RESUMO
Cholinergic signaling is essential to mediate the auditory prepulse inhibition (PPI), an operational measure of sensorimotor gating, that refers to the reduction of the acoustic startle reflex (ASR) when a low-intensity, non-startling acoustic stimulus (the prepulse) is presented just before the onset of the acoustic startle stimulus. The cochlear root neurons (CRNs) are the first cells of the ASR circuit to receive cholinergic inputs from non-olivocochlear neurons of the ventral nucleus of the trapezoid body (VNTB) and subsequently decrease their neuronal activity in response to auditory prepulses. Yet, the contribution of the VNTB-CRNs pathway to the mediation of PPI has not been fully elucidated. In this study, we used the immunotoxin anti-choline acetyltransferase (ChAT)-saporin as well as electrolytic lesions of the medial olivocochlear bundle to selectively eliminate cholinergic VNTB neurons, and then assessed the ASR and PPI paradigms. Retrograde track-tracing experiments were conducted to precisely determine the site of lesioning VNTB neurons projecting to the CRNs. Additionally, the effects of VNTB lesions and the integrity of the auditory pathway were evaluated via auditory brain responses tests, ChAT- and FOS-immunohistochemistry. Consequently, we established three experimental groups: 1) intact control rats (non-lesioned), 2) rats with bilateral lesions of the olivocochlear bundle (OCB-lesioned), and 3) rats with bilateral immunolesions affecting both the olivocochlear bundle and the VNTB (OCB/VNTB-lesioned). All experimental groups underwent ASR and PPI tests at several interstimulus intervals before the lesion and 7, 14, and 21 days after it. Our results show that the ASR amplitude remained unaffected both before and after the lesion across all experimental groups, suggesting that the VNTB does not contribute to the ASR. The%PPI increased across the time points of evaluation in the control and OCB-lesioned groups but not in the OCB/VNTB-lesioned group. At the ISI of 50 ms, the OCB-lesioned group exhibited a significant increase in%PPI (p < 0.01), which did not occur in the OCB/VNTB-lesioned group. Therefore, the ablation of cholinergic non-olivocochlear neurons in the OCB/VNTB-lesioned group suggests that these neurons contribute to the mediation of auditory PPI at the 50 ms ISI through their cholinergic projections to CRNs. Our study strongly reinforces the notion that auditory PPI encompasses a complex mechanism of top-down cholinergic modulation, effectively attenuating the ASR across different interstimulus intervals within multiple pathways.
Assuntos
Estimulação Acústica , Vias Auditivas , Inibição Pré-Pulso , Reflexo de Sobressalto , Corpo Trapezoide , Animais , Inibição Pré-Pulso/fisiologia , Masculino , Corpo Trapezoide/metabolismo , Corpo Trapezoide/fisiologia , Vias Auditivas/fisiologia , Vias Auditivas/metabolismo , Ratos Sprague-Dawley , Saporinas/metabolismo , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Proteínas Inativadoras de Ribossomos Tipo 1 , Potenciais Evocados Auditivos do Tronco Encefálico , Imunotoxinas , Nervo Coclear/metabolismo , Nervo Coclear/fisiologia , RatosRESUMO
NEW FINDINGS: What is the central question of this study? C1 neurons innervate pontine noradrenergic cell groups, including the A5 region: do A5 noradrenergic neurons contribute to the activation of sympathetic and respiratory responses produced by selective activation of the C1 group of neurons. What is the main finding and its importance? The increase in sympathetic and respiratory activities elicited by selective stimulation of C1 neurons is reduced after blockade of excitatory amino acid within the A5 region, suggesting that the C1-A5 pathway might be important for sympathetic-respiratory control. ABSTRACT: Adrenergic C1 neurons innervate and excite pontine noradrenergic cell groups, including the ventrolateral pontine noradrenergic region (A5). Here, we tested the hypothesis that C1 activates A5 neurons through the release of glutamate and this effect is important for sympathetic and respiratory control. Using selective tools, we restricted the expression of channelrhodopsin2 under the control of the artificial promoter PRSx8 to C1 neurons (69%). Transduced catecholaminergic terminals within the A5 region are in contact with noradrenergic A5 neurons and the C1 terminals within the A5 region are predominantly glutamatergic. In a different group of animals, we performed retrograde lesion of C1 adrenergic neurons projecting to the A5 region with unilateral injection of the immunotoxin anti-dopamine ß-hydroxylase-saporin (anti-DßH-SAP) directly into the A5 region during the hypoxic condition. As expected, hypoxia (8% O2 , 3 h) induced a robust increase in fos expression within the catecholaminergic C1 and A5 regions of the brainstem. Depletion of C1 cells projecting to the A5 regions reduced fos immunoreactivity induced by hypoxia within the C1 region. Physiological experiments showed that bilateral injection of kynurenic acid (100 mM) into the A5 region reduced the rise in mean arterial pressure, and sympathetic and phrenic nerve activities produced by optogenetic stimulation of C1 cells. In conclusion, the C1 neurons activate the ventrolateral pontine noradrenergic neurons (A5 region) possibly via the release of glutamate and might be important for sympathetic and respiratory outputs in anaesthetized rats.
Assuntos
Neurônios Adrenérgicos , Neurônios Adrenérgicos/metabolismo , Animais , Tronco Encefálico/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Bulbo/fisiologia , Ratos , Respiração , Saporinas/farmacologiaRESUMO
The pontine A5 noradrenergic group contributes to the maturation of the respiratory system before birth in rats. These neurons are connected to the neural network responsible for respiratory rhythmogenesis. In the present study, we investigated the participation of A5 noradrenergic neurons in neonates (P7-8 and P14-15) in the control of ventilation during hypoxia and hypercapnia in in vivo experiments using conjugated saporin anti-dopamine beta-hydroxylase (DßH-SAP) to specifically ablate noradrenergic neurons. Thus, DßH-SAP (420 ng/µL) or saporin (SAP, control) was injected into the A5 region of neonatal male Wistar rats. Hypoxia reduced respiratory variability in control animals; however, A5 lesion prevented this effect in P7-8 rats. Our data suggest that noradrenergic neurons of the A5 region in neonate rats do not participate in the control of ventilation under baseline and hypercapnic conditions, but exert an inhibitory modulation on breathing variability under hypoxic challenge in early life (P7-8).
Assuntos
Neurônios Adrenérgicos/metabolismo , Tronco Encefálico/citologia , Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Respiração , Neurônios Adrenérgicos/efeitos dos fármacos , Neurônios Adrenérgicos/fisiologia , Animais , Animais Recém-Nascidos , Tronco Encefálico/crescimento & desenvolvimento , Tronco Encefálico/fisiopatologia , Dopamina beta-Hidroxilase/farmacologia , Masculino , Ratos , Ratos Wistar , Saporinas/farmacologiaRESUMO
The stage-specific embryonic antigen-4 (SSEA-4) is a cell surface glycosphingolipid antigen expressed in early stages of human development. This surface marker is downregulated during the differentiation process but is found re-expressed in several types of tumors, including breast cancer. This feature makes SSEA-4 an attractive target for the development of therapeutic antibodies against tumors. In this work, we first studied the binding and intracellular fate of the monoclonal antibody MC-813-70 directed against SSEA-4. MC-813-70 was found to be rapidly internalized into triple-negative breast cancer cells following binding to its target at the plasma membrane, and to accumulate in acidic organelles, most likely lysosomes. Given the internalization feature of MC-813-70, we next tested whether the antibody was able to selectively deliver the saporin toxin inside SSEA-4-expressing cells. Results show that the immunotoxin complex was properly endocytosed and able to reduce cell viability of breast cancer cells in vitro, either alone or in combination with chemotherapeutic drugs. Our findings indicate that the MC-813-70 antibody has the potential to be developed as an alternative targeted therapeutic agent for cancer cells expressing the SSEA-4 glycolipid.
Assuntos
Imunotoxinas/farmacologia , Saporinas/farmacologia , Antígenos Embrionários Estágio-Específicos/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Feminino , Humanos , Imunotoxinas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Saporinas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
KEY POINTS: A strong association between disordered breathing patterns, elevated sympathetic activity, and enhanced central chemoreflex drive has been shown in experimental and human heart failure (HF). The aim of this study was to determine the contribution of catecholaminergic rostral ventrolateral medulla catecholaminergic neurones (RVLM-C1) to both haemodynamic and respiratory alterations in HF. Apnoea/hypopnoea incidence (AHI), breathing variability, respiratory-cardiovascular coupling, cardiac autonomic control and cardiac function were analysed in HF rats with or without selective ablation of RVLM-C1 neurones. Partial lesion (â¼65%) of RVLM-C1 neurones reduces AHI, respiratory variability, and respiratory-cardiovascular coupling in HF rats. In addition, the deleterious effects of central chemoreflex activation on cardiac autonomic balance and cardiac function in HF rats was abolished by ablation of RVLM-C1 neurones. Our findings suggest that RVLM-C1 neurones play a pivotal role in breathing irregularities in volume overload HF, and mediate the sympathetic responses induced by acute central chemoreflex activation. ABSTRACT: Rostral ventrolateral medulla catecholaminergic neurones (RVLM-C1) modulate sympathetic outflow and breathing under normal conditions. Heart failure (HF) is characterized by chronic RVLM-C1 activation, increased sympathetic activity and irregular breathing patterns. Despite studies showing a relationship between RVLM-C1 and sympathetic activity in HF, no studies have addressed a potential contribution of RVLM-C1 neurones to irregular breathing in this context. Thus, the aim of this study was to determine the contribution of RVLM-C1 neurones to irregular breathing patterns in HF. Sprague-Dawley rats underwent surgery to induce volume overload HF. Anti-dopamine ß-hydroxylase-saporin toxin (DßH-SAP) was used to selectively lesion RVLM-C1 neurones. At 8 weeks post-HF induction, breathing pattern, blood pressures (BP), respiratory-cardiovascular coupling (RCC), central chemoreflex function, cardiac autonomic control and cardiac function were studied. Reduction (â¼65%) of RVLM-C1 neurones resulted in attenuation of irregular breathing, decreased apnoea-hypopnoea incidence (11.1 ± 2.9 vs. 6.5 ± 2.5 events h-1 ; HF+Veh vs. HF+DßH-SAP; P < 0.05) and improved cardiac autonomic control in HF rats. Pathological RCC was observed in HF rats (peak coherence >0.5 between breathing and cardiovascular signals) and was attenuated by DßH-SAP treatment (coherence: 0.74 ± 0.12 vs. 0.54 ± 0.10, HF+Veh vs. HF+DßH-SAP rats; P < 0.05). Central chemoreflex activation had deleterious effects on cardiac function and cardiac autonomic control in HF rats that were abolished by lesion of RVLM-C1 neurones. Our findings reveal that RVLM-C1 neurones play a major role in irregular breathing patterns observed in volume overload HF and highlight their contribution to cardiac dysautonomia and deterioration of cardiac function during chemoreflex activation.
Assuntos
Catecolaminas/metabolismo , Insuficiência Cardíaca/fisiopatologia , Bulbo/metabolismo , Neurônios/fisiologia , Respiração , Animais , Masculino , Bulbo/citologia , Bulbo/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Reflexo , Saporinas/toxicidadeRESUMO
Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.
Assuntos
Hipersensibilidade/patologia , Inflamação/patologia , Nociceptores/patologia , Peptídeos/metabolismo , Animais , Dinoprostona/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Hipersensibilidade/complicações , Hipersensibilidade/fisiopatologia , Inflamação/complicações , Inflamação/fisiopatologia , Lectinas/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Fibras Nervosas Amielínicas/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Dor/complicações , Dor/fisiopatologia , Saporinas/farmacologiaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra compacta (SNpc) and the only risk factor is aging. We showed that in 6-hydroxydopamine (6-OHDA)-model of PD there is a reduction in the neuronal profile within the brainstem ventral respiratory column with a decrease in the hypercapnic ventilatory response. Here we tested the involvement of orexin cells from the lateral hypothalamus/perifornical area (LH/PeF) on breathing in a 6-OHDA PD model. In this model of PD, there is a reduction in the total number of orexinergic neurons and in the number of orexinergic neurons that project to the RTN, without changing the number of CO2-activated orexinergic neurons during the dark phase. The ventilation at rest and in response to hypercapnia (7% CO2) was assessed in animals that received 6-OHDA or vehicle injections into the striatum and saporin anti-Orexin-B or IgG saporin into the LH/PeF during the sleep and awake states. The experiments showed a reduction of respiratory frequency (fR) at rest during the light phase in PD animals only during sleep. During the dark phase, there was an impaired fR response to hypercapnia in PD animals with depletion of orexinergic neurons in awake and sleeping rats. In conclusion, the degeneration of orexinergic neurons in this model of PD can be related to impaired chemoreceptor function in the dark phase.
Assuntos
Hipotálamo/patologia , Neurônios/metabolismo , Orexinas/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Respiração , Animais , Escuridão , Modelos Animais de Doenças , Eletroencefalografia , Eletromiografia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Oxidopamina/toxicidade , Doença de Parkinson/etiologia , Ventilação Pulmonar/fisiologia , Ratos , Ratos Wistar , Saporinas/farmacologia , Estilbamidinas/metabolismo , Simpatolíticos/toxicidadeRESUMO
BACKGROUND & OBJECTIVE: Adult neurogenesis, a specific form of brain plasticity in mammals that occurs in the subventricular zone, is subject to complex regulation. Hypocretin/orexin neurons are implicated in the regulation of sleep and arousal states, among other functions. Here we report for the first time the presence of orexinergic projections within the adult rat subventricular zone. Post-mortem retrograde tracing combined with immunofluorescence indicated orexinergic projections toward the subventricular zone. To establish the relationship between the depletion of orexin neurons and the number of proliferating cells in the subventricular zone, we labeled mitotic cells. Histological analysis revealed proliferating cells to be in close contact with orexinergic fibers. Neurotoxinlesioning of orexin neurons in the lateral hypothalamus significantly activated precursor cell proliferation in the subventricular zone. Furthermore, cell proliferation in both normal and lesioned animals failed to reveal newly born orexin neurons in the lateral hypothalamus. CONCLUSION: Based on these findings, we suggest that the adult subventricular zone is affected by orexinergic signaling, the functional implication of which must be further elucidated.
Assuntos
Proliferação de Células , Ventrículos Laterais/citologia , Neurogênese/fisiologia , Neurônios/metabolismo , Orexinas/deficiência , Saporinas/deficiência , Animais , Região Hipotalâmica Lateral/efeitos dos fármacos , Região Hipotalâmica Lateral/fisiologia , Masculino , Vias Neurais/fisiologia , Técnicas de Rastreamento Neuroanatômico , Neurogênese/efeitos dos fármacos , RatosRESUMO
The A5 area at the ventrolateral pons contains noradrenergic neurons connected with other medullary areas involved in the cardiorespiratory control. Its contribution to the cardiorespiratory regulation was previously evidenced in anesthetized conditions. In the present study, we investigated the involvement of the A5 noradrenergic neurons to the basal and chemoreflex control of the sympathetic and respiratory activities in unanesthetized conditions. A5 noradrenergic neurons were lesioned using microinjections of anti-dopamine ß-hydroxylase saporin (anti-DßH-SAP). After 7-8days, we evaluated the arterial pressure levels, heart rate and minute ventilation in freely moving adult rats (280-350g) as well as recorded from thoracic sympathetic (tSN) and phrenic nerves (PN) using the arterially perfused in situ preparation of juvenile rats (80-90g). Baseline cardiovascular, sympathetic and respiratory parameters were similar between control (n=7-8) and A5-lesioned rats (n=5-6) in both experimental preparations. In adult rats, lesions of A5 noradrenergic neurons did not modify the reflex cardiorespiratory adjustments to hypoxia (7% O2) and hypercapnia (7% CO2). In the in situ preparations, the sympatho-excitation, but not the PN reflex response, elicited by either the stimulation of peripheral chemoreceptors (ΔtSN: 110±12% vs 58±8%, P<0.01) or hypercapnia (ΔtSN: 9.5±1.4% vs 3.9±1.7%, P<0.05) was attenuated in A5-lesioned rats compared to controls. Our data demonstrated that A5 noradrenergic neurons are part of the circuitry recruited for the processing of sympathetic response to hypoxia and hypercapnia in unanesthetized conditions.
Assuntos
Neurônios Adrenérgicos/fisiologia , Hipercapnia/fisiopatologia , Ponte/citologia , Sistema Nervoso Simpático/fisiologia , Vigília , Análise de Variância , Animais , Anticorpos Monoclonais/toxicidade , Pressão Sanguínea/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Hipercapnia/induzido quimicamente , Masculino , Ponte/efeitos dos fármacos , Ponte/lesões , Ventilação Pulmonar/fisiologia , Ratos , Ratos Wistar , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Saponinas/toxicidade , Saporinas , Sistema Nervoso Simpático/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The stimuli that commonly activate the catecholaminergic C1 neurons (nociception, hypotension, and hypoxia) also increase breathing. Pharmacogenetic evidence suggests that catecholaminergic neurons regulate breathing. Therefore, we evaluated whether the loss of C1 cells affects cardiorespiratory control during resting, hypoxic (8% O2) and hypercapnic (7% CO2) conditions. A bilateral injection of the immunotoxin anti-dopamine ß-hydroxylase-saporin (anti-DßH-SAP; 2.4ng/100nl) or saline was performed in adult male Wistar rats (270-300g, N=5-8/group). Histology revealed a 60-75% loss of C1 neurons in anti-DßH-SAP-treated rats, but no significant changes or C1 cell loss was observed in sham-treated rats or those with off-target injection sites. Bilateral depletion of C1 neurons did not alter cardiorespiratory variables during rest and hypercapnia (7% CO2), but it did affect the response to hypoxia. Specifically, the increase in ventilation, the number of sighs, and the tachycardia were reduced, but unexpectedly, the mean arterial pressure increased during hypoxia (8% O2). The present study indicates that C1 neurons contribute to cardiorespiratory control during hypoxia rather than at rest or during hypercapnia.
Assuntos
Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Bulbo/fisiopatologia , Neurônios/citologia , Animais , Anticorpos Monoclonais/farmacologia , Pressão Sanguínea/fisiologia , Estado de Consciência/fisiologia , Modelos Animais de Doenças , Hipóxia/patologia , Masculino , Neurônios/efeitos dos fármacos , Ratos Wistar , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Taquicardia/induzido quimicamenteRESUMO
Noradrenergic neurons in the caudal ventrolateral medulla (CVLM; A1 group) contribute to cardiovascular regulation. The present study assessed whether specific lesions in the A1 group altered the cardiovascular responses that were evoked by hypertonic saline (HS) infusion in non-anesthetized rats. Male Wistar rats (280-340 g) received nanoinjections of antidopamine-ß-hydroxylase-saporin (A1 lesion, 0.105 ng.nL(-1)) or free saporin (sham, 0.021 ng.nL(-1)) into their CVLMs. Two weeks later, the rats were anesthetized (2% halothane in O2) and their femoral artery and vein were catheterized and led to exit subcutaneously between the scapulae. On the following day, the animals were submitted to HS infusion (3 M NaCl, 1.8 ml ⢠kg(-1), b.wt., for longer than 1 min). In the sham-group (nâ=â8), HS induced a sustained pressor response (ΔMAP: 35±3.6 and 11±1.8 mmHg, for 10 and 90 min after HS infusion, respectively; P<0.05 vs. baseline). Ten min after HS infusion, the pressor responses of the anti-DßH-saporin-treated rats (nâ=â11)were significantly smaller(ΔMAP: 18±1.4 mmHg; P<0.05 vs. baseline and vs. sham group), and at 90 min, their blood pressures reached baseline values (2±1.6 mmHg). Compared to the sham group, the natriuresis that was induced by HS was reduced in the lesioned group 60 min after the challenge (196±5.5 mM vs. 262±7.6 mM, respectively; P<0.05). In addition, A1-lesioned rats excreted only 47% of their sodium 90 min after HS infusion, while sham animals excreted 80% of their sodium. Immunohistochemical analysis confirmed a substantial destruction of the A1 cell group in the CVLM of rats that had been nanoinjected withanti-DßH-saporin. These results suggest that medullary noradrenergic A1 neurons are involved in the excitatory neural pathway that regulates hypertensive and natriuretic responses to acute changes in the composition of body fluid.
Assuntos
Neurônios Adrenérgicos , Hipernatremia/complicações , Hipernatremia/fisiopatologia , Hipertensão/etiologia , Hipertensão/fisiopatologia , Natriurese , Neurônios Adrenérgicos/efeitos dos fármacos , Animais , Barorreflexo , Pressão Sanguínea , Frequência Cardíaca , Hemoglobinas/metabolismo , Rim/metabolismo , Rim/fisiopatologia , Masculino , Ratos , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Solução Salina Hipertônica/administração & dosagem , Solução Salina Hipertônica/farmacologia , Saporinas , Sódio/sangueRESUMO
Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.
Assuntos
Anticorpos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Gangliosídeos/metabolismo , Imunotoxinas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Animais , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Endossomos/metabolismo , Gangliosídeos/imunologia , Humanos , Imunotoxinas/farmacocinética , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Inativadoras de Ribossomos Tipo 1/farmacocinética , Saporinas , Sais de Tetrazólio , TiazóisRESUMO
Renal vasodilation and sympathoinhibition are recognized responses induced by hypernatremia, but the central neural pathways underlying such responses are not yet entirely understood. Several findings suggest that A2 noradrenergic neurons, which are found in the nucleus of the solitary tract (NTS), play a role in the pathways that contribute to body fluid homeostasis and cardiovascular regulation. The purpose of this study was to determine the effects of selective lesions of A2 neurons on the renal vasodilation and sympathoinhibition induced by hypertonic saline (HS) infusion. Male Wistar rats (280-350 g) received an injection into the NTS of anti-dopamine-beta-hydroxylase-saporin (A2 lesion; 6.3 ng in 60 nl; nâ=â6) or free saporin (sham; 1.3 ng in 60 nl; nâ=â7). Two weeks later, the rats were anesthetized (urethane 1.2 gâ kg(-1) b.wt., i.v.) and the blood pressure, renal blood flow (RBF), renal vascular conductance (RVC) and renal sympathetic nerve activity (RSNA) were recorded. In sham rats, the HS infusion (3 M NaCl, 1.8 mlâ kg(-1) b.wt., i.v.) induced transient hypertension (peak at 10 min after HS; 9±2.7 mmHg) and increases in the RBF and RVC (141±7.9% and 140±7.9% of baseline at 60 min after HS, respectively). HS infusion also decreased the RSNA (-45±5.0% at 10 min after HS) throughout the experimental period. In the A2-lesioned rats, the HS infusion induced transient hypertension (6±1.4 mmHg at 10 min after HS), as well as increased RBF and RVC (133±5.2% and 134±6.9% of baseline at 60 min after HS, respectively). However, in these rats, the HS failed to reduce the RSNA (115±3.1% at 10 min after HS). The extent of the catecholaminergic lesions was confirmed by immunocytochemistry. These results suggest that A2 noradrenergic neurons are components of the neural pathways regulating the composition of the extracellular fluid compartment and are selectively involved in hypernatremia-induced sympathoinhibition.
Assuntos
Hipernatremia/fisiopatologia , Rim/fisiopatologia , Neurônios/metabolismo , Norepinefrina/metabolismo , Núcleo Solitário/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipernatremia/induzido quimicamente , Hipernatremia/metabolismo , Rim/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Circulação Renal/fisiologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Solução Salina Hipertônica/farmacologia , Saporinas , Núcleo Solitário/efeitos dos fármacos , Núcleo Solitário/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The aim of this work was to study the effect of molsidomine (MOLS), a nitric oxide (NO) donor, on the nitrergic system changes in an experimental model of cholinergic damage induced by 192 IgG saporin (SAP). Male rats were injured by intraseptal administration of SAP (0.22 µg), after seven days, rats were administered with MOLS (4 mg/kg, i.p.) 60 min before sacrifice. Prefrontal cortex (PC), striatum (S) and hippocampus (HC) were dissected out. Results showed significant recovery of the constitutive NOS activity (cNOS) in PC and S regions by MOLS but not in HC compared against controls. SAP reduced the cellular population in the lesion site and MOLS was able to avoid the progression of damage in this area. NO donor is able to modulate the nitrergic status in an experimental model induced by SAP.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Molsidomina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Animais , Anticorpos Monoclonais/toxicidade , Colinérgicos/toxicidade , Masculino , Ratos , Ratos Wistar , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , SaporinasRESUMO
The 24-h distribution of rapid eye movement (REM) sleep is known to be deeply reshaped among albino rats with neurotoxic lesions in the lateral hypothalamus (LH) or among rodent models of human narcolepsy-cataplexy, with selective damage of orexinergic neurones. We explored the hypothesis that this phenomenon is explained by an enhancement of REM sleep photic masking, as a consequence of damage in the LH. Orexin-B-saporin neurotoxic lesions were induced in the LH of male Sprague-Dawley rats. LH-lesioned and control rats were sleep-recorded successively under 12:12 light/dark (LD) and skeleton photoperiod. Compared to controls, lesioned rats exhibited 50% less and 82% more REM sleep during rest and active phases, respectively, under the 12:12 LD schedule. After transference to a skeleton photoperiod, lesioned rats exhibited an 88% increase in REM sleep during the rest phase, recovering the characteristic rest phase preference of REM sleep observed among control rats. The increase in rest phase REM sleep during the skeleton photoperiod was correlated positively with the magnitude of the LH lesion. Our results suggest that changes in the temporal organization of sleep-wake states observed among rats with neurotoxic lesions in the lateral hypothalamus and rodent models of narcolepsy-cataplexy may be explained by the enhancement of photic masking.
Assuntos
Hipotálamo/patologia , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neuropeptídeos/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Sono REM/fisiologia , Animais , Contagem de Células , Eletroencefalografia , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiopatologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Orexinas , Estimulação Luminosa , Ratos , Ratos Sprague-Dawley , Saporinas , Transtornos do Sono do Ritmo Circadiano/patologia , Transtornos do Sono do Ritmo Circadiano/fisiopatologia , Vigília/fisiologiaRESUMO
We assessed the role of NK-1 receptors (NK1R) expressing neurons in the locus coeruleus (LC) on cardiorespiratory responses to hypercapnia. To this end, we injected substance P-saporin conjugate (SP-SAP) to kill NK-1 immunoreactive (NK1R-ir) neurons or SAP alone as a control. Immunohistochemistry for NK1R, tyrosine hydroxylase (TH-ir) and Glutamic Acid Decarboxylase (GAD-ir) were performed to verify if NK1R-expressing neurons, catecholaminergic and/or GABAergic neurons were eliminated. A reduced NK1R-ir in the LC (72%) showed the effectiveness of the lesion. SP-SAP lesion also caused a reduction of TH-ir (66%) and GABAergic neurons (70%). LC SP-SAP lesion decreased by 30% the ventilatory response to 7% CO(2) and increased the heart rate (fH) during hypercapnia but did not affect MAP. The present data suggest that different populations of neurons (noradrenergic, GABAergic, and possibly others) in the LC express NK1R modulating differentially the hypercapnic ventilatory response, since catecholaminergic neurons are excitatory and GABAergic ones are inhibitory. Additionally, NK1R-ir neurons in the LC, probably GABAergic ones, seem to modulate fH during CO(2) exposure, once our previous data demonstrated that catecholaminergic lesion does not affect this variable.
Assuntos
Sistema Cardiovascular , Hipercapnia/fisiopatologia , Locus Cerúleo/patologia , Neurônios/fisiologia , Ventilação Pulmonar/fisiologia , Receptores da Neurocinina-1/metabolismo , Animais , Sistema Cardiovascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Glutamato Descarboxilase/metabolismo , Fatores Imunológicos/farmacologia , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/lesões , Masculino , Neurônios/efeitos dos fármacos , Ventilação Pulmonar/efeitos dos fármacos , Ratos , Ratos Wistar , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Saporinas , Substância P/análogos & derivados , Substância P/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Ribosome-inactivating proteins (RIPs) from plants inhibit protein synthesis by inactivating ribosomes. Some two-chain (type 2) RIPs are highly toxic and may play a role in plant defense. The lower toxicity of single-chain (type 1) RIPs reflects the lack of a protein domain able to bind to, and translocate the toxin across cell membranes. We studied the effect of single-chain RIPs, lychnin, momordin, gelonin, PAP-S and saporin S-6, in larvae of Anticarsia gemmatalis and Spodoptera frugiperda. After ingesting a total dose of 20 or 40 microg of the toxins, weight gain, survival rate, lesions in DNA and oxidative status (catalase and superoxide dismutase activities and lipidic peroxidation) of RIP-treated insects were assayed. Momordin was the less toxic in the biossays. S. frugiperda had a more pronounced weight loss on the 4th day of treatment and A. gemmatalis on the 10th day. RIP-induced mortality reached 57.13% for A. gemmatalis and 29.45% for S. frugiperda. RIP-treated insects showed a 2-3-fold increase in DNA lesions as assessed by the comet assay, but there were no correlations between stress markers and DNA damage. We conclude that single-chain RIPs are entomotoxic to lepidopteran insects causing extensive DNA lesions.
Assuntos
Mariposas/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Animais , Catalase/metabolismo , Dano ao DNA/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/enzimologia , Peroxidação de Lipídeos , Mariposas/enzimologia , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Saporinas , Superóxido Dismutase/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
Several findings suggest that catecholaminergic neurons in the caudal ventrolateral medulla (CVLM) contribute to body fluid homeostasis and cardiovascular regulation. From the CVLM other areas in central nervous system involved in cardiovascular regulation and hydroelectrolyte balance can be activated. Therefore, the aim of the present study was to investigate the effects of lesions of these neurons on 0.3M NaCl and water intake induced by subcutaneous injection of furosemide (FURO)+captopril (CAP) or 36 h of water deprivation/partial hydration with only water (WD/PR). Male Wistar rats (320-360 g) were submitted to medullary catecholaminergic neuron lesions by microinjection of anti-dopamine-beta-hydroxylase-saporin (anti-DbetaH-saporin; 6.3 ng in 60 nl) into the CVLM (SAP-rats). Sham rats received microinjections of free saporin (1.3 ng in 60 nl) in the same region. In SAP-rats, the 0.3M NaCl intake was increased after FURO+CAP (6.8+/-1.0 ml/2h, vs. sham: 3.7+/-0.7 ml/2h) as well as after WD/PR (11.1+/-1.3 ml/2h vs. sham: 6.1+/-1.8 ml/2h). Conversely, in SAP-rats, the water intake induced by FURO+CAP (14.8+/-1.3 ml/2h, vs. sham: 14.1+/-1.6 ml/2h) or by WD/PR (3.6+/-0.9 ml/2h, vs. sham: 3.2+/-1.1 ml/2h) was not different from sham rats. Immunohistochemical analysis indicates that microinjections of anti-DbetaH-saporin produced extensive destruction within the A1 cell groups in the CVLM. These results suggest an inhibitory role for medullary catecholaminergic neurons on sodium appetite.
Assuntos
Catecolaminas/metabolismo , Bulbo/metabolismo , Neurônios/metabolismo , Cloreto de Sódio/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Regulação do Apetite/efeitos dos fármacos , Captopril/administração & dosagem , Captopril/farmacologia , Dopamina beta-Hidroxilase/antagonistas & inibidores , Ingestão de Líquidos/efeitos dos fármacos , Combinação de Medicamentos , Furosemida/administração & dosagem , Furosemida/farmacologia , Imuno-Histoquímica , Injeções Subcutâneas , Masculino , Bulbo/efeitos dos fármacos , Bulbo/patologia , Microinjeções/métodos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Wistar , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Saporinas , Cloreto de Sódio/administração & dosagem , Privação de ÁguaRESUMO
The nucleus of the solitary tract (NTS) receives primary afferents involved in cardiovascular regulation. We investigated the role of NK(1)-receptor bearing neurons in the NTS on cardiovascular reflexes in awake rats fitted with chronic venous and arterial cannulae. These neurons were lesioned selectively with saporin conjugated with substance P (SP-SAP, 2 microM, bilateral injections of 20 nL in the subpostremal NTS, or 200 nL in both the subpostremal and the commissural NTS). Before, and 7 and 14 days after injection of SP-SAP, we measured changes in blood pressure and heart rate induced by i.v. injection of phenylephrine and nitroprusside (baroreceptor reflex), cyanide (arterial chemoreceptor reflex), and phenylbiguanide (Bezold-Jarisch reflex). The smaller injections with SP-SAP completely abolished NK1 receptor staining in the subpostremal NTS. The larger injections abolished NK1 receptor immunoreactivity in an area that extended from the commissural NTS to the rostral end of the subpostremal NTS. The lesions seemed to affect only a limited number of neurons, since neutral red stained sections did not show any obvious reduction in cell number. The smaller lesions reduced the gain of baroreflex bradycardia and the hypotension induced by phenylbiguanide. The larger lesions completely abolished the response to phenylbiguanide, blocked the baroreflex bradycardia induced by phenylephrine, severely blunted the baroreflex tachycardia, and blocked the bradycardia and reduced the hypertension induced by cyanide. Thus, these responses depend critically on NK(1)-receptor bearing neurons in the NTS.
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Coração/inervação , Neurônios Aferentes/metabolismo , Receptores da Neurocinina-1/metabolismo , Núcleo Solitário/metabolismo , Fibras Aferentes Viscerais/metabolismo , Animais , Barorreflexo/efeitos dos fármacos , Barorreflexo/fisiologia , Biguanidas/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Bradicardia/induzido quimicamente , Bradicardia/fisiopatologia , Células Quimiorreceptoras/efeitos dos fármacos , Células Quimiorreceptoras/fisiologia , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/fisiologia , Hipotensão/induzido quimicamente , Hipotensão/fisiopatologia , Masculino , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Neurônios Aferentes/citologia , Neurotoxinas , Parassimpatectomia , Cianeto de Potássio/farmacologia , Ratos , Ratos Wistar , Proteínas Inativadoras de Ribossomos Tipo 1 , Saporinas , Agonistas do Receptor de Serotonina/farmacologia , Núcleo Solitário/citologia , Substância P/análogos & derivados , Fibras Aferentes Viscerais/citologia , Vigília/fisiologiaRESUMO
Several findings suggest that catecholaminergic neurones in the caudal ventrolateral medulla (CVLM) contribute to body fluid homeostasis and cardiovascular regulation. The present study sought to determine the effects of lesions of these neurones on the cardiovascular responses induced by changes in circulating volume. All experiments were performed in male Wistar rats (320-360 g). Medullary catecholaminergic neurones were lesioned by microinjection of anti-dopamine beta-hydroxylase-saporin (6.3 ng in 60 nl; SAP rats, n = 14) into the CVLM, whereas sham rats received microinjections of free saporin (1.3 ng in 60 nl, n = 15). Two weeks later, rats were anaesthetized (urethane, 1.2 g kg(-1), i.v.), instrumented for measurement of mean arterial pressure (MAP), renal blood flow (RBF) and renal vascular conductance (RVC), and infused with hypertonic saline (HS; 3 m NaCl, 0.18 ml (100 g body weight)(-1), i.v.) or an isotonic solution (volume expansion, VE; 4% Ficoll, 1% of body weight, i.v.). In sham rats, HS induced sustained increases in RBF and RVC (155 +/- 7 and 145 +/- 6% of baseline, at 20 min after HS). In SAP rats, RBF responses to HS were blunted (125 +/- 6%) and RVC increases were abolished (108 +/- 5%) 20 min after HS. Isotonic solution increased RBF and RVC in sham rats (149 +/- 10 and 145 +/- 12% of baseline, respectively, at 20 min). These responses were reduced in SAP rats (131 +/- 6 and 126 +/- 5%, respectively, at 20 min). Pressor responses to HS were larger in SAP rats than in sham rats (17 +/- 5 versus 9 +/- 2 mmHg, at 20 min), whereas during VE these responses were similar in both groups (6 +/- 3 versus 4 +/- 6 mmHg, at 20 min). Immunohistochemical analysis indicates that microinjections of anti-DbetaH-saporin produced extensive destruction within the A1/C1 cell groups in the CVLM. These results suggest that catecholaminergic neurones mediate the cardiovascular responses to VE or increases in plasma sodium levels.