Distribution and co-expression of adrenergic receptor-encoding mRNA in the mouse inferior colliculus.

Adrenergic receptors are mediators of adrenergic and noradrenergic modulation throughout the brain. Previous studies have provided evidence for the expression of adrenergic receptors in the midbrain auditory nucleus, the inferior colliculus (IC), but have not examined the cellular patterns of expression in detail. Here, we utilize multichannel fluorescent in situ hybridization to detect the expression of adrenergic receptor-encoding mRNA in the inferior colliculus of male and female mice. We found expression of α1 , α2A , and ß2 receptor-encoding mRNA throughout all areas of the IC. While we observed similar levels of expression of α1 receptor-encoding mRNA across the subregions of the IC, α2A and ß2 receptor-encoding mRNA was expressed differentially. To account for developmental changes in noradrenergic receptor expression, we measured expression levels in mice aged P15, P20, and P60. We observed little change in levels of expression across these ages. To ascertain the modulatory potential of multiple adrenergic receptor subtypes in a single IC cell, we measured co-expression of α1 , α2A , and ß2 receptor-encoding mRNA. We found greater proportions of cells in the IC that expressed no adrenergic receptor-encoding mRNA, α1 and α2A adrenergic receptor-encoding mRNA, and α1, α2A, and ß2 receptor-encoding mRNA than would be predicted by independent expression of each receptor subtype. These data suggest a coordinated pattern of adrenergic receptor expression in the IC and provide the first evidence for adrenergic receptor expression and co-expression in the subregions of the mouse auditory midbrain.

Localization of α-adrenoceptors: JR Vane Medal Lecture.

UNLABELLED: This review is based on the JR Vane Medal Lecture presented at the BPS Winter Meeting in December 2011 by J.C. McGrath. A recording of the lecture is included as supporting information. It covers his laboratory's work from 1990 to 2010 on the localization of vascular α1 -adrenoceptors in native tissues, mainly arteries. MAIN POINTS: (i) α1 -adrenoceptors are present on several cell types in arteries, not only on medial smooth muscle, but also on adventitial, endothelial and nerve cells; (ii) all three receptor subtypes (α1 A , α1 B , α1 D ) are capable of binding ligands at the cell surface, strongly indicating that they are capable of function and not merely expressed. (iii) all of these cell types can take up an antagonist ligand into the intracellular compartments to which endocytosing receptors move; (iv) each individual subtype can exist at the cell surface and intracellularly in the absence of the other subtypes. As functional pharmacological experiments show variations in the involvement of the different subtypes in contractions of different arteries, it is concluded that the presence and disposition of α1 -adrenoceptors in arteries is not a simple guide to their involvement in function. Similar locations of the subtypes, even in different cell types, suggest that differences between the distribution of subtypes in model systems do not directly correlate with those in native tissues. This review includes a historical summary of the alternative terms used for adrenoceptors (adrenergic receptors, adrenoreceptors) and the author's views on the use of colours to illustrate different items, given his partial colour-blindness.

Noradrenergic modulation of neuronal responses to n-methyl-d-aspartate in the vestibular nuclei: an electrophysiological and immunohistochemical study.

Excitatory responses evoked by N-methyl-d-aspartate (NMDA) in the vestibular nuclei (VN) of the rat were studied in vivo during microiontophoretic application of noradrenaline (NA) and/or its agonists and antagonists. Ejection of NA-modified excitatory responses mediated by NMDA receptors (NMDAR) in all neurons tested; the effect was enhancement in 59% of cases and depression in the remaining 41%. Enhancements prevailed in all VN with the exception of the lateral vestibular nucleus, where both effects were recorded in an equal number of cases. The enhancing action of NA on NMDAR-mediated responses was mimicked by the noradrenergic beta-receptor agonist isoproterenol, the beta1 specific agonist denopamine and the alpha2 agonist clonidine. These effects were blocked respectively by the generic beta-receptor antagonist timolol, the beta1 antagonist atenolol and the alpha2 antagonist yohimbine. In contrast, application of the alpha1 receptor agonist cirazoline and the specific alpha1 antagonist prazosin respectively mimicked and partially antagonized the depression of NMDAR-mediated excitations induced by NA. Double-labeling immunohistochemical techniques demonstrated broad colocalization of NMDAR (specifically NR1 and NR2 subunits) with noradrenergic receptors (alpha1, alpha2 and beta1) in many VN neurons; only minor differences were found between nuclei. These results indicate that NA can produce generalized modulation of NMDAR-mediated excitatory neurotransmission in VN, which may in turn modify synaptic plasticity within the nuclei.

Polimorfismos genéticos como indicadores de la vulnerabilidad individual a la adicción al tabaco / Genetic polymorphisms as tool indicators for individual vulnerability to smoking behaviour

Fundamento y objetivo Estudios genéticos y farmacológicos sugieren que algunas vías de neurotransmisores influyen en la dependencia nicotínica. En esta línea se investigó el rol de 4 polimorfismos genéticos en genes relacionados con el control de impulsos: OPRM1, TPH1, ADRA2A y HTR1B. Se analizó el genotipo de una población de 490 individuos no emparentados de Cataluña y se compararon las frecuencias génicas de fumadores, ex fumadores y no fumadores con el fin de descubrir correlaciones. Material y método Todos los polimorfismos se genotiparon en cada grupo de población y se analizaron estadísticamente. Resultados Los datos obtenidos muestran que existe una relación entre el sexo, la edad y el locus TPH1, que indica una tendencia hacia una menor frecuencia del genotipo AA en los ex fumadores. Conclusiones El polimorfismo del triptófano «TPH1» parece desempeñar un papel como indicador de pronóstico en la deshabituación al tabaquismo (AU)

Background and objectives In line with genetic and pharmacological studies suggesting that neurotransmitter pathways play a role in nicotine dependence, research was conducted in connection with 4 genetic polymorphisms: OPRM1, TPH1, ADRA2A and HTR1B. This study compares the genotype and allele frequencies in 3 groups (non-smokers, former smokers and smokers) of unrelated individuals (n=490) from Catalonia (north east Spain) in order to find any relationship. Material and methods All polymorphisms were genotyped in each population group and statistical analysis was performed. Results Data obtained show that there is a relationship between sex, age and the TPH1 locus, indicating a trend towards a lower frequency of the AA genotype in former smokers for the TPH1 locus. Conclusions The results indicate that a role is played by the TPH1 polymorphism as an indicator of therapeutic failure in smoking cessation (AU)

Different expression of adrenoceptors and GRKs in the human myocardium depends on heart failure etiology and correlates to clinical variables.

Downregulation of ß(1)- adrenergic receptors (ß(1)-ARs) and increased expression/function of G-protein-coupled receptor kinase 2 (GRK2) have been observed in human heart failure, but changes in expression of other ARs and GRKs have not been established. Another unresolved question is the incidence of these compensatory mechanisms depending on heart failure etiology and treatment. To analyze these questions, we quantified the mRNA/protein expressions of six ARs (α(1A), α(1B), α(1D), ß(1), ß(2), and ß(3)) and three GRKs (GRK2, GRK3, and GRK5) in left (LV) and right ventricle (RV) from four donors, 10 patients with ischemic cardiomyopathy (IC), 14 patients with dilated cardiomyopathy (DC), and 10 patients with nonischemic, nondilated cardiopathies (NINDC). We correlated the changes in the expressions of ARs and GRKs with clinical variables such as left ventricular ejection fraction (LVEF) and left ventricular end-systolic and left ventricular end-diastolic diameter (LVESD and LVEDD, respectively). The main findings were 1) the expression of the α(1A)-AR in the LV positively correlates with LVEF; 2) the expression of GRK3 and GRK5 inversely correlates with LVESD and LVEDD, supporting previous observations about a protective role for both kinases in failing hearts; and 3) ß(1)-AR expression is downregulated in the LV and RV of IC, in the LV of DC, and in the RV of NINDC. This difference, better than an increased expression of GRK2 (not observed in IC), determines the lower LVEF in IC and DC vs. NINDC.

Maprotiline treatment differentially influences cardiac β -adrenoreceptors expression under normal and stress conditions

Alterations in cardiac function were observed in antidepressants treated patients and published in several clinical reports. These detected changes could be either a consequence of the treatment or of depression itself, which has already been proved to be a risk factor in heart diseases. In order to determine a possible influence of chronic treatment with norepinephrinergic reuptake inhibitor, maprotiline, on the heart, we investigated gene expression of cardiac β-adrenoceptors both in controls and in animals with signs of depression. The rats were divided into two groups, unstressed controls and those exposed to chronic unpredictable mild stress (CUMS). The groups were further divided into two subgroups, one receiving daily intraperitoneal injections of vehicle (sterile water) and another one maprotiline (10 mg/kg) for four weeks. Tissue samples were collected after the last application. Gene expression of cardiac β1- and β2-adrenoceptor was determined using Real-time RT-PCR analysis. Our results show that in control animals expression of both adrenoreceptors was decreased in the right atria after 4 weeks of maprotiline application. Contrary, the same treatment led to a significant increase in expression of cardiac β1-adrenoceptor in the stressed rats, with no change in the characteristics of β2-adrenoceptor. Our findings might reflect the that molecular mechanisms are underlying factors involved in the development of cardiovascular diseases linked with antidepressant treatment.

Vários relatórios clínicos observaram alterações de funcionamento cardíaco de pacientes depressivos que foram tratados com os antidepressivos. As alterações detectadas podem ser consequência do tratamento ou, por outro lado, da depressão que, como se tem provado, é um fator de risco no caso de doenças cardíacas. De modo a determinar a possível influência de tratamento crônico com o inibidor da recaptação de norepinefrina, maprotilina, no coração, foi investigada a expressão do gene aos receptores β-adrenérgicos cardíacos dos animais em grupos de controle e em grupos com sinais de depressão. Os ratos foram divididos em grupos de controle não estressados e os grupos de ratos submetidos ao estresse crônico moderado imprevisível (CUMS). Os grupos foram, ainda, divididos em dois subgrupos, que, durante quatro semanas, diariamente receberam injeções intraperitoneais de placebo (água estéril) ou de maprotilina (10 mg/kg). As amostras de tecido foram coletadas após a última aplicação. A expressão do gene aos receptores adrenérgicos β1 e β2 foi determinada utilizando a análise PCR quantitativa em tempo real (RT-PCR). Os nossos resultados demonstram a diminuição de expressão dos ambos os receptores adrenérgicos no átrio direito dos animais do grupo de controle depois de quatro semanas de aplicação de maprotilina. Em contraste, o mesmo tratamento conduziu ao aumento significativo na expressão do receptor β1-adrenérgico no coração dos ratos estressados, sem qualquer alteração nas características do receptor β2-adrenérgico. Estes resultados podem refletir os mecanismos moleculares envolvidos no desenvolvimento de doenças cardiovasculares associadas ao tratamento com os antidepressivos.

[From lab to clinical activity: adrenergic receptors and human uro-genital tissues]. / Dal laboratorio alla clinica: recettori adrenergici e tessuti umani dell'apparato uro-genitale.

BACKGROUND: Nowadays translational medicine is acquiring a more and more important role in connecting laboratory experimental results on human tissues to clinical findings and drug employment. We want to underline the importance of in vitro studies, which have been extensively performed on animal organs, but few studies have been performed on human tissues. Nevertheless, a more accurate result when compared to the in vivo use of drugs can be given only by testing the very same human tissues in a lab. We related clinical treatments of different pathologies with the results obtained in laboratory studying in vitro fragments of human organs extracted during surgery exposed to different mediators and drugs. METHODS: Fragments of urethers, bladder (detrusorial muscle and bladder neck muscle fibers), corpora cavernosa, and vas deferens were extracted during demolitive surgery trying not to traumatize the tissue, in order to keep it alive and not to ruin its contractile fibers. The fragments were then put into polisaline solution and, once in the laboratory, fixed on suitable isolated organ support, fixed at one side of the thermostatic pool and on the other side connected to a digital monitoring system. The contractility was then studied after adding different mediators. RESULTS: The urethers have shown a stronger response to NE and PGF2a, with a different contractility in their distal part due to a major concentration of alpha-receptors; the bladder neck has also shown a strong contractile response to NE and PGF2a, and is inhibited by alpha-blockers; the bladder detrusor, instead, responds to ACH (acetylcholine) and PGF2a; the vas deferens shows a different type of contractility in the prostatic part compared to the epididimary part when stimulated with noradrenaline and PGF2a; the corpora cavernosa respond to NE and PGF2a. CONCLUSIONS: The results obtained after stimulating the fragments can explain and prove the receptorial activity of inner mediators and of commonly used drugs which have, for years, been used empirically; the simplicity and repetitivity of the method can be considered and used not only to research the physiological functioning of different organs, but also the functioning of new drugs before testing them on patients, being more reliable and accurate than tests on animal tissues. This experimental work has shown that using human tissues in testing specific mediators is the most reliable laboratory method.

Manifestaciones adrenérgicas de los tumores del tejido cromafín extraadrenal / High percentage of adrenergic symtoms in extraadrenal chromaffin toumors

ResumenEstudio descriptivo retrospectivo de 14 pacientes con un diagnóstico histológico de paraganglioma (PGL) recogidos en el Hospital Universitario de Vigo, Pontevedra, durante los últimos 25 años. Hemos analizado las características clínicas y la evolución de esta serie de enfermos.ResultadosLa forma de presentación de los 14 pacientes fue por efecto masa en 10, clínica adrenérgica en 3 e incidental en uno. Algunos enfermos con PGL simpáticos (PGLS) (6/9) y PGL parasimpáticos (PGLPS) (2/5) presentaron clínica de hiperproducción adrenérgica antes del diagnóstico. La determinación de catecolaminas urinarias fue positiva en los 4 enfermos con PGLPS en quienes se determinó. El tamaño tumoral medio fue de 37,8±18,9mm, sin diferencias entre los PGLS y los PGLPS. Dos PGLS fueron malignos. La evolución resultó favorable en 11 pacientes, 2 pacientes con PGLS fallecieron y el tumor de un paciente con PGLPS intracraneal no se pudo extirpar en su totalidad.ConclusionesLos PGL se presentan con frecuencia clínica adrenérgica secundaria a la producción de catecolaminas. Las características de estos tumores aconsejan un estudio detallado en un ámbito especializado antes de la cirugía(AU)

AbstractDescriptive retrospective study of 14 patients with paragangliomas (PGL) attended in the University Hospital of Vigo (Pontevedra) during the last 25 years to evaluating their characteristics and neuroendocrine potential.Results71.4% were diagnosed due to mass tumoral effect, 21.4% due to adrenergic symptoms and 7.1% incidentally. Regarding to symptoms and signs 66.7% of PGL Simpatic (PGLS) and 40% of PGL Parasimpatic (PGLPS) presented adrenergic symptoms. Urine catecholamine analysis was carried out to 4 PGLS and high levels were found in all patients. The tumoral size reached a mean value of 37.8±18.9mm, there were not differences found between both types of tumors. Inmunohistoquimia showed positive Chromogranine A stain in all patients. Two PGLS were maligns. Eleven patients had positive outcome, 2 PGLS died, and 1 PGLPS remains not cured.ConclusionsHigh percentage of patients had clinical manifestations related to catecholamine hyperproduction. We believe that due to the risk related to surgical treatment, malignance, multiple location and family associations, it would be advisable to carry out a complete examination prior to surgery(AU)

A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors.

The intrinsic structural determinants for export trafficking of G protein-coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The alpha(2B)-adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates alpha(2B)-AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of alpha(2B)-AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of beta(2)-AR, alpha(1B)-AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.

Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors.

Commercially available antisera against five subtypes of muscarinic receptors and nine subtypes of adrenoceptors showed highly distinct immunohistochemical staining patterns in rat ureter and stomach. However, using the M(1-4) muscarinic receptor subtypes and alpha(2B)-, beta(2)-, and beta(3)-adrenoceptors as examples, Western blots with membranes prepared from cell lines stably expressing various subtypes of muscarinic receptors or adrenoceptors revealed that each of the antisera recognized a set of proteins that differed between the cell lines used but lacked specificity for the claimed target receptor. We propose that receptor antibodies need better validation before they can reliably be used.

Innervation and receptor profiles of the human apocrine (epitrichial) sweat gland: routes for intervention in bromhidrosis.

BACKGROUND: Human apocrine (epitrichial) sweat glands secrete in response to local or systemic administration of catecholamines and cholinergic agonists. As the process of secretion in human apocrine glands is not fully understood and no literature detailing the expression of adrenergic, cholinergic and purinergic receptors is available, there is a need to know the receptor types. Such data could provide new approaches for the treatment of axillary bromhidrosis. OBJECTIVES: To investigate the localization of nerve fibres, adrenergic, cholinergic and purinergic receptors in human axillary apocrine sweat glands by immunohistochemistry. METHODS: Human axillary apocrine sweat glands were investigated by serial sectioning of paraffin wax-embedded skin samples from volunteers. Sections were examined by light microscopy and immunohistochemistry, using antibodies against neurofilament, alpha- and beta-adrenoceptors, P2Y(1), P2Y(2) and P2Y(4) purinoceptors, and M(3) cholinoceptors. RESULTS: Neurofilaments were found near the eccrine but not the apocrine gland. Apocrine glands demonstrated the presence of beta-2 and beta-3 adrenoceptors in the secretory coil of the gland, but not alpha-1, beta-1 or M(3) receptors. Glandular purinergic staining (P2Y(1), P2Y(2) and P2Y(4)) was found in what looked like myoepithelial cells, while P2Y(1) and P2Y(2) staining was found on apical membranes and diffusely throughout secretory cells. Eccrine gland staining acted as internal positive controls. CONCLUSIONS: No nerve fibres were found near the apocrine gland, suggesting that any catecholamine influence is through humoral effects and that glands could be influenced by beta-adrenoceptor subtypes and purinoceptors. Blockage of both these types of receptors offers a route to controlling apocrine secretion from axillary glands and reducing the opportunity for the development of bromhidrosis.

Differential effects of the tricyclic antidepressant desipramine on the density of adrenergic receptors in juvenile and adult rats.

Although the tricyclic antidepressants, such as desipramine (DMI), are among the most efficacious treatments for adult depression, they are not effective in treating childhood and adolescent depression. Because the adrenergic nervous system is not fully developed until late adolescence, we hypothesized that the mechanisms regulating receptor density may not yet be mature in young mammals. To test this hypothesis, the effects of DMI treatment on cortical alpha-1-, alpha-2-, and beta-adrenergic receptors were compared in juvenile and adult rats. DMI was delivered either by 4 days of twice daily injections to postnatal day 9 to 13 (4 and 7 mg/kg/day) and adult (20 mg/kg/day) rats, or by 2 weeks of continual drug infusion (osmotic minipumps) to postnatal day 21-35 (15 mg/kg/day) and adult (10 mg/kg/day) rats. These delivery paradigms gave juvenile brain concentrations of DMI similar to those in adult rats. The beta-adrenergic receptor was down-regulated with both treatment paradigms in both juvenile and adult rats. By contrast, in the postnatal day 9 to 13 rats, there was a dose-dependent up-regulation of the alpha-1 in the cortex and alpha-2-adrenergic receptor in the prefrontal cortex, whereas there was no change in density in adult rats. These differences in the alpha-adrenergic receptor regulation after DMI treatment suggest that the lack of efficacy of tricyclic antidepressants in treating childhood depression may be related to immature regulatory mechanisms for these receptors.

Studies on the importance of sympathetic innervation, adrenergic receptors, and a possible local catecholamine production in the development of patellar tendinopathy (tendinosis) in man.

Changes in the patterns of production and in the effects of signal substances may be involved in the development of tendinosis, a chronic condition of pain in human tendons. There is no previous information concerning the patterns of sympathetic innervation in the human patellar tendon. In this study, biopsies of normal and tendinosis patellar tendons were investigated with immunohistochemical methods, including the use of antibodies against tyrosine hydroxylase (TH) and neuropeptide Y, and against alpha1-, alpha2A-, and beta1-adrenoreceptors. It was noticed that most of the sympathetic innervation was detected in the walls of the blood vessels entering the tendon through the paratendinous tissue, and that the tendon tissue proper of the normal and tendinosis tendons was very scarcely innervated. Immunoreactions for adrenergic receptors were noticed in nerve fascicles containing both sensory and sympathetic nerve fibers. High levels of these receptors were also detected in the blood vessel walls; alpha1-adrenoreceptor immunoreactions being clearly more pronounced in the tendinosis tendons than in the tendons of controls. Interestingly, immunoreactions for adrenergic receptors and TH were noted for the tendon cells (tenocytes), especially in tendinosis tendons. The findings give a morphological correlate for the occurrence of sympathetically mediated effects in the patellar tendon and autocrine/paracrine catecholamine mechanisms for the tenocytes, particularly, in tendinosis. The observation of adrenergic receptors on tenocytes is interesting, as stimulation of these receptors can lead to cell proliferation, degeneration, and apoptosis, events which are all known to occur in tendinosis. Furthermore, the results imply that a possible source of catecholamine production might be the tenocytes themselves

Adrenergic receptor density in brown adipose tissue of active and hibernating hamsters and ground squirrels.

The ligand-binding characteristics (B(max) and K(D)) of alpha(1)- and beta(1)/beta(2)-adrenoceptors were investigated in membranes prepared from brown adipose tissue (BAT) of warm-acclimated, cold-acclimated, hibernating and arousing ground squirrels (Spermophillus undulatus) and hamsters (Mesocricetus auratus) by specific binding of [(3)H]prazosin and [(3)H]CGP-12177, respectively. The physiological state did not change the affinity for the adrenoceptors in the BAT of ground squirrels and hamsters. There was a significant decrease in alpha(1)-receptor density in arousing ground squirrels and a significant decrease in beta(1)/beta(2) density in hibernating ground squirrels. The level of alpha(1)-receptors was in all conditions higher than that of beta(1)/beta(2) receptors. The results indicate a possible change in balance of adrenoceptor density in the processes of cold acclimation, hibernation and arousal. The balance between the various adrenoceptor subtypes may be important for the final effect of catecholamines in BAT in different physiological states.

Lymphocytic infiltration and goblet cell marker alteration in the conjunctiva of the MRL/MpJ-Fas(lpr) mouse model of Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disorder characterized by a progressive, immune-mediated destruction of mucosal tissues such as the lacrimal and salivary glands, leading to ocular and oral dryness. The MRL/MpJ-Fas(lpr) mouse is one of the animal models used to study this disease. However, little is known about the potential alterations in the conjunctiva in this murine model. The purpose of this study was to determine: (1) whether the conjunctiva is infiltrated by T lymphocytes, (2) characterize the type, amount and temporal sequence of the inflammatory infiltrates, and (3) investigate whether the amount of conjunctival goblet cells is altered in this murine model of Sjögren's syndrome. Female 4-, 9-, 13-, 16-, and 18-/20-wk-old MRL/MpJ-Fas(lpr) (lpr, diseased) and congenic MRL/MpJ (+/+, control) mice were used. Right eyes were either fixed, frozen, cryosectioned, and studied by immunofluorescence microscopy or the conjunctiva was removed, homogenized and analyzed by electrophoresis and Western blot analysis. The following antibodies were used: anti-CD3 (specific T lymphocyte marker), anti-cytokeratin 7 (CK-7), anti-PKD (formerly known as PKCmu, both markers of goblet cell bodies), anti-PGP 9.5 (pan-neuronal marker), anti-VIP and TH (markers for parasympathetic and sympathetic nerves, respectively), anti-adrenergic (alpha(1) and beta(1-3)) and muscarinic (M(1)-M(3)) receptor subtypes (markers for neurotransmitter receptors of the sympathetic and parasympathetic pathways, respectively). Left eyes were fixed, embedded, sectioned, and stained. Hematoxylin/eosin, Giemsa, or alcian blue/periodic acid Schiff's reagent were used to study lymphocyte infiltration; to determine the presence of eosinophils, neutrophils, and mast cells; and to count the number of goblet cells, respectively. By immunofluorescence microscopy, lymphocytes were detected in the conjunctiva of 9-wk-old lpr, but not +/+, mice. The lymphocytic infiltration became more extensive as the animals aged, with 16- and 18-/20-wk lpr mice appearing to have a greater lymphocytic infiltration than +/+ mice at the same age. By Western blot analysis, the amount of CD3 was enhanced in lpr compared to +/+ mice by the 16th wk, but not by the 9th wk. No major differences in the presence of eosinophils, neutrophils and degranulated mast cells between lpr and +/+ mice were observed. By light microscopy, a significant increase in goblet cell number was found in lpr mice compared to +/+ mice at 16 wks on. By Western blotting, the amount of CK-7 was significantly increased at 9 wks on and the amount of PKD was significantly increased at 16 wks. By immunofluorescence microscopy, there were no major differences in distribution of sympathetic and parasympathetic nerves present in the lpr conjunctiva compared to that of +/+ mice at any ages, although slight differences were observed with increased age. Muscarinic receptor expression was decreased, as less M(3) receptor subtype-associated immunofluorescence was detected in older lpr mice compared to +/+ mice and confirmed by Western blot analysis. No differences in the localization or the amount of alpha(1)- or beta(1-3)-adrenergic receptor immunodetection were observed between lpr and +/+ mice. We conclude that the conjunctiva is a target tissue in Sjögren's syndrome-related inflammation in this murine model.

Atrioventricular ring reentry in embryonic mouse hearts.

BACKGROUND: During development, AV conduction switches from base-to-apex to apex-to-base conduction after emergence of the conduction system. We hypothesize that after this transition, the bulk of the AV ring, although no longer required for AV conduction, remains transiently able to conduct, providing a potential arrhythmia substrate. We studied AV conduction during this transition and its sensitivity to autonomic modulation. METHODS AND RESULTS: Simultaneous voltage and Ca2+ mapping with RH-237 and Rhod-2 was performed with 2 CCD cameras in embryonic mouse hearts (n = 43). Additionally, isolated calcium mapping was performed in 309 hearts with fluo-3AM. Propagation patterns in voltage and Ca2+ mapping coincided. Arrhythmias were uncommon under basal conditions, with AV block in 14 (4%) and junctional rhythms in 4 (1%). Arrhythmias increased after stimulation with isoproterenol-junctional rhythm in 9 (3%) and ventricular rhythms in 22 (6%)-although AV block decreased (3 hearts, 1%). Adding carbachol after isoproterenol caused dissociated antegrade and retrograde AV ring conduction in 30 (8.6%) of E10.5 and E11.5 hearts, occurring preferentially in the right and left sides of the ring, respectively. In 2 cases, reentry occurred circumferentially around the AV ring, perpendicular to normal propagation. Reentry persisted for multiple beats, lasting from 3 to 22 minutes. No episodes of AV ring reentry occurred in E9.5 hearts. CONCLUSIONS: AV ring reentry can occur by spatial dissociation of antegrade and retrograde conduction during combined adrenergic and muscarinic receptor stimulation. Critical maturation (> E9.5) seems to be required to sustain reentry.

Epithelial distribution of neural receptors in the guinea pig small intestine.

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.

Immunomodulation by the autonomic nervous system: therapeutic approach for cancer, collagen diseases, and inflammatory bowel diseases.

The distribution of leukocytes is regulated by the autonomic nervous system in humans and animals. The number and function of granulocytes are stimulated by sympathetic nerves whereas those of lymphocytes are stimulated by parasympathetic nerves. This is because granulocytes bear adrenergic receptors, but lymphocytes bear cholinergic receptors on the surface. These regulations may be beneficial to protect the body of living beings. However, when the autonomic nervous system deviates too much to one direction, we fall victim to certain diseases. For example, severe physical or mental stress --> sympathetic nerve activation --> granulocytosis --> tissue damage, including collagen diseases, inflammatory bowel diseases, and cancer. If we introduce the concept of immunomodulation by the autonomic nervous system, a new approach for collagen diseases, inflammatory bowel diseases, and even cancer is raised. With this approach, we believe that these diseases are no longer incurable.

Benign prostatic hyperplasia: correlations between receptor density and binding affinity of alpha(1)-adrenoceptors and several clinical parameters.

The aim of the study was to determine whether relations do exist between the concentration and activity of alpha(1)-adrenoceptors, both inside the prostatic adenoma and the periurethral zone corresponding to the bladder neck, and clinical and biological parameters such as symptoms, evaluated by the American Urological Association (AUA) score, age, weight of the prostate, PSA, and the flow rate. Twenty patients with symptomatic benign prostatic hyperplasia were selected for an open prostatectomy. One gram of tissue was dissected from inside the adenoma and 1 g from the periurethral zone corresponding to the bladder neck. The alpha(1)-adrenoceptors were evaluated for the apparent dissociation constant (K(d)) and the maximal number of binding sites (B(max)). A correlation seems to exist between receptor density inside the adenoma and the bladder neck and an inverse correlation between receptor density and the AUA total symptoms score. Finally, a highly significant difference was found in patients with an AUA score of <15 or >15. No relationship was found between receptor binding affinity and the considered clinical parameters.

Norepinephrine content and adrenoceptor function in the bladder of cats with feline interstitial cystitis.

PURPOSE: Interstitial cystitis is a chronic syndrome affecting humans and domestic animals, including cats (feline interstitial cystitis). The aggravation of interstitial cystitis symptoms by stress suggests involvement of the sympathetic nervous system. Studies have identified increased sympathetic nervous system activity in patients with interstitial cystitis but to our knowledge effects on bladder function have not been reported. To address this question we measured bladder norepinephrine (NE) content, the electrical field stimulation flux of NE and acetylcholine (ACh), and the effects of feline interstitial cystitis on adrenoceptor (AR) mediated bladder strip contractility. MATERIALS AND METHODS: Bladders were obtained from healthy cats and cats with feline interstitial cystitis. In experiment 1 bladder tissue NE content was determined and the simultaneous release of 3H-NE and 14C-ACh in perfusion bath effluent after electrical field stimulation was measured. NE and ACh release was calculated from the area under the efflux curve. In experiment 2 electrical field stimulation induced contractility of bladder body strips was measured in the presence of 100 nM. to 25 microM. NE only or combined with atipamezole (an alpha2-AR antagonist), propranolol (a beta-AR antagonist) or phentolamine (an alpha-AR antagonist). Antagonists were added to the bath at least 15 minutes before stimulation, after which NE was added in cumulative doses and dose response curves were constructed. RESULTS: Significant increases in NE content and efflux in the absence of alterations in ACh efflux were identified. In the bladder strip studies decreased alpha2 and beta1-AR function was found in strips from cats with feline interstitial cystitis, whereas beta3 or atypical beta-ARs were tentatively identified. CONCLUSIONS: These results support and extend previous studies by identifying an effect of increased sympathetic activity on bladder function in cats with feline interstitial cystitis.