Overexpression of the alpha1B-adrenergic receptor causes apoptotic neurodegeneration: multiple system atrophy.

Progress toward elucidating the function of alpha1B-adrenergic receptors (alpha1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate alpha1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active alpha1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in alpha1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the alpha1AR antagonist terazosin, indicating that alpha1AR signaling participated directly in the pathology. Our results indicate that overstimulation of alpha1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.

Delay discounting of different outcomes: Review and theory.

Steep delay discounting is characterized by a preference for small immediate outcomes relative to larger delayed outcomes and is predictive of drug abuse, risky sexual behaviors, and other maladaptive behaviors. Nancy M. Petry was a pioneer in delay discounting research who demonstrated that people discount delayed monetary gains less steeply than they discount substances with abuse liability. Subsequent research found steep discounting for not only drugs, but other nonmonetary outcomes such as food, sex, and health. In this systematic review, we evaluate the hypotheses proposed to explain differences in discounting as a function of the type of outcome and explore the trait- and state-like nature of delay discounting. We found overwhelming evidence for the state-like quality of delay discounting: Consistent with Petry and others' work, nonmonetary outcomes are discounted more steeply than monetary outcomes. We propose two hypotheses that together may account for this effect: Decreasing Future Preference and Decreasing Future Worth. We also found clear evidence that delay discounting has trait-like qualities: People who steeply discount monetary outcomes steeply discount nonmonetary outcomes as well. The implication is that changing delay discounting for one outcome could change discounting for other outcomes.

Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function.

The alpha 1-adrenergic receptors activate a phospholipase C enzyme by coupling to members of the large molecular size (approximately 74 to 80 kilodaltons) G alpha h family of guanosine triphosphate (GTP)-binding proteins. Rat liver G alpha h is now shown to be a tissue transglutaminase type II (TGase II). The transglutaminase activity of rat liver TGase II expressed in COS-1 cells was inhibited by the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) or by alpha 1-adrenergic receptor activation. Rat liver TGase II also mediated alpha 1-adrenergic receptor stimulation of phospholipase C activity. Thus, G alpha h represents a new class of GTP-binding proteins that participate in receptor signaling and may be a component of a complex regulatory network in which receptor-stimulated GTP binding switches the function of G alpha h from transglutamination to receptor signaling.

A retrospective review of conjunctival melanoma: Presentation, treatment and evolution. / Revisión retrospectiva del melanoma conjuntival: presentación, tratamiento y evolución.

OBJECTIVE: The purpose of the present study is to describe the clinical characteristics and outcomes of patients diagnosed and treated for conjunctival melanoma in the Ocular Oncology Unit of the University Hospital Clinic of Valladolid. METHODS: A retrospective observational case series study was conducted on patients diagnosed with conjunctival melanoma in the Ocular Oncology Unit of University Hospital Clinic of Valladolid, from January 1992 to December 2017. Demographic information and tumour features were recorded in a Microsoft Access database. RESULTS: Among a total of 462 consecutive patients, the tumour was classified as melanocytic in 252 cases (54.5%), with 27 patients having the pathological diagnosis of conjunctival melanoma. The mean age at diagnosis was 59.2years (16-88), and there were 41% males and 59% females, with a mean follow-up of 6.1±6.8years. As regards the origin of conjunctival melanoma, 16 cases (59%) arose from primary acquired melanosis, 26% from nevus, and 15% developed de novo. The treatment performed was incisional or excisional biopsy in all patients, local adjuvant chemotherapy in 15 cases (56%) and brachytherapy in 5 patients (18%). The median survival was 18years and the probability of survival at 5 and 10years was 89% and 69%, respectively. CONCLUSIONS: Conjunctival melanoma is a rare disease, usually undervalued by the patient as well as being underdiagnosed, leading to insufficient and delayed treatment. Early diagnosis and treatment are essential to prevent recurrences and systemic extension, as well as to preserve vision and life.

Behaviour of the main nonsteroidal anti-inflammatory drugs in a membrane bioreactor treating urban wastewater at high hydraulic- and sludge-retention time.

The behaviour and removal efficiency of ibuprofen (IBU), diclofenac (DCF), ketoprofen (KPF), and naproxen (NPX) during the real urban wastewater treatment by an experimental full-scale MBR working at high sludge and hydraulic retention time (SRT, HRT) were determined. The MBR worked in denitrification/nitrification conformation at 35.4h of HRT (Q=0.45m3/h), 37 d of SRT and a recirculation flow rate of 4Q. The experiments were made under steady-state conditions, reaching a biodegradable organic matter removal higher than 99.5%. The MBR system showed similar removal capacity for IBU, NPX, and KTP (>95%), whose main transformation occurred in the aerobic reactor with a low contribution from the anoxic reactor. The system worked with complete nitrification, also achieving an effective retention of the unbiodegradable organic matter due to recirculation. DCF removal was low with negative removal yields for several samplings. Both removal and increase transformation of DCF also occurred in the aerobic reactor, this not being observed in the anoxic one. DCF tends to accumulate in the system and to be recirculated. Thus, during the sampling in which DCF influent concentration decreases, removal yields turn negative. The increase of DCF concentration in the aerobic bioreactor also contributes to the negative removal yields.

Avance de tuberosidad tibial con heteroimplante óseo para el tratamiento de ruptura de ligamento cruzado anterior en caninos / Advance of tibial tuberosity with bone heteroimplant for previous crossed ligament in canine

RESUMEN Las evaluaciones clínica y radiológica son técnicas que se emplean para el diagnóstico de ruptura de ligamento cruzado anterior (RLCA) y para la evaluación posoperatoria de cirugías correctivas. Se planteó como objetivo describir los resultados posoperatorios de la técnica avance de tuberosidad tibial en caninos con RLCA utilizando hueso de origen bovino conservado en glicerina al 98%. Fueron intervenidos 5 caninos con la patología y sin cambios radiológicos marcados en las columnas óseas relacionadas. El heteroim-plante óseo se cortó a manera de cuña y, para determinar su tamaño, se tuvo en cuenta la medida prequirúrgica que necesitaba la rodilla para alcanzar el ángulo de avance de la cresta tibial. Seguidamente se fijó al receptor local con implantes metálicos (agujas de Kirschner y alambre ortopédico). En el posoperatorio, se evaluaron por 90 días signos clínicos como claudicación, dolor al movimiento flexión-extensión e inflamación, según la circunferencia de la rodilla, y se evaluó por 120 días el ángulo de avance tibial y la radiopacidad del heteroimplante con respecto al hueso circunvecino (tuberosidad tibial y tibia caudal). El estudio estableció que los parámetros clínicos y radiológicos tienen un efecto estadísticamente significativo; sin embargo, la evaluación del ángulo de avance tibial no tiene un efecto significativo entre el paciente y los días posquirúrgicos. Durante el periodo de evaluación el heteroimplante óseo bovino demostró ser efectivo para estabilizar una rodilla con RLCA en caninos.

ABSTRACT The clinical and radiological evaluations are techniques used for the diagnosis of the anterior cruciate ligament rupture (LCAR) and the post-operatory assessment of corrective surgeries. It was proposed as an objective to describe the post-operatory results of the advance technique of tibial tuberosity in canines with LCAR using bovine bone conserved in 98% glycerin. Five canines were intervened and with no other radiological changed in the related bone columns. The bone heteroimplant was cut in a wedge-like manner and for its size it was taken into account the pre-operatory measurement that the knee needed to reach the advancement angle of the tibial crest. Next, the bone implant was fixated to the local receptor with metallic implants (Kirschner needles and orthopedic wire). In the post-op, the canines were evaluated for 90 days for clinical signs such as claudication, pain when flexing and extending and inflammation according to the knee circumference, and for 120 days the advancement angle of the tibia and the radiopacitry of the heteroimplant in relation to the surrounding bone. The trial established that the clinical and radiological parameters have a statistically significant effect; however, the evaluation of the tibial advancement angle does not have a significant effect between the patient and the post-operatory days. During the evaluation period, the heteroimplant bovine bone proved to be effective to stabilize the knee in canines with LCAR.

Recent advances in the molecular pharmacology of the alpha 1-adrenergic receptors.

This review is intended to discuss recent developments in the molecular pharmacology of the alpha 1-adrenergic receptor (alpha 1-AR) subtypes. After a brief historical development, we will focus on the more contemporary issues having to do with this receptor family. Emphasis will be put on recent data regarding the cloning, nomenclature, signalling mechanisms, and genomic organization of the alpha 1-AR subtypes. We will also highlight recent mutational studies that identify key amino acid residues involved in ligand binding, as well as the role of the alpha 1-AR subtypes in regulating physiologic processes.

Bovine beta-lactoglobulin: interaction studies with palmitic acid.

Bovine beta-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

A comparison of saliva testing to urinalysis in an arrestee population.

Past studies have concluded that individuals under criminal justice supervision often underreport their recent use of illicit drugs. To address this underreporting, objective biological measures, such as urine, saliva, and hair testing, have been used to gain better estimates of illegal drug use. While urinalysis is generally recognized as the reference standard, a method recently introduced in nonlaboratory settings for ascertaining drug use-saliva testing-may offer an alternative to urinalysis. To date, however, no studies have compared saliva testing to urinalysis among criminal justice populations. In the current study, urine and saliva specimens were collected from 114 adult arrestees interviewed as part of Maryland's Substance Abuse Need for Treatment among Arrestees (SANTA) project. With urinalysis as the reference standard, analysis of the saliva test results indicated sensitivity of 100% and specificity of 99% for cocaine and sensitivity of 88% and specificity of 100% for heroin. For marijuana, however, the saliva results indicated a sensitivity of only 5%. Anecdotal reports from the field suggest that saliva may have some advantages over urine because of the ease of collection, invulnerability to adulteration, and minimal personal invasiveness. These findings suggest that a more comprehensive study to evaluate the efficacy of saliva testing in field research may be warranted.

The influence of utterance length upon bilabial closure duration of selected deaf children.

Measurements were made of intraoral air pressure associated with bilabial closure durations of VCV syllables in sentence-like frames of increasing length for nine deaf and nine matched normal hearing chidren. Results demonstrated significantly longer absolute closure durations at all utterance lengths for the deaf, but with the same relative decrease in closure durations with increases in utterance length for both groups. The deaf children showed lower peak intraoral air pressure values with more variability than the normal children. In addition, the deaf children had longer air-pressure pulse durations than did the normals. Clinical application of these findings to speech management are discussed.

Caracterización de la condición periodontal de pacientes adultos con obesidad / Characterization of the periodontal condition in obese adults

La periodontitis es una enfermedad inflamatoria que puede evolucionar hasta la pérdida dentaria. En la presente investigación se caracterizó la condición periodontal de 30 adultos con obesidad, atendidos en el Instituto de Investigaciones en Nutrición (INVESNUT) de la Facultad de Ciencias de la Salud, Universidad de Carabobo y de un grupo control conformado por 15 adultos normo-peso. Los adultos fueron evaluados clínica y odontológicamente, mediante antropometría (Talla, Peso, IMC, circunferencia abdominal) y los índices: índice de higiene oral simplificado (IHOS), índice de gingivitis (IG), índice de sangramiento gingival (IGB "gingival bleeding index"). El grupo de estudio mostró diferencias significativas para IG, IGB IHOS con respecto al control, el análisis de correlación de Pearson para las variables IMC, circunferencia abdominal y las variables sobre la condición periodontal (IG, IGB) mostraron asociación positiva y significativa, aunque se no se observó correlación para las variables IMC y circunferencia abdominal con IHOS. Se concluye que los pacientes obesos presentan una peor condición periodontal y que su higiene oral no corresponde con el estado inflamatorio del periodonto...

Periodontitis is an inflammatory disease that may progress to tooth loss. The objective of this research was to characterize the periodontal condition of 30 obese adults treated at the Nutrition Research Institute (INVESNUT), Faculty of Health Sciences, University of Carabobo and of a control group of 15 normal-weight adults. All participants were assessed clinically and dentally by anthropometry (Height, weight, BMI, abdominal circumference) and indexes: simplified oral hygiene index (OHI-S), gingivitis index (GI) gingival bleeding index (GBI). The study group showed significant differences for GI, GBI, OHI-S relative to the control, and a significant positive association (Pearson correlation analysis) for BMI, abdominal circumference, GI and GBI, showed, although no correlation was observed to the variables BMI, abdominal circumference with OHI-S. It is concluded that obese patients have a worse periodontal condition, but oral hygiene does not correspond to the inflammatory state of the periodontium...

Participación de los mediadores de la respuesta inmune inflamatoria en la resorción del hueso alveolar durante la periodontitis crónica: revisión de la literatura / Participation of the immune response mediators in inflamatory alveolar bone resortion during chronic periodontitis: a literature review

La enfermedad pulmonar obstructiva crónica (EPOC) abarca todas aquellas enfermedades respiratorias que cursan con obstrucción no totalmente reversible del flujo aéreo. La limitación es progresiva y está asociada a una respuesta inflamatoria. La denominación de fenotipo se utiliza para referirse a formas clínicas de los pacientes con EPOC, describiéndose: 1. No agudizador, con enfisema o bronquitis crónica, 2. Mixto EPOC-asma, 3. Agudizador con enfisema y 4. Agudizador con bronquitis crónica. La superposición de los síntomas hace difícil el diagnóstico, y para la mayoría de los pacientes, el tabaquismo es el factor etiológico más importante. La obstrucción de las vías bronquiales en el asma es esencialmente reversible, pero muchos años de exacerbaciones recurrentes puede producir una obstrucción permanente debido al remodelado de las vías respiratorias. La inflamación crónica esta asociada a un aumento en la hiperreactividad de la vía aérea que conduce a episodios recurrentes de sibilancias, disnea, opresión torácica y tos, particularmente en la noche o temprano en la mañana. Estos episodios se asocian generalmente a la obstrucción generalizada pero variable en el flujo aéreo pulmonar que es frecuentemente reversible espontáneamente o con tratamiento

Chronic obstructive pulmonary disease (COPD) includes all those respiratory diseases that curse with not fully reversible obstruction of the airflow. The limitation is progressive and its associated with a inflammatory response. The denomination of phenotype is used to refer to clinical forms of COPD patients, describing: 1. No peaking, emphysema or chronic bronchitis, 2. Mixed COPD-asthma, 3. Peaking with emphysema and 4. Peaking with chronic bronchitis. The superposition of the symptoms makes the diagnosis difficult, and for most patients, smoking is the most important etiologic factor. The bronchial airway obstruction in asthma is essentially reversible, but many years of recurrent exacerbations can produce a permanent obstruction due to airway remodelling. Chronic inflammation is associated with increased airway hyper responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing, particularly at night or early in the morning. These episodes are usually associated with widespread but variable obstruction in lung airflow that is often reversible either spontaneously or with treatment

La respuesta inmunológica innata y la destrucción del tejido óseo en la enfermedad periodontal: revisión de la literatura / The innate immune response and tissue destruction in periodontal disease bone. A literatura review

La periodontitis desencadena una respuesta inmunológica del huésped contra los microorganismos de la placa dentobacteriana y sus productos. La activación de los receptores Toll-like (TLR) y Nod-like (NLR) en presencia de antígenos bacterianos inducen la respuesta inflamatoria y celular, con la consecuente expresión de citocinas inflamatorias como interleucina 1 (IL-1), interleucina 6 (IL-6), interleucina-8 (IL-8), interleucina 11 (IL-11), interleucina 18 (IL-18) y el factor de necrosis tumoral alfa (TNF-?), así como moléculas antibacterianas como b-defensinas, catelicidina y calprotectina. La IL-1 se asocia con la activación severa de las metaloproteinasas de la matriz extracelular (MMPs) que promueve la perdida de los tejidos de sostén del diente. La IL-6 estimula la diferenciación de los osteoclastos y estimula la síntesis de IL-1. El TNF-? induce la diferenciación de osteoclastos, la resorción ósea y tiene actividad sinérgica con IL-1?. Los neutrófilos forman una barrera entre el epitelio de unión y la placa dentobacteriana cuya función sinérgica es la actividad secretora de especies de oxigeno reactivas y proteínas bactericidas con el mecanismo fagocítico cuya actividad afecta la integridad de los tejidos del periodonto. Los derivados lipídicos del ácido araquidónico como Prostanglandina E2 (PGE2), han sido estrechamente relacionados con la pérdida del hueso alveolar por estimulación de los osteoclastos. Todo esto sugiere que la persistencia de las bacterias, una excesiva respuesta inflamatoria y/o una inadecuada resolución de la inflamación pueden afectar la estructura del tejido óseo durante la enfermedad periodontal.

Periodontitis triggers a host immune response against plaque microorganisms and their products. Activation of Toll-like receptors (TLRs) and Nod-like (NLR) by bacterial antigens triggers an inflammatory response and cell, with the expression of inflammatory cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) , interleukin-8 (IL-8), interleukin 11 (IL-11), interleukin 18 (IL-18) and tumor necrosis factor alpha (TNF-?) and antibacterial molecules as b-defensins, cathelicidin and calprotectin . Among the cytokines IL-1? is associated with severe activation of extracellular matrix metalloproteinases (MMPs) that promotes the loss of the tooth-supporting tissues. IL-6 stimulates osteoclast differentiation and stimulates the synthesis of IL-1. TNF-? induces the differentiation of osteoclasts, bone resorption and has synergistic activity with IL-1?. In the cellular response, the neutrophils form a barrier between the junctional epithelium and plaque and secrete reactive oxygen species and bactericidal proteins with the phagocytic mechanism whose activity affects the integrity of the periodontal tissues. Also an arachidonic acid lipid derivative as prostaglandin E2 (PGE2) has been closely related to the loss of alveolar bone by stimulating osteoclast. This suggests that the persistence of bacteria, excessive inflammatory response and / or an inadequate resolution of the inflammation can affect the structure of the bone tissue during periodontal disease.

The unique nature of the serine interactions for alpha 1-adrenergic receptor agonist binding and activation.

Activation of the beta2- and alpha2-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine. With the beta2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the alpha2a-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the beta-AR) appears to contribute partially to agonist-binding and activation. Because the alpha1a-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine- substitution mutants that lacked either Ser188 (Ser188--> Ala) or Ser192 (Ser192-->Ala). With the substitution of both serines in the double mutant, Ser188/192-->Ala, binding of all three ligands was significantly reduced (10- 100-fold) consistent with a single hydrogen bond interaction. However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser188-->Ala mutation and not with Ser192-->Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192-->Ala increased 7-fold over that observed with either the wild type receptor or the Ser188-->Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192-->Ala mutation. In addition, when combined with a recently described constitutively active alpha1a-AR mutation (Met292-->Leu), only the Ser188-->Ala mutation and not Ser192-->Ala relieved the high affinity binding and increased agonist potency observed with the Met292-->Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV alpha-helix, whereas Ser204 and Ser207 of the beta2-AR are separated by only two residues, the orientation of the catechol ring in the alpha1-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta2-AR.

Influence of a lysine 331 counterion on the pK(a) of aspartic acid 125: evidence for a salt-bridge interaction and role in alpha(1b)-adrenergic receptor activation.

We have hypothesized previously that a salt-bridge constraint exists in the alpha(1b)-adrenergic receptor (AR). Docking of the agonist epinephrine can disrupt this constraint via competition of its protonated amine, leading to an agonist-induced activation of second messengers. The amino acids, K331 and D125, which comprise this salt-bridge, should be closely associated with each other in the unbound form of the alpha(1b)-AR. This ionic association should stabilize the negative charge of D125, leading to an increase in its acid strength or a decreased pK(a). If the charged state of D125 is important for agonist binding, then changing the type of amino acid at position 331 should decrease the acid strength of D125, leading to epinephrine affinity changes for the alpha(1b)-AR. To test this hypothesis, site-directed mutagenesis was performed at position 331 of the alpha(1b)-AR. The effect these substitutions had on D125 acid strength was quantitated via epinephrine affinity changes calculated from competition binding experiments performed at different pH values. For all mutations of the alpha(1b)-AR where the positive charge at position 331 was eliminated, there was a significant increase in the pK(a) ( congruent with 0.73) of an acidic amino acid(s). In addition, there was an increase in the binding affinity of epinephrine for these mutants that was associated with a gain in the basal production of inositol triphosphates. These results are consistent with an aspartic acid residue as the counterion for K331 of the salt-bridge constraint, which disrupted, is a part of the receptor activation process. Moreover, changes in the pK(a) of D125 were not dependent on the type of amino acid substituted at position 331. This suggests a mechanism in which K331 is no longer influencing D125 after salt-bridge disruption in the wild-type alpha(1b)-AR, but may move to another stabilized position, analogous to what has been suggested for bacteriorhodopsin. Differences from the wild-type receptor in D125 pK(a) for the K331 mutations were used to estimate the free-energy potential of the constraining salt-bridge. This free energy ( congruent with 1 kcal/mol) is significant, but weak enough to be consistent with an activational mechanism where docking of the receptor agonist has sufficient free energy to cause disruption of the salt-bridge.

Characteristics for a salt-bridge switch mutation of the alpha(1b) adrenergic receptor. Altered pharmacology and rescue of constitutive activity.

Agonist-dependent activation of the alpha(1)-adrenergic receptor is postulated to be initiated by disruption of an interhelical salt-bridge constraint between an aspartic acid (Asp-125) and a lysine residue (Lys-331) in transmembrane domains three and seven, respectively. Single point mutations that disrupt the charges of either of these residues results in constitutive activity. To validate this hypothesis, we used site-directed mutagenesis to switch the position of these amino acids to observe, if possible, regeneration of the salt-bridge reverses that the constitutive activity of the single point mutations. The transiently expressed switch mutant receptor displayed an altered pharmacological profile. The affinity of selective alpha(1b)-adrenergic receptor antagonists for the switch mutant (D125K/K331D) was no different from the wild-type alpha(1b)-adrenergic receptor, suggesting that both receptors are maintaining similar tertiary structures in the cell membrane. However, there was a significant 4-6-fold decrease in the affinity of protonated amine receptor agonists and a 3-6-fold increase in the affinity of carboxylated catechol derivatives for the switch mutant compared with the wild-type alpha(1b)-adrenergic receptor. This pharmacology is consistent with a reversed charge at position 125 in transmembrane domain three. Interestingly, the ability of either a negatively or positively charged agonist to generate soluble inositol phosphates was similar for both types of receptors. Finally, the switch mutant (D125K/K331D) displayed similar basal signaling activity as the wild-type receptor, reversing the constitutive activity of the single point mutations (D125K and K331D). This suggests an ionic constraint has been reformed in the switch mutant analogous to the restraint previously described for the wild-type alpha(1b)-adrenergic receptor. These results strongly establish the disruption of an electrostatic interaction as an initial step in the agonist-dependent activation of alpha(1)-adrenergic receptors.

Alpha1-adrenergic receptors: new insights and directions.

The adrenergic receptors play a key role in the modulation of sympathetic nervous system activity as well as a site of action for many therapeutic agents. The alpha1-adrenergic receptor subtypes (alpha1A-, alpha1B-, alpha1D) are the prime mediators of smooth muscle contraction and hypertrophic growth, but their characterization in both binding and function have lagged the other adrenergic family members. Although they are derived from a related ancestral gene and all nine adrenergic receptor family members bind the endogenous ligands, epinephrine and norepinephrine, with roughly similar affinities, there are major differences in the mode of binding, second messenger utilization, and physiological effects of the alpha1-subtypes compared with beta- or alpha2-subtypes. Here, we review the recent literature on aspects of its binding pocket and how it differs from the beta-adrenergic receptor paradigms. We also review the signaling components and aspects of its function and provide new insights into its roles in smooth muscle, growth, neurological, and cardiovascular function.