitro by protein kinase C (PKC) [J. Biol. Chem. 267:14470-14476 (1992); Neuron 12:1081-1095 (1994)]. To determine the physiological consequences of phosphorylation of GABAR isoforms containing the beta 1 and gamma 2L subtypes, the specific serine residues phosphorylated by PKC (beta 1 S409, gamma 2L S327 and S343) were changed to alanines through site-directed mutagenesis. Wild-type (alpha 1 beta 1 gamma 2L GABARs) and three mutant GABAR isoforms [alpha 1 beta 1 gamma 2L(S327A, S343A), alpha 1 beta 1(S409A) gamma 2L, and alpha 1 beta 1(S409A) gamma 2L(S327A, S343A) GABARs) were expressed in mouse L929 fibroblasts through transient cotransfection. Recordings were obtained from each cell with the use of the whole-cell patch-clamp technique. The initial recording was made with the use of control intrapipette solution, and a second recording from the same cell was obtained with pipettes containing either constitutively active PKC [protein kinase M (PKM)] or control solution to obtain paired GABA concentration-response relationships. All GABAR isoforms studied had equivalent maximal GABA currents and similar GABA concentration-response profiles under the control condition. Intracellular PKM treatment increased the maximal current and EC50 value in cells expressing wild-type GABARs. However, PKM reimpalement did not significantly change these parameters in cells expressing any of the mutant GABAR isoforms, indicating that the mutation of either the beta 1 or gamma 2L subtype alone was sufficient to prevent enhancement of GABAR current by PKM. No significant changes were obtained during control reimpalement recordings of wild-type or mutant receptors. Furthermore, PKM treatment did not after the time constants of GABA current desensitization kinetics measured from cells expressing wild-type or mutant receptors. These data thus suggest that PKC phosphorylation of the beta 1 and gamma 2L subtypes enhances GABAR current and that both subtypes are required for complete PKC-mediated enhancement of alpha 1 beta 1 gamma 2L GABAR current.

  View details for Web of Science ID A1996UY02700024

  View details for PubMedID 8700112

• Functional expression of recombinant GABAA receptor channels in L929 fibroblasts. Epilepsy research. Supplement Macdonald, R. L., Saxena, N. C., ANGELOTTI, T. P. 1996; 12: 177-185

  Abstract

  While GABAergic inhibition plays a major role in the regulation of neuronal excitability, a role for altered GABAergic inhibition in the pathogenesis of epilepsy remains to be proven. The demonstration that GABAA receptors are composed of multiple subunits and that the properties and pharmacology of GABAA receptors are different for different subunit combinations, suggests that GABAA receptor heterogeneity may be of importance in determining the properties of GABAergic inhibition in different regions of the nervous system. While it is clear that GABAA receptor heterogeneity is present in the nervous system, a role for receptor heterogeneity in the pathogenesis of epilepsy remains uncertain. GABAA receptor heterogeneity may have implications for the treatment of epilepsy. It is quite possible that drugs which regulate GABAergic function may have variable efficacy in different regions of the nervous system due to expression of receptors with subunits that have different sensitivity to allosteric regulators. In situ hybridization studies indicate the colocalization of alpha 1 beta 1 gamma 2L and delta subunit mRNAs in hippocampal dentate gyrus granule cells while only the alpha 1, beta 1 and gamma 2L and not the delta subunit mRNAs colocalize in the pyramidal cells of the hippocampus. The reduced rate of acute desensitization and the slow recovery of GABA-evoked currents typical of delta-containing subunit combinations could generate tonic inhibition via long-lasting IPSPs in the dentate gyrus and thus play a role in preventing seizures. By the same rationale, a reduction in the level of expression of the delta subunit mRNA in the dentate gyrus or its absence as in the hippocampal pyramidal cells could be associated with a reduced seizure threshold. Furthermore, it is likely that there are developmental changes in the stoichiometry or subunit composition of GABAA receptors rendering the developing nervous system more or less sensitive to the effects of GABAergic anticonvulsant drugs. In addition to the heterogeneous expression of GABAA receptors, other issues concerning the regulation of GABAergic function are of potential importance. The regulatory events that control the expression of specific receptor subtypes and levels of GABA receptors are unknown. To understand the role of GABAA receptor heterogeneity in the pathogenesis of epilepsy will require the combination of biophysical and molecular biological techniques. It will be important to determine not only whether the properties of GABAA receptors have been altered in a specific form of epilepsy but also whether gene expression has been altered.

  View details for PubMedID 9302516

• THE TYPE-II ISOFORM OF CGMP-DEPENDENT PROTEIN-KINASE IS DIMERIC AND POSSESSES REGULATORY AND CATALYTIC PROPERTIES DISTINCT FROM THE TYPE-I ISOFORMS JOURNAL OF BIOLOGICAL CHEMISTRY Gamm, D. M., Francis, S. H., ANGELOTTI, T. P., Corbin, J. D., Uhler, M. D. 1995; 270 (45): 27380-27388

  Abstract

  The type I cGMP-dependent protein kinases (cGK I alpha and I beta) form homodimers (subunit M(r) approximately 76,000), presumably through conserved, amino-terminal leucine zipper motifs. Type II cGMP-dependent protein kinase (cGK II) has been reported to be monomeric (M(r) approximately 86,000), but recent cloning and sequencing of mouse brain cGK II cDNA revealed a leucine zipper motif near its amino terminus. In the present study, recombinant mouse brain cGK II was expressed, purified, and characterized. Sucrose gradient centrifugation and gel filtration chromatography were used to determine M(r) values for holoenzymes of cGK I alpha (168,000) and cGK II (152,500), which suggest that both are dimers. Native cGK I alpha possessed significantly lower K alpha values for cGMP (8-fold) and beta-phenyl-1,N2-etheno-cGMP (300-fold) than did recombinant cGK II. Conversely, the Sp- and Rp-isomers of 8-(4-chloro-phenylthio)-guanosine-3',5'-cyclic monophosphorothioate demonstrated selectivity toward cGK II in assays of kinase activation or inhibition, respectively. A peptide substrate derived from histone f2B had a 20-fold greater Vmax/Km ratio for cGK I alpha than for cGK II, whereas a peptide based upon a cAMP response element binding protein phosphorylation site exhibited a greater Vmax/Km ratio for cGK II. Finally, gel filtration of extracts of mouse intestine partially resolved two cGK activities, one of which had properties similar to those demonstrated by recombinant cGK II. The combined results show that both cGK I and cGK II form homodimers but possess distinct cyclic nucleotide and substrate specificities.

  View details for Web of Science ID A1995TE58300102

  View details for PubMedID 7593002

• Fluorescent b-galactosidase staining of transfected cells prior to patch clamp recording. AxoBits Angelotti, T., Macdonald RL 1994; 15 (15): 6-8
• ENHANCEMENT OF RECOMBINANT GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR CURRENTS BY CHRONIC ACTIVATION OF CAMP-DEPENDENT PROTEIN-KINASE MOLECULAR PHARMACOLOGY ANGELOTTI, T. P., Uhler, M. D., Macdonald, R. L. 1993; 44 (6): 1202-1210

  Abstract

  alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.

  View details for Web of Science ID A1993MN46600016

  View details for PubMedID 8264557

• NATIVE AND RECOMBINANT GABA(A) RECEPTOR CHANNELS CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Macdonald, R. L., ANGELOTTI, T. P. 1993; 3 (5-6): 352-373

  View details for Web of Science ID A1993LX26500009

• ASSEMBLY OF GABA(A) RECEPTOR SUBUNITS - ALPHA-1-BETA-1-SUBUNIT AND ALPHA-1-BETA-1-GAMMA-2S-SUBUNIT PRODUCE UNIQUE ION CHANNELS WITH DISSIMILAR SINGLE-CHANNEL PROPERTIES JOURNAL OF NEUROSCIENCE ANGELOTTI, T. P., Macdonald, R. L. 1993; 13 (4): 1429-1440

  Abstract

  Recent experimental evidence has led to the hypothesis that GABAA receptor channel (GABAR) heterogeneity or receptor channel subtypes may occur by differential assembly of a given set of subunits into various configurations. Alternatively, assembly of subunits into mature GABARs may arise from an ordered process to produce a preferred form of the receptor channel, as seen for nicotinic ACh receptors. In the preceding article, we demonstrated that transient expression of GABAR alpha 1 and beta 1 subunits in mouse L929 fibroblast cells produced two different types of GABARs, when coexpressed with and without the gamma 2S subunit. Not only did these GABARs differ in their GABA and diazepam pharmacology, but initial single-channel recordings suggested that the two types of GABARs (alpha 1 beta 1 and alpha 1 beta 1 gamma 2S) had different conductance and gating properties. It also appeared that alpha 1 beta 1 gamma 2S GABARs were preferentially formed over alpha 1 beta 1 GABARs, but it was not completely shown if both forms of GABARs were produced when a cell expressed all three subunits. To characterize further the assembly process and determine the preferred form, if it existed, it was necessary to obtain a kinetic "fingerprint" for both alpha 1 beta 1 and alpha 1 beta 1 gamma 2S GABARs. Thus, single-channel patch-clamp recording and kinetic analysis of receptor channel gating were performed. For both alpha 1 beta 1 and alpha 1 beta 1 gamma 2S GABARs, GABA evoked single-channel openings to both a main conductance (15 and 29 pS, respectively) and a subconductance level (10 and 21 pS, respectively) with greater than 90% of the total current through the main conductance level openings. The two GABAR populations were further differentiated by their open and burst properties. On average, alpha 1 beta 1 gamma 2S GABARs opened for almost three times the duration as alpha 1 beta 1 GABARs (6.0 vs 2.3 msec, respectively) and had three openings per burst. alpha 1 beta 1 GABARs opened predominantly as single opening bursts. Using the conductance and gating properties to differentiate the two GABAR populations, we determined that alpha 1 beta 1 GABARs were rarely, if ever, formed upon coexpression of all three subunits, suggesting that alpha 1 beta 1 gamma 2S GABARs were the preferred final form of the receptor channel. Also, the homogeneity of the conductance and gating properties of alpha 1 beta 1 gamma 2S GABARs among the different patches studied implied that a single preferred configuration of GABARs may exist.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1993KV71100010

  View details for PubMedID 7681870

• ASSEMBLY OF GABA(A) RECEPTOR SUBUNITS - ANALYSIS OF TRANSIENT SINGLE-CELL EXPRESSION UTILIZING A FLUORESCENT SUBSTRATE MARKER GENE TECHNIQUE JOURNAL OF NEUROSCIENCE ANGELOTTI, T. P., Uhler, M. D., Macdonald, R. L. 1993; 13 (4): 1418-1428

  Abstract

  GABAA receptor channels (GABARs) composed of varying combinations of alpha 1, beta 1, and gamma 2S subunits were transiently expressed in mammalian cell lines. The whole-cell patch-clamp recording technique was used to determine which combinations of GABAR subunits produced functional receptor channels and whether assembly of GABAR subunits into receptor channels followed a random or preferred sequence. To identify rapidly cells expressing GABARs, mammalian cell lines were cotransfected with combinations of GABAR subunit cDNAs and the Escherichia coli beta-galactosidase gene as a transfection marker. Positively transfected cells were identified by staining with the enzyme substrate fluorescein di-beta-galactopyranoside. Using this technique, we confirmed that functional alpha 1 beta 1 and alpha 1 beta 1 gamma 2S GABARs were assembled in transfected mouse L929 fibroblast cells, but surprisingly, functional alpha 1 gamma 2S and beta 1 gamma 2S GABARs were not expressed. It was determined that after transient transfection, levels of expressed receptors varied little among individual cells permitting comparison of absolute whole-cell GABA-evoked current values. Whole-cell currents recorded from cells coexpressing alpha 1 beta 1 gamma 2S subunits were three to four times larger than those recorded from cells coexpressing alpha 1 beta 1 subunits, and they were always enhanced by coapplied diazepam. The increase in whole-cell current was due in part to the larger single-channel current of the alpha 1 beta 1 gamma 2S GABARs. GABARs comprised of alpha 1 beta 1 gamma 2S subunits were formed preferentially over GABARs of alpha 1 beta 1 subunits alone, since only after substantially increasing the ratio of the beta 1 expression vector over the alpha 1 and gamma 2S subunit expression vectors were alpha 1 beta 1 GABARs formed in the presence of the gamma 2S subunit. These findings suggest that assembly of GABARs from constituent subunits did not proceed randomly to form all possible combinations, but that certain subunit combinations were preferred intermediates during the assembly process.

  View details for Web of Science ID A1993KV71100009

  View details for PubMedID 7681869

• MOLECULAR AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF A ALLELIC VARIANT OF THE RAT ALPHA-6 GABA-A RECEPTOR SUBUNIT MOLECULAR BRAIN RESEARCH ANGELOTTI, T. P., Tan, F., Chahine, K. G., Macdonald, R. L. 1992; 16 (1-2): 173-178

  Abstract

  A 1.45 kb DNA sequence encoding the rat alpha 6 GABAA receptor subunit (nucleotides 33-1483) was cloned from a Sprague-Dawley rat brain cDNA library by PCR amplification. Dideoxy sequencing of two individual clones revealed that the nucleotide sequence differed at only one basepair (T480-->G) from that published previously. This difference altered the deduced amino acid sequence, producing a conservative amino acid substitution (His121-->Gln). A Gln residue is present at the same location in the bovine alpha 6 subunit. Restriction endonuclease analysis of the total PCR product demonstrated that this variant of the rat alpha 6 subunit was the only allele found in this particular rat brain library, the original allele was not present. These results were further verified by RNAse protection assays performed with RNA isolated from individual rat cerebella. alpha 6, beta 1, and gamma 2S subunits were transiently expressed in L929 cells for electrophysiological analysis. Whole-cell recordings obtained from the cells demonstrated that GABAA receptor channels with the expected GABA and benzodiazepine pharmacology were produced. Excised outside out single channel recordings from the same cells revealed that GABA elicited brief duration openings to a 33 pS main conductance level and to at least one smaller (approximately 21 pS) subconductance level. Thus this allelic variant of rat alpha 6 subunit could assemble with other subunits to form a functional GABAA receptor channel with similar properties to the original allelic form.

  View details for Web of Science ID A1992JW77400023

  View details for PubMedID 1281255

• KINETIC-PROPERTIES OF ALPHA-1-BETA-1-GAMMA-AMINOBUTYRIC ACID(A) RECEPTOR CHANNELS EXPRESSED IN CHINESE-HAMSTER OVARY CELLS - REGULATION BY PENTOBARBITAL AND PICROTOXIN MOLECULAR PHARMACOLOGY Porter, N. M., ANGELOTTI, T. P., Twyman, R. E., Macdonald, R. L. 1992; 42 (5): 872-881

  Abstract

  Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (GABA) type A (GABAA) receptor channel alpha 1 and beta 1 subunit cDNAs. The predominant or main conductance level recorded had a 17-pS chord conductance. There were minor contributions made from 25-pS and 11-pS conductance levels. Average open duration, burst duration, and openings/burst did not change as the GABA concentration was increased from 5 to 25 microM. However, opening frequency increased from 11.0 to 19.5 openings/sec. Pentobarbital increased average channel open duration without increasing opening frequency, whereas picrotoxin slightly reduced average channel open duration and reduced opening frequency. Open duration frequency distributions were fitted best with the sum of two exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had at least two open states. The time constants and relative proportions of the two components did not vary when GABA concentration was increased from 5 to 25 microM. Closed duration distributions of closures between main conductance level openings were fitted best with multiple exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had several closed states. Burst duration frequency distributions were fitted best with two exponential functions whose time constants and relative proportions did not change with GABA concentration. A gating kinetic scheme for the alpha 1 beta 1 GABAA receptor channel was proposed that consisted of a single binding site for GABA and at least two open and five closed states. The kinetic properties of the alpha 1 beta 1 main conductance level differed from those of the spinal cord neuron (native) 27-pS main conductance level and the 19-pS subconductance level. The native main conductance and subconductance levels were characterized by longer openings and at least three open states. Based on the aforementioned observations, it appears that different subunit combinations produce receptor channels with different kinetic properties, but the basic mechanism of regulation by pentobarbital and picrotoxin may be similar for the different receptor channels. Also, it is unlikely that the 19-pS substate of the native GABAA receptor is produced by an alpha 1 beta 1 dimer.

  View details for Web of Science ID A1992JY34200019

  View details for PubMedID 1331767

• REGULATION OF GABA-A-RECEPTOR CHANNELS BY ANTICONVULSANT AND CONVULSANT DRUGS AND BY PHOSPHORYLATION WORKSHOP ON MOLECULAR NEUROBIOLOGY OF EPILEPSY Macdonald, R. L., Twyman, R. E., RYANJASTROW, T., ANGELOTTI, T. P., Wilson, W. A., Fisher, R. S., Mody, I., Bowery, N. G., Ticku, M. K. ELSEVIER SCIENCE BV. 1992: 265–277

  Abstract

  The GABAA receptor channel is a highly regulated receptor. The function of the receptor may be modified by drugs which alter the rates of binding of GABA, modify the gating of the channel or block the channel. It is also likely that phosphorylation of the receptor subunits modifies the biophysical properties, stability or assembly of the receptor. While GABAergic inhibition plays a major role in the regulation of neuronal excitability, a role for altered GABAergic inhibition in the pathogenesis of epilepsy remains to be proven. The demonstration that GABAA receptors are composed of multiple subunits and that the properties and pharmacology of GABAA receptors are different for different subunit combinations suggests that GABAA receptor heterogeneity may be of importance in determining the properties of GABAergic inhibition in different regions of the nervous system. While it is clear that GABAA receptor heterogeneity is present in the nervous system, a role for receptor heterogeneity in the pathogenesis of epilepsy remains uncertain. GABAA receptor heterogeneity may have implications for the treatment of epilepsy. It is quite possible that drugs which regulate GABAergic function may have variable efficacy in different regions of the nervous system due to expression of receptors with subunits that have different sensitivity to allosteric regulators. Furthermore, it is likely that there are developmental changes in the stoichiometry or subunit composition of GABAA receptors rendering the developing nervous system more or less sensitive to the effects of GABAergic anticonvulsant drugs. In addition to the heterogeneous expression of GABAA receptors, other issues concerning the regulation of GABAergic function are of potential importance. The regulatory events that control the expression of specific receptor subtypes and levels of GABA receptors are unknown. The post-translational events that regulate GABAA receptor function are uncertain. It is possible that post-translational regulation of GABAA receptors by phosphorylation may contribute to altered GABAA receptor function in epilepsy. To understand the role of GABAA receptor heterogeneity in the pathogenesis of epilepsy will require the combination of biophysical and molecular biological techniques. It will be important to determine not only whether the properties of GABAA receptors have been altered in a specific form of epilepsy, but also whether gene expression has been altered.

  View details for Web of Science ID A1992KH38100027

  View details for PubMedID 1337441

• BIOTINYLATED GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR ANALOGS - EFFECT OF LINKAGE CHEMISTRY ON ACTIVITY AND BINDING BIOCONJUGATE CHEMISTRY ANGELOTTI, T. P., Clarke, M. F., Longino, M. A., Emerson, S. G. 1991; 2 (6): 466-474

  Abstract

  Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF) and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF) were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein (EC50 = 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be useful nonradioactive probes with which to study GM-CSF receptor cytochemistry and receptor modulation by flow cytometry.

  View details for Web of Science ID A1991GR93700016

  View details for PubMedID 1839606