RESUMEN
Despite the high incidence of metaphyseal bone fractures in patients, the mechanisms underlying the healing processes are poorly understood due to the lack of suitable experimental animal models. Hence, the present study was conducted to establish and characterise a clinically relevant large-animal model for metaphyseal bone healing. Six female adult Merino sheep underwent full wedge-shaped osteotomy at the distal left femur metaphysis. The osteotomy was stabilised internally with a customised anatomical locking titanium plate that allowed immediate post-operative full-weight bearing. Bone healing was evaluated at 12 weeks post-fracture relative to the untouched right femur. Histological and quantitative micro-computed tomography results revealed an increased mineralised bone mass with a rich bone microarchitecture. New trabeculae healed by direct intramembranous ossification, without callus and cartilaginous tissue formation. Stiffness at the cortical and trabecular regions was comparable in both groups. Functional morphological analysis of the osteocyte lacunae revealed regularly arranged spherically shaped lacunae along with the canalicular network. Bone surface biochemical analysis using time-of-flight secondary-ion mass spectrometry showed high and homogeneously distributed levels of calcium and collagenous components. Ultrastructure imaging of the new trabeculae revealed a characteristic parallel arrangement of the collagen fibrils, evenly mineralised by the dense mineral substance. The specialised bone cells were also characterised by their unique structural features. Bone remodelling in the fractured femur was evident in the higher expression levels of prominent bone formation and resorption genes. In conclusion, the novel metaphyseal fracture model is beneficial for studying healing and treatment options for the enhancement of metaphyseal bone defects.
Asunto(s)
Fracturas del Fémur/fisiopatología , Fémur/fisiopatología , Curación de Fractura/fisiología , Animales , Callo Óseo/metabolismo , Callo Óseo/fisiopatología , Calcio/metabolismo , Modelos Animales de Enfermedad , Femenino , Fracturas del Fémur/metabolismo , Fémur/metabolismo , Osteogénesis/fisiología , Osteotomía/métodos , OvinosRESUMEN
Bioresorbable implants may serve as an alternative option for the fixation of bone fractures. Because of their minor inherent mechanical properties and insufficient anchorage within bone bioresorbable implants have so far been limited to mechanically nondemanding fracture types. By briefly liquefying the surface of the biomaterial during insertion, bioresorbable implants can be ultrasonically fused with bone to improve their mechanical fixation. The objective of this study was to investigate the biomechanical fixation performance and in vivo biocompatibility of an ultrasonically fused bioresorbable polymeric pin (SonicPin). First, we biomechanically compared the fused pin with press fitted metallic and bioresorbable polymeric implants for quasi-static and fatigue strength under shear and tensile loading in a polyurethane foam model. Second, fused implants were inserted into cancellous bovine bone and tested biomechanically to verify the reproducibility of their fusion behavior. Finally, the fused pins were tested in a lapine model of femoral condyle osteotomies and were histologically examined by light and transmission electron microscopy. While comparable under static shear loads, fixation performance of ultrasonically fused pins was significantly (p = 0.001) stronger under tensile loading than press fit implants and showed no pull-out. Both bioresorbable implants withstood comparable fatigue shear strength, but less than the K-wire. In bovine bone the ultrasonic fusion process worked highly reproducible and provided consistent mechanical fixation. In vivo, the polymeric pin produced no notable foreign body reactions or resorption layers. Ultrasonic fusion of polymeric pins achieved adequate and consistent mechanical fixation with high reproducibility and exhibits good short-term resorption and biocompatibility.
Asunto(s)
Implantes Absorbibles , Clavos Ortopédicos , Regeneración Ósea , Fracturas del Fémur/cirugía , Ensayo de Materiales , Ondas Ultrasónicas , Animales , Bovinos , Fracturas del Fémur/patología , ConejosRESUMEN
Staphylococcus aureus is the most clinically relevant pathogen regarding implant-associated bone infection and its capability to invade osteoblasts is well known. The aim of this study was to investigate firstly whether S. aureus is not only able to invade but also to proliferate within osteoblasts, secondly to delineate the mechanism of invasion and thirdly to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival of S. aureus. The SAOS-2 osteoblast-like cell line and human primary osteoblasts were infected with S. aureus EDCC5055 and S. aureus Rosenbach 1884. Both S. aureus strains were able to invade efficiently and to proliferate within human osteoblasts. Immunofluorescence microscopy showed intracellular invasion of S. aureus and transmission electron microscopy images could demonstrate bacterial division as a sign of intracellular proliferation as well as cytosolic bacterial persistence. Cytochalasin D, the major actin depolymerisation agent, was able to significantly reduce S. aureus invasion, suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 µg/mL of rifampicin was able to inhibit bacterial survival in SAOS-2 cells with almost complete elimination of bacteria after 4 h. Gentamicin could also kill intracellular S. aureus in a dose-dependent manner, an effect that was significantly lower than that observed using rifampicin. In conclusion, S. aureus is not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication of S. aureus whereas gentamicin only poorly eliminates intracellularly replicating bacteria.
Asunto(s)
Antibacterianos/farmacología , Proliferación Celular , Gentamicinas/farmacología , Osteoblastos/microbiología , Rifampin/farmacología , Staphylococcus aureus/efectos de los fármacos , Línea Celular , Humanos , Staphylococcus aureus/fisiologíaRESUMEN
OBJECTIVES: Bone is innervated by autonomic nervous system that consists of sympathetic and parasympathetic nerves that were recently identified in bone. Thus we asked whether parasympathetic nerves occur in bone defects and at the interface of substitution materials that were implanted for stabilization and improvement of healing in an osteoporosis animal model. METHODS: Osteoporosis was induced in rats by ovariectomy and deficiency diet. A wedge-shaped osteotomy was performed in the metaphyseal area of femur. Eight different implants were inserted that were based on calcium phosphate cement, iron, silica-mineralized collagen, and modifications with strontium. Nerves were identified by immunohistochemistry with antibodies against vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and protein gene product 9.5 (PGP 9.5) as neuronal marker. RESULTS: Cholinergic nerves identified with VAChT immunostaining were detected in defects filled with granulation tissue and in surrounding mast cells. No immunolabeling of cholinergic nerves was found after implantation. The general presence of nerves was reduced after implantation as shown by PGP 9.5. Sympathetic nerves identified by TH immunolabeling were increased in strontium functionalized materials. CONCLUSION: Since cholinergic innervation was diminished after implantation a further increase in the compatibility of substitution materials to nerves could improve defect healing especially in osteoporotic bone.
Asunto(s)
Sustitutos de Huesos/efectos adversos , Huesos/inervación , Fibras Colinérgicas/efectos de los fármacos , Osteoporosis Posmenopáusica , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Ovariectomía , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Osteoporosis is a widespread disease characterised by low bone mass and structural deterioration of bone resulting in an increased susceptibility to fractures. Osteoporosis affects women more frequently than men; every second woman older than 50 years suffers from an osteoporotic fracture, frequently a vertebral fracture. The aim of this study was to induce osteoporosis in rats to establish an osteoporotic small-animal model that simulates the human pathology particularly in the spine. Therefore, bone density parameters, which are routinely determined in the spine of osteoporotic patients, were investigated by Dual-Energy X-ray Absorptiometry (DEXA). MATERIALS AND METHODS: Fourteen-week-old female Sprague-Dawley rats (n = 50) were either sham-operated (control group: sham) or ovariectomised (experimental group). Ovariectomised rats were further divided into two groups; one received calcium/vitamin D2/D3 deficient diet (OVX + diet), and the other received subcutaneous steroid injections (dexamethasone 0.3 mg/kg body weight) twice a month (OVX + steroid). Rats were scanned by DEXA at three time points (Month = M, 0 M, 1 M and 3 M). DEXA measurement of the spine delivered T-value, Z-value, bone mineral content (BMC), and the scanned area. Fifteen female patients at an age of 57-72 years were scanned in 8-10 regions of the spine (150 measurements). T-values and Z-values were pre-calculated based on patient databases. Statistical analysis was performed using two-way ANOVA followed by Bonferroni correction, with significance considered at p < 0.05. RESULTS: T-value and Z-value of both rat groups were compared with the patient data as well as with each others. Both treated rat groups revealed significantly lower T- and Z-values than controls. Despite the significant difference, the reference line (-2.5 for T-value and -1.5 for Z-value) was only reached by the OVX + diet group. On the other hand, the sham group showed an increase in BMC over time, while no change was seen in OVX + diet or OVX + steroid. Bone area demonstrated a significant increase up to M3. However, the increase in bone area within the OVX + diet group was notably higher than in both sham and OVX + steroid groups. Patients showed significantly lower T- and Z-values than sham and OVX + steroid but insignificant ones when compared with OVX + diet. CONCLUSION: A reproducible vertebral osteoporosis can be generated in a rat model by combination of ovariectomy with administration of a calcium/vitamin D3 deficient diet. T- and Z-values of this experimental group mimicked values obtained from osteoporotic patients, reflecting a simulation of their pathology. Interestingly, the increase in bone area over time with the steady BMC results in lower mineral density (BMD) of the OVX + diet group. Therefore, this rat model presents a reliable experimental set-up that may serve as a tool to better understand and treat osteoporosis.
Asunto(s)
Calcio/deficiencia , Colecalciferol/deficiencia , Modelos Animales de Enfermedad , Ergocalciferoles/deficiencia , Osteoporosis/diagnóstico por imagen , Ovariectomía , Enfermedades de la Columna Vertebral/diagnóstico por imagen , Animales , Femenino , Osteoporosis/fisiopatología , Radiografía , Ratas , Ratas Sprague-Dawley , Enfermedades de la Columna Vertebral/fisiopatologíaRESUMEN
Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT-PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3ß4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, ß2-4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR - α3(∗), α6(∗) and α7(∗) - and of additional combinations with ß-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP-nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP-nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP-nAChRα3 were observed, with eGFP-nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.
Asunto(s)
Neuronas Aferentes/metabolismo , Receptores Nicotínicos/genética , Vejiga Urinaria/inervación , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trazadores del Tracto Neuronal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Nicotínicos/metabolismo , Vejiga Urinaria/metabolismoRESUMEN
BACKGROUND: This investigation describes experimental tests of the biomechanical features of a new resorbable bone adhesive based on methacrylate-terminated oligolactides enhanced with osteoconductive ß-tricalcium phosphate. MATERIAL AND METHODS: 51 New Zealand white rabbits were randomised to an adhesive group (n = 29) and a control group (n = 22). An extra-articular bone cylinder was taken from the proximal tibia, two stripes of adhesive were applied and the cylinders were replanted. After 10 and 21 days, 3 and 12 months tibial specimens were harvested and the cylinder pull-out test was performed with a servo-hydraulic machine. Additionally the pull-out force was evaluated with the bone-equivalent Ebazell® after 5, 10 and 360 minutes in 14 specimens each. RESULTS: Average pull-out forces in the adhesive group were 28 N after 10 days (control: 57 N), 155 N after 21 days (216 N), 184 N after 3 months (197 N) and 205 N after 12 months (185 N). Investigations with Ebazell® showed almost identical pull-out forces after 5 min, 15 min and 360 min. Adhesive forces were as high as 125 N/cm (2) of adhesive surface and more than 1200 N/g of adhesive mass. CONCLUSIONS: The adhesive investigated here has a very good primary adhesive power, compared to the literature data, achieved after only 5 minutes. Even in moist surroundings the adhesive capacity remains sufficient. The adhesive has to prove its resorptive properties in further investigations and in first line its medium-term and long-lasting biocompatibility. Furthermore, biomechanical features will have to be compared to those of conventional fixation techniques.
Asunto(s)
Materiales Biocompatibles , Fenómenos Biomecánicos , Cementos para Huesos , Trasplante Óseo , Fosfatos de Calcio , Tibia/fisiopatología , Tibia/cirugía , Animales , Tomografía Computarizada de Haz Cónico , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Técnicas In Vitro , Oseointegración/fisiología , Resistencia a la Tracción , Tibia/diagnóstico por imagenRESUMEN
The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three ß subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.
Asunto(s)
Acetilcolina/metabolismo , Colina O-Acetiltransferasa/metabolismo , Testículo/metabolismo , Animales , Células Cultivadas , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Inmunohistoquímica , Hibridación in Situ , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WF , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Células de Sertoli/citología , Células de Sertoli/metabolismo , Espermatogénesis , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/citología , Testículo/inervación , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/metabolismoRESUMEN
Ciliary beating of airway epithelial cells drives the removal of mucus and particles from the airways. Mucociliary transport and possibly airway epithelial development are governed by muscarinic acetylcholine receptors but the precise roles of the subtypes involved are unknown. This issue was addressed by determining cilia-driven particle transport, ciliary beat frequency, and the composition and ultrastructural morphology of the tracheal epithelium in M1-M5 muscarinic receptor gene-deficient mice. Knockout of M3 muscarinic receptors prevented an increase in particle transport speed and ciliary beat frequency in response to muscarine. Furthermore, the ATP response after application of muscarine was blunted. Pretreatment with atropine before application of muscarine restored the response to ATP. Additional knockout of the M2 receptor in these mice partially restored the muscarine effect, most likely through the M1 receptor, and normalised the ATP response. M1, M4 and M5 receptor-deficient mice exhibited normal responses to muscarine. None of the investigated mutant mouse strains had any impairment of epithelial cellular structure or composition. In conclusion, M3 receptors stimulate whereas M2 receptors inhibit cilia-driven particle transport. The M1 receptor increases cilia-driven particle transport if the M3 and M2 receptors are missing. None of the receptors is necessary for epithelial development.
Asunto(s)
Cilios/fisiología , Receptores Muscarínicos/deficiencia , Tráquea/fisiología , Adenosina Trifosfato/farmacología , Animales , Cilios/ultraestructura , Inmunohistoquímica , Ratones , Ratones Noqueados , Depuración Mucociliar , Muscarina/farmacología , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no ParamétricasRESUMEN
Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types--ciliated, basal and secretory--are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.
Asunto(s)
Acetilcolina/metabolismo , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Simportadores/metabolismo , Acetilcolina/biosíntesis , Animales , Colina O-Acetiltransferasa/biosíntesis , Colina O-Acetiltransferasa/genética , Células Epiteliales/ultraestructura , Humanos , Neuronas/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Mucosa Respiratoria/ultraestructura , Tráquea/metabolismo , Tráquea/ultraestructuraRESUMEN
Smoking during pregnancy causes low birth weight, premature delivery, neonatal morbidity, and mortality. Nicotine is a main pathogenic compound of cigarette smoke, and depresses active amino-acid uptake by human placental villi. It binds to the acetylcholine binding site of the alpha-subunits of nicotinic acetylcholine receptors (nAChR). Eight different neuronal nAChR alpha-subunits have been identified in mammals. Here, we investigated their localisation and distribution in the human and rat placenta by RT-PCR and immunofluorescence. The mRNAs of all alpha-subunits are expressed in the human and rat placenta. Immunohistochemically, subunits alpha2-5, alpha7, alpha9 and alpha10 are localised in different combinations in rat cytotrophoblast, human and rat syncytiotrophoblast, vascular smooth muscle cells, endothelial cells, Hofbauer cells, human amnion epithelium and rat visceral yolk sac epithelium. Thus, all human and rat placental cell types exhibit receptor subunits with binding sites for the endogenous ligand ACh and nicotine. ACh is suggested to be an important placental signalling molecule that, through stimulation of nAChR, controls the uptake of nutrients, blood flow and fluid volume in placental vessels, and the vascularisation during placental development. Chronic stimulation of nAChR by nicotine might result in unbalanced receptor activation or functional desensitisation followed by the known pathological effects of smoking.
Asunto(s)
Placenta/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Femenino , Técnica del Anticuerpo Fluorescente Directa , Humanos , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Receptores Nicotínicos/biosíntesis , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The pro-inflammatory cytokine interleukin-6 (IL-6) together with its soluble receptor (sIL-6R) induces and maintains thermal hyperalgesia. It facilitates the heat-induced release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Here we report that exposure of nociceptive neurons to the IL-6-sIL-6R complex or the gp130-stimulating designer IL-6-sIL-6R fusion protein Hyper-IL-6 (HIL-6) resulted in a potentiation of heat-activated inward currents (I(heat)) and a shift of activation thresholds towards lower temperatures without affecting intracellular calcium levels. The Janus tyrosine kinase inhibitor AG490, the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide 1 (BIM1), as well as rottlerin, a selective blocker of the PKCdelta isoform, but not the cyclooxygenase inhibitor indomethacin, effectively reduced the effect. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed expression of mRNA for the signal-transducing beta subunit of the receptor gp130 in neuronal somata, rather than satellite cells in rat dorsal root ganglia. Together, the results suggest that IL-6-sIL-6R acts directly on sensory neurons. It increases their susceptibility to noxious heat via the gp130/Jak/PKCdelta signalling pathway.
Asunto(s)
Ganglios Espinales/fisiología , Calor/efectos adversos , Interleucina-6/farmacología , Neuronas Aferentes/fisiología , Receptores de Interleucina-6/metabolismo , Acetofenonas/farmacología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Benzopiranos/farmacología , Calcio/metabolismo , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Receptor gp130 de Citocinas , Femenino , Ganglios Espinales/efectos de los fármacos , Hibridación in Situ , Indoles/farmacología , Indometacina/farmacología , Interleucina-6/genética , Janus Quinasa 1 , Maleimidas/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Neuronas Aferentes/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-delta , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de Interleucina-6/genética , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Umbral Sensorial/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tirfostinos/farmacologíaRESUMEN
The contribution of nicotinic acetylcholine receptors (nAChRs) to stimulation of NO-production was investigated in isolated rat dorsal root ganglion (DRG) neurons utilizing an NO-sensitive fluorescent indicator 4,5-diaminofluorescein-diacetate (DAF-2DA) and appropriate channel blockers. RT-PCR and immunohistochemical analysis of NOS isoforms in cultured neurons revealed the expression of eNOS in the vast majority of neurons, nNOS in about 5-10%, and iNOS only exceptionally. Application of nicotine resulted in an abrupt increase in DAF-2T fluorescence in 65% of neuronal cell bodies that was fully sensitive to the general nAChR antagonist mecamylamine. Methyllycaconitine reduced the number of nicotine-sensitive neurons and the extent of NO-generation. Thus, alpha7- and/or alpha9/10-nAChRs are required for nicotine-induced NO-production in a subpopulation of DRG neurons, and appear to be partially involved in the remaining, larger subpopulation.
Asunto(s)
Ganglios Espinales/enzimología , Neuronas Aferentes/enzimología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/biosíntesis , Receptores Nicotínicos/metabolismo , Animales , Células Cultivadas , Femenino , Fluoresceína , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Nicotina/farmacología , Antagonistas Nicotínicos/farmacología , Óxido Nítrico Sintasa/efectos de los fármacos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Nicotínicos/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Calor , Canales Iónicos/fisiología , Adenilil Ciclasas/biosíntesis , Animales , Electrofisiología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Ganglios Espinales/citología , Ganglios Espinales/enzimología , Ganglios Espinales/fisiología , Hibridación in Situ , Canales Iónicos/efectos de los fármacos , Ionomicina/farmacología , Ionóforos/farmacología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous binding studies have suggested the presence of a so far unknown nicotinic acetylcholine receptor subunit in dorsal root ganglia (Pugh et al., 1995). Here, we investigated whether the most recently identified subunit, alpha10, and its potential interaction partner, alpha9 (Elgoyhen et al., 2001), are expressed in these ganglia. All neurons of rat dorsal root ganglia, but no glial cells, expressed both alpha9 and alpha10 mRNA in in situ hybridization, and exhibited alpha10 immunoreactivity using a newly raised antibody. These findings were confirmed by RT-PCR and western blotting. The data show that dorsal root ganglion neurons coexpress alpha9 and alpha10 nicotinic receptor subunits, thereby providing the first example of neuronal expression of this receptor subunit pair.
Asunto(s)
Ganglios Espinales/metabolismo , Neuronas/metabolismo , Subunidades de Proteína/biosíntesis , Receptores Nicotínicos/biosíntesis , Secuencia de Aminoácidos , Animales , Femenino , Ganglios Espinales/química , Cobayas , Masculino , Datos de Secuencia Molecular , Neuronas/química , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Conejos , Ratas , Ratas Wistar , Receptores Nicotínicos/análisis , Receptores Nicotínicos/genéticaRESUMEN
The duplication of genes for recognition molecules and the ensuing diversification of the members of such families generate complex groups of homologous proteins. One example are galactoside-specific lectins whose sequences display constant features related to sugar binding, the galectins. Based on the inverse abundance of the chicken galectins CG-14 and CG-16 in adult intestine and liver, these two lectins represent a model to comparatively study expression of the related proteins and the galectin-reactive sites (glycoproteins and glycolipids) biochemically and histochemically. Functional overlap and/or acquisition of distinct functions would be reflected in qualitative and/or quantitative aspects of ligand display. Using five different stages of embryogenesis, differential regulation of the two galectins was detected in liver and intestine. The clear preference for one galectin (CG-14) was observed in intestine already at rather early stages, whereas equivalence for both proteins was noted in liver from day 12 to day 18 prior to hatching, as seen by ELISA assays and Western blot analysis. Presentation of galectin-reactive glycoproteins showed a tendency for gradual increase in both organs. Galectin-blotting analysis revealed primarily very similar patterns of positive bands at the different stages of development and only few quantitative and qualitative changes. The reactivity of glycolipids in a solid-phase assay was more variable, even surpassing the response of extracts of the adult organ at several embryonic stages. While the localization patterns of the galectins and galectin-reactive sites were nearly indistinguishable in the liver, intestinal tissue differed with respect to the placement and accessibility of binding sites. Thus, the results suggest a differential regulation of galectin activities in the two organs. As a sum they resemble the course of development of availability of glycoprotein ligands in vitro. These findings support the notion for a partial functional redundancy in this family. The described approach to employ galectin-specific antibodies and the labeled galectins as tools to assess presentation of ligands is suggested to be of general relevance to address the question of distinct vs. overlapping functions of related recognition molecules.
Asunto(s)
Glucolípidos/metabolismo , Glicoproteínas/metabolismo , Hemaglutininas/metabolismo , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Animales , Embrión de Pollo , Pollos , Galectinas , Intestinos/embriología , Intestinos/patología , Hígado/embriología , Hígado/patologíaRESUMEN
The display of cellular oligosaccharide chains is known to undergo marked developmental changes, as monitored histochemically with plant lectins. In conjunction with endogenous lectins respective ligand structures may have a functional role during fetal development. The assumption of a recognitive, functionally productive interplay prompts the study of the expression of a tissue lectin and of lectin-reactive glycoconjugates concomitantly. Focusing on common beta-galactosides as constituents of oligosaccharide chains and the predominant member of the family of galectins in mammals, namely galectin-1, the question therefore is addressed as to whether expression of lectin and lectin-reactive glycoconjugates exhibits alterations, assessed in three morphologically defined fetal stages and in adult bovine organs. Using a sandwich ELISA, the level of the rather ubiquitous galectin-1 is mostly increased in adult organs relative to respective fetal stages, except for the case of kidney. This developmental course is seen rather seldom, when the amounts of lectin-reactive glycoproteins or glycolipids are quantitated in solid-phase assays after tissue homogenization. Western blotting, combined with probing by labeled galectin-1, discloses primarily quantitative changes in the reactivity of individual glycoproteins. Performing the same assays on extract aliquots with a plant agglutinin, namely the galactoside-binding mistletoe lectin, whose fine specificity is different from galectin-1, its reduced extent of binding in solid-phase assays and the disparate profile of lectin-reactive glycoproteins reveal a non-uniform developmental alteration within the group of structural variants of beta-galactosides. Although sample preparation can affect ligand preservation and/or presentation and thus restricts the comparability of biochemical and histochemical results, especially for soluble reactants, the histochemical studies on frozen and paraffin-embedded sections of bovine heart, kidney and liver demonstrate that the localization of the galectin and of lectin-reactive epitopes can show a similar distribution, as seen in liver and heart, with organ-typical quantitative changes of a rather similar staining profile (heart, kidney) or notable changes in the spatial distribution (liver) in the course of development. This report emphasizes the potential value of combined monitoring of the lectin and its potential in vivo ligands to contribute to eventually unravel organ-related function(s) of a tissue lectin.