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1.
Exp Eye Res ; 211: 108760, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487726

RESUMO

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.


Assuntos
Células Acinares/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Catepsinas/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Fenilefrina/farmacologia , Via Secretória/efeitos dos fármacos , Lágrimas/metabolismo , Células Acinares/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Carbacol/farmacologia , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Modelos Animais de Doenças , Feminino , Inativação Gênica , Aparelho Lacrimal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , beta-N-Acetil-Hexosaminidases/metabolismo , Proteínas rab3 de Ligação ao GTP/genética
2.
Nat Commun ; 12(1): 5475, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531380

RESUMO

Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.


Assuntos
Acetilcolina/metabolismo , Região CA1 Hipocampal/fisiologia , Córtex Entorrinal/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Retroalimentação Fisiológica/fisiologia , Transmissão Sináptica/fisiologia , Animais , Região CA1 Hipocampal/citologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Córtex Entorrinal/citologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Interneurônios/metabolismo , Interneurônios/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Transmissão Sináptica/efeitos dos fármacos
3.
World J Gastroenterol ; 27(33): 5566-5574, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34588752

RESUMO

BACKGROUND: Oral intake is dependent on the gastric ability to accommodate the food bolus. Comparatively, neonates have a smaller gastric capacity than adults and this may limit the volume of their milk intake. Yet, we previously reported that the newborn rat gastric milk volume is greatest after birth and, when normalized to body weight, decreases with postnatal age. Such age-dependent changes are not the result of intake differences, but greater gastric accommodation and reduced emptying rate. AIM: Hypothesizing that breastmilk-derived adiponectin is the factor regulating gastric accommodation in neonates, we comparatively evaluated its effects on the rat fundic muscle tone at different postnatal ages. METHODS: In freshly dispersed smooth muscle cells (SMC), we measured the adiponectin effect on the carbachol-induced length changes. RESULTS: Adiponectin significantly reduced the carbachol-stimulated SMC shortening independently of age. In the presence of the inhibitor iberiotoxin, the adiponectin effect on SMC shortening was suppressed, suggesting that it is mediated via large-conductance Ca2+ sensitive K+ channel activation. Lastly, we comparatively measured the newborn rat gastric milk curd adiponectin content in one- and two-week-old rats and found a 50% lower value in the latter. CONCLUSION: Adiponectin, a major component of breastmilk, downregulates fundic smooth muscle contraction potential, thus facilitating gastric volume accommodation. This rodent's adaptive response maximizes breastmilk intake volume after birth.


Assuntos
Adiponectina , Músculo Liso , Animais , Animais Recém-Nascidos , Carbacol/farmacologia , Esvaziamento Gástrico , Contração Muscular , Ratos
4.
Methods Mol Biol ; 2268: 289-304, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085276

RESUMO

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).


Assuntos
Cálcio/metabolismo , Carbacol/farmacologia , Microfluídica/métodos , Pirenzepina/farmacologia , Receptor Muscarínico M1/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Agonistas Colinérgicos/farmacologia , Células HEK293 , Humanos , Antagonistas Muscarínicos/farmacologia , Transdução de Sinais
5.
Sci Rep ; 11(1): 12465, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127743

RESUMO

Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.


Assuntos
Diabetes Mellitus Tipo 2/complicações , Neuropatias Diabéticas/fisiopatologia , Frequência Cardíaca/fisiologia , Proteínas RGS/metabolismo , Nó Sinoatrial/metabolismo , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Diabetes Mellitus Tipo 2/genética , Neuropatias Diabéticas/etiologia , Modelos Animais de Doenças , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Sistema Nervoso Parassimpático , Proteínas RGS/antagonistas & inibidores , Nó Sinoatrial/citologia , Nó Sinoatrial/efeitos dos fármacos , Nó Sinoatrial/inervação
6.
J Pharmacol Sci ; 146(2): 116-119, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33941322

RESUMO

We aimed to investigate detrusor function in a previously developed rat neurogenic voiding dysfunction model that we have developed previously. We performed sham or bilateral accessory nerve injury (BACNI) surgeries on ten-week-old male Wistar/ST rats. One week after surgery, we evaluated detrusor contractility in the bladder using isometric tension and mRNA expression assays. Cholinergic contraction was attenuated in the injury model, whereas carbachol-evoked contraction was enhanced, and mRNA expression of the cholinergic receptor increased. These findings suggest that there was a reduction in neurotransmitter release causing detrusor underactivity.


Assuntos
Traumatismos do Nervo Acessório/complicações , Bexiga Inativa/complicações , Animais , Carbacol/farmacologia , Modelos Animais de Doenças , Expressão Gênica , Contração Isométrica/efeitos dos fármacos , Contração Isométrica/genética , Masculino , Neurotransmissores/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Receptores Colinérgicos/metabolismo , Bexiga Urinária/fisiopatologia , Bexiga Urinaria Neurogênica , Bexiga Inativa/fisiopatologia
7.
Neurourol Urodyn ; 40(6): 1470-1478, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34015163

RESUMO

AIMS: To determine the effect of prostatic radiation therapy (RT) on bladder contractility and morphology, and axon, or neuron profiles within the detrusor and major pelvic ganglia (MPG) in male rats. METHODS: Male Sprague-Dawley rats (8 weeks) received a single dose of prostatic RT (0 or 22 Gy). Bladders and MPG were collected 2- and 10-weeks post-RT. Detrusor contractile responses to carbachol and electrical field stimulation (EFS) were measured. Bladders were stained with Masson's trichrome, and antibodies for nonspecific neuronal marker, cholinergic nerve marker choline acetyltransferase (ChAT), and alpha-smooth muscle actin. MPG gene expression was assessed by quantitative polymerase chain reaction for ubiquitin carboxy-terminal hydrolase L1 (Uchl1) and Chat. RESULTS: At 2 weeks post-RT, bladder smooth muscle, detrusor cholinergic axon profiles, and MPG Chat gene expression were increased (p < .05), while carbachol and EFS-mediated contractions were decreased (p < .05). In contrast, at 10 weeks post-RT, nerve-mediated contractions were increased compared with control (p < .05), while bladder smooth muscle, detrusor cholinergic axon profiles, MPG Chat expression, and carbachol contractions had normalized. At both 2- and 10-weeks post-RT, there was no change in detrusor nonspecific axon profiles and MPG Uchl1 expression. CONCLUSION: In a rat model, RT of the prostate and MPG was associated with early changes in MPG Chat gene expression, and bladder cholinergic axon profiles and smooth muscle content which resolved over time. After RT recovery, bladder contractility decreased early and increased by 10 weeks. Long-term changes to the MPG and increased bladder cholinergic axons may contribute to RT-induced bladder dysfunction in prostate cancer survivors.


Assuntos
Contração Muscular , Bexiga Urinária , Animais , Carbacol/farmacologia , Masculino , Músculo Liso , Ratos , Ratos Sprague-Dawley
8.
Am J Physiol Cell Physiol ; 320(6): C1074-C1087, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33852365

RESUMO

Voltage-gated Kv7 (KCNQ family) K+ channels are expressed in many neuronal populations and play an important role in regulating membrane potential by generating a hyperpolarizing K+ current and decreasing cell excitability. However, the role of KV7 channels in the neural regulation of intestinal epithelial Cl- secretion is not known. Cl- secretion in mouse distal colon was measured as a function of short-circuit current (ISC), and pharmacological approaches were used to test the hypothesis that activation of KV7 channels in enteric neurons would inhibit epithelial Cl- secretion. Flupirtine, a nonselective KV7 activator, inhibited basal Cl- secretion in mouse distal colon and abolished or attenuated the effects of drugs that target various components of enteric neurotransmission, including tetrodotoxin (NaV channel blocker), veratridine (NaV channel activator), nicotine (nicotinic acetylcholine receptor agonist), and hexamethonium (nicotinic antagonist). In contrast, flupritine did not block the response to epithelium-targeted agents VIP (endogenous VPAC receptor ligand) or carbachol (nonselective cholinergic agonist). Flupirtine inhibited Cl- secretion in both full-thickness and seromuscular-stripped distal colon (containing the submucosal, but not myenteric plexus) but generated no response in epithelial T84 cell monolayers. KV7.2 and KV7.3 channel proteins were detected by immunofluorescence in whole mount preparations of the submucosa from mouse distal colon. ICA 110381 (KV7.2/7.3 specific activator) inhibited Cl- secretion comparably to flupirtine. We conclude that KV7 channel activators inhibit neurally driven Cl- secretion in the colonic epithelium and may therefore have therapeutic benefit in treating pathologies associated with hyperexcitable enteric nervous system, such as irritable bowel syndrome with diarrhea (IBS-D).


Assuntos
Cloretos/metabolismo , Colo/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios/metabolismo , Aminopiridinas/farmacologia , Animais , Carbacol/farmacologia , Linhagem Celular Tumoral , Agonistas Colinérgicos/farmacologia , Colo/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
9.
Neurosci Lett ; 751: 135827, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33727128

RESUMO

The role of the ventral tegmental area (VTA) and the lateral hypothalamus (LH) in the modulation of formalin-induced nociception is well documented individually. The present study aimed to investigate the role of dopamine receptors of the VTA in the modulation of the LH stimulation-induced antinociception during both phases of the formalin test as an animal model of tonic pain. In this study, male Wistar rats were unilaterally implanted with two guide cannulae in the VTA and LH. In two separate groups, animals received different doses (0.25, 1, and 4 µg/rat) of D1- or D2-like dopamine receptor antagonists (SCH-23,390 or Sulpiride, respectively) into the VTA before intra-LH injection of carbachol (22.83 ng/rat) following formalin injection (50 µL; s.c.) into their contralateral hind paws. The blockade of these two receptors reduced intra-LH carbachol-induced antinociception during both phases of the formalin test. This reduction during the late phase of the formalin test was more than that of the early phase. The results indicated that LH stimulation-induced antinociception was mediated by D1- and D2-like dopamine receptors in the VTA, and so, the neural pathway projecting from the LH to the VTA contributes to the modulation of formalin-induced nociception in the rats.


Assuntos
Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Nociceptividade , Sulpirida/farmacologia , Área Tegmentar Ventral/metabolismo , Analgésicos não Narcóticos/farmacologia , Animais , Carbacol/farmacologia , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Vias Neurais/fisiologia , Ratos , Ratos Wistar , Receptores Dopaminérgicos/metabolismo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologia
10.
J Vet Med Sci ; 83(4): 622-629, 2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33583865

RESUMO

Excessive stress response causes disability in social life. There are many diseases caused by stress, such as gastrointestinal motility disorders, depression, eating disorders, and cardiovascular diseases. Transient receptor potential (TRP) channels underlie non-selective cation currents and are downstream effectors of G protein-coupled receptors. Ca2+ influx is important for smooth muscle contraction, which is responsible for gastrointestinal motility. Little is known about the possible involvement of TRP channels in the gastrointestinal motility disorders due to stress. The purpose of this study was to measure the changes in gastrointestinal motility caused by stress and to elucidate the mechanism of these changes. The stress model used the water immersion restraint stress. Gastrointestinal motility, especially the ileum, was recorded responses to electric field stimulation (EFS) by isometric transducer. EFS-induced contraction was significantly reduced in the ileum of stressed mouse. Even under the conditions treated with atropine, EFS-induced contraction was significantly reduced in the ileum of stressed mouse. In addition, carbachol-induced, neurokinin A-induced, and substance P-induced contractions were all significantly reduced in the ileum of stressed mouse. Furthermore, the expression of TRPC3 was decreased in the ileum of stressed mouse. These results suggest that the gastrointestinal motility disorders due to stress is associated with specific non-selective cation channel.


Assuntos
Músculo Liso , Canais de Potencial de Receptor Transitório , Animais , Carbacol/farmacologia , Estimulação Elétrica , Motilidade Gastrointestinal , Íleo , Camundongos , Contração Muscular
11.
J Physiol Sci ; 71(1): 8, 2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33622238

RESUMO

Colonic motor activity is important for the formation and propulsion of feces. The production of prostaglandins (PGs) in colonic tissue is considered to play a critical role in the generation and regulation of colonic motility. In this study, we investigated the inhibitory effects of PGE2 and selective agonists of four EP receptors on the spontaneous phasic contractions, called 'giant contractions' (GCs), of mucosa-free circular smooth muscle strips from the rat middle colon. Neural blockade with tetrodotoxin (TTX) increased the frequency and amplitude of the GCs by about twofold. However, inhibiting PG production with piroxicam reduced the GC frequency in the presence of TTX, but did not affect the GC amplitude. In the presence of both TTX and piroxicam, exogenous PGE2 and each EP receptor agonist were cumulatively added to the tissue bath. In this setting, PGE2, the EP2 agonist ONO-AE1-259, and the EP4 agonist ONO-AE1-329, but not the EP1 agonist ONO-AE-DI-004 or the EP3 agonist ONO-AE-248, concentration-dependently reduced the GC frequency and amplitude. The PGE2-induced inhibition of GC frequency and amplitude was inhibited by the EP4 antagonist ONO-AE3-208, but not by the EP1/2 antagonist AH6809. Immunohistochemistry revealed the EP2 and EP4 receptors were localized in perinuclear sites in circular smooth muscle cells. EP2 immunoreactivity was also located in GFAP-immunoreactive enteroglia, whereas EP4 immunoreactivity was also located in HU (embryonic lethal, abnormal vision [ELAV] protein; a marker of all myenteric neurons)-immunoreactive myenteric nerve cell bodies. These results suggest that the PGs produced in the colonic tissue inhibit the GC frequency and amplitude of circular muscle in the rat middle colon, and is mediated by EP4 receptors expressed in the smooth muscle cells.


Assuntos
Colo/efeitos dos fármacos , Dinoprostona/farmacologia , Motilidade Gastrointestinal/fisiologia , Músculo Liso/efeitos dos fármacos , Piroxicam/farmacologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Colo/fisiologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP1 , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4/agonistas , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Proteínas de Peixe-Zebra
12.
Nutrients ; 13(2)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562242

RESUMO

High salt intake has been reported as a risk factor for urinary storage symptoms. However, the association between high salt intake and detrusor muscle contraction is not clear. Therefore, we investigated the effects of high salt intake on the components of detrusor muscle contraction in rats. Six-week-old male Dahl salt-resistant (DR; n = 5) and Dahl salt-sensitive (DS; n = 5) rats were fed a high salt (8% NaCl) diet for one week. The contractile responses of the detrusor muscle to the cumulative administration of carbachol and electrical field stimulation (EFS) with and without suramin and atropine were evaluated via isometric tension study. The concentration-response curves of carbachol were shifted more to the left in the DS group than those in the DR group. Contractile responses to EFS were more enhanced in the DS group than those in the DR group (p < 0.05). Cholinergic component-induced responses were more enhanced in the DS group than those in the DR group (p < 0.05). High salt intake might cause urinary storage symptoms via abnormalities in detrusor muscle contraction and the enhancement of cholinergic signals. Excessive salt intake should be avoided to preserve bladder function.


Assuntos
Contração Isométrica/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cloreto de Sódio na Dieta/efeitos adversos , Cloreto de Sódio na Dieta/farmacologia , Bexiga Urinária/efeitos dos fármacos , Animais , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Masculino , Ratos Endogâmicos Dahl , Suramina/farmacologia
13.
Int J Mol Sci ; 22(4)2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562815

RESUMO

Altered lacrimal gland (LG) secretion is a feature of autoimmune dacryoadenitis in Sjögren's syndrome (SS). Cathepsin S (CTSS) is increased in tears of SS patients, which may contribute to disease. Rab3D and Rab27a/b isoforms are effectors of exocytosis in LG, but Rab27a is poorly studied. To investigate whether Rab27a mediates CTSS secretion, we utilized quantitative confocal fluorescence microscopy of LG from SS-model male NOD and control male BALB/c mice, showing that Rab27a-enriched vesicles containing CTSS were increased in NOD mouse LG. Live-cell imaging of cultured lacrimal gland acinar cells (LGAC) transduced with adenovirus encoding wild-type (WT) mCFP-Rab27a revealed carbachol-stimulated fusion and depletion of mCFP-Rab27a-enriched vesicles. LGAC transduced with dominant-negative (DN) mCFP-Rab27a exhibited significantly reduced carbachol-stimulated CTSS secretion by 0.5-fold and ß-hexosaminidase by 0.3-fold, relative to stimulated LGAC transduced with WT mCFP-Rab27a. Colocalization of Rab27a and endolysosomal markers (Rab7, Lamp2) with the apical membrane was increased in both stimulated BALB/c and NOD mouse LG, but the extent of colocalization was much greater in NOD mouse LG. Following stimulation, Rab27a colocalization with endolysosomal membranes was decreased. In conclusion, Rab27a participates in CTSS secretion in LGAC though the major regulated pathway, and through a novel endolysosomal pathway that is increased in SS.


Assuntos
Catepsinas/metabolismo , Aparelho Lacrimal/citologia , Síndrome de Sjogren/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carbacol/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Endossomos/metabolismo , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Proteínas rab27 de Ligação ao GTP/genética
14.
Life Sci ; 265: 118735, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33166589

RESUMO

AIMS: To investigates the effects of water avoidance stress on voiding behaviour and functional bladder responses in mice. MAIN METHODS: Mice in the Stress group were exposed to water avoidance stress (WAS) for 1 h/day for 10 days, Controls were age-matched and housed normally. Voiding behaviour was measured periodically throughout the stress protocol and bladders were isolated 24-h after final stress exposure to measure bladder compliance, spontaneous phasic activity, contractile responses, and release of urothelial mediators. KEY FINDINGS: Repeated stress exposure induced a significant increase in plasma corticosterone levels in the WAS group compared to control. An overactive bladder phenotype was observed in WAS mice, causing a significant increase in the number of voiding events observed from as early as day-3, and a 7-fold increase following 10-days' stress. This increase in voiding frequency was associated with a significant decrease in void size, an increase in the number of small voids, but no change in total voided volume. Bladders from stressed mice showed a significant increase in the maximum responses to the muscarinic agonist carbachol (p < 0.01), in addition to enhanced pressure responses to the purinergic agonists ATP (p < 0.05) and αß-mATP (p < 0.05), and non-receptor mediated contractions to KCl (p < 0.05) compared to controls. Nerve-mediated bladder contractions to electric field stimulation were not significantly affected by stress, nor were spontaneous phasic contractions or release of urothelial ATP and acetylcholine. SIGNIFICANCE: Repeated exposure to water avoidance stress produced an overactive bladder phenotype, confirmed by increased voiding frequency, and associated with enhanced bladder contractile responses.


Assuntos
Contração Muscular/fisiologia , Estresse Psicológico/metabolismo , Bexiga Urinária Hiperativa/fisiopatologia , Acetilcolina , Animais , Carbacol/farmacologia , Corticosterona , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Agonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Agonistas Purinérgicos/farmacologia , Estresse Psicológico/fisiopatologia , Bexiga Urinária/patologia , Bexiga Urinária Hiperativa/metabolismo , Micção/efeitos dos fármacos
15.
Toxicol Appl Pharmacol ; 411: 115368, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33338514

RESUMO

Cannabidiol (CBD) is suggested to possess cardioprotective properties. We examined the influence of chronic (10 mg/kg once daily for 2 weeks) CBD administration on heart structure (e.g. cardiomyocyte width) and function (e.g. stimulatory and inhibitory responses induced by ß-adrenoceptor (isoprenaline) and muscarinic receptor (carbachol) activation, respectively). Experiments were performed on hearts and/or left atria isolated from spontaneously (SHR) and deoxycorticosterone (DOCA-salt) hypertensive rats; Wistar-Kyoto (WKY) and sham-operated rats (SHAM) served as the respective normotensive controls. CBD diminished the width of cardiomyocytes in left ventricle and reduced the carbachol-induced vasoconstriction of coronary arteries both in DOCA-salt and SHR. However, it failed to affect left ventricular hypertrophy and even aggravated the impaired positive and negative lusitropic effects elicited by isoprenaline and carbachol, respectively. In normotensive hearts CBD led to untoward structural and functional effects, which occurred only in WKY or SHAM or, like the decrease in ß1-adrenoceptor density, in either control strain. In conclusion, due to its modest beneficial effect in hypertension and its adverse effects in normotensive hearts, caution should be taken when using CBD as a drug in therapy.


Assuntos
Anti-Hipertensivos/toxicidade , Canabidiol/toxicidade , Tamanho Celular/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertrofia Ventricular Esquerda/etiologia , Miócitos Cardíacos/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Disfunção Ventricular Esquerda/etiologia , Função Ventricular Esquerda/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Carbacol/farmacologia , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Hipertensão/complicações , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Preparação de Coração Isolado , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Vasoconstritores/farmacologia , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia
16.
J Neurophysiol ; 125(1): 248-255, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33296614

RESUMO

Pathological synchronization of neurons is associated with symptoms of movement disorders, such as Parkinson's disease and essential tremor. High-frequency deep brain stimulation (DBS) suppresses symptoms, presumably through the desynchronization of neurons. Coordinated reset (CR) delivers trains of high-frequency stimuli to different regions in the brain through multiple electrodes and may have more persistent therapeutic effects than conventional DBS. As an alternative to CR, we present a closed-loop control setup that desynchronizes neurons in brain slices by inducing clusters using a single electrode. Our setup uses calcium fluorescence imaging to extract carbachol-induced neuronal oscillations in real time. To determine the appropriate stimulation waveform for inducing clusters in a population of neurons, we calculate the phase of the neuronal populations and then estimate the phase response curve (PRC) of those populations to electrical stimulation. The phase and PRC are then fed into a control algorithm called the input of maximal instantaneous efficiency (IMIE). By using IMIE, the synchrony across the slice is decreased by dividing the population of neurons into subpopulations without suppressing the oscillations locally. The desynchronization effect is persistent 10 s after stimulation is stopped. The IMIE control algorithm may be used as a novel closed-loop DBS approach to suppress the symptoms of Parkinson's disease and essential tremor by inducing clusters with a single electrode.NEW & NOTEWORTHY Here, we present a closed-loop controller to desynchronize neurons in brain slices by inducing clusters using a single electrode using calcium imaging feedback. Phase of neurons are estimated in real time, and from the phase response curve stimulation is applied to achieve target phase differences. This method is an alternative to coordinated reset and is a novel therapy that could be used to disrupt synchronous neuronal oscillations thought to be the mechanism underlying Parkinson's disease.


Assuntos
Encéfalo/fisiologia , Estimulação Encefálica Profunda/métodos , Neurônios/fisiologia , Algoritmos , Animais , Encéfalo/citologia , Ondas Encefálicas , Cálcio/metabolismo , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Estimulação Encefálica Profunda/instrumentação , Eletrodos Implantados , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Long-Evans
17.
Mol Vis ; 26: 780-788, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33311973

RESUMO

Purpose: Vasoactive intestinal peptide (VIP) is an important regulator of lacrimal gland (LG) function although the effect of VIP on ductal fluid secretion is unknown. Therefore, the aim of the present study was to investigate the role of VIP in the regulation of fluid secretion of isolated LG ducts and to analyze the underlying intracellular mechanisms. Methods: LGs from wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) knockout (KO) mice were used. Immunofluorescence was applied to confirm the presence of VIP receptors termed VPAC1 and VPAC2 in LG duct cells. Ductal fluid secretion evoked by VIP (100 nM) was measured in isolated ducts using videomicroscopy. Intracellular Ca2+ signaling underlying VIP stimulation was investigated with microfluorometry. Results: VIP stimulation resulted in a robust and continuous fluid secretory response in isolated duct segments originated from WT mice. In contrast, CFTR KO ducts exhibited only a weak pulse-like secretion. A small but statistically significant increase was detected in the intracellular Ca2+ level [Ca2+]i during VIP stimulation in the WT and in CFTR KO ducts. VIP-evoked changes in [Ca2+]i did not differ considerably between the WT and CFTR KO ducts. Conclusions: These results suggest the importance of VIP in the regulation of ductal fluid secretion and the determining role of the adenylyl cyclase-cAMP-CFTR route in this process.


Assuntos
Aparelho Lacrimal/metabolismo , Lágrimas/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Carbacol/farmacologia , Quelantes/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Espaço Intracelular/metabolismo , Camundongos Knockout , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo
18.
Nat Commun ; 11(1): 5555, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144559

RESUMO

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.


Assuntos
Relógios Biológicos , AMP Cíclico/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Nó Sinoatrial/patologia , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/patologia , Relógios Biológicos/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Bradicardia/complicações , Bradicardia/patologia , Carbacol/farmacologia , Eletrocardiografia , Feminino , Células HEK293 , Coração/efeitos dos fármacos , Coração/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Nó Sinoatrial/fisiopatologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiopatologia
19.
Behav Pharmacol ; 31(8): 759-767, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32925229

RESUMO

Recent studies have shown that orexin neurons in the lateral hypothalamus send a compelling project to the ventral tegmental area (VTA). Besides, orexin-1 (OX1) and orexin-2 (OX2) in the VTA are necessary for the development of morphine-induced place preference. Also, sensitivity to morphine can reinforce the rewarding effects of morphine. The current study aims to determine the role of VTAs orexin receptors in morphine sensitization in rats. In 84 adult male albino Wistar rats, two separate cannulae bilaterally implanted into the VTA. They received intra-VTA infusions of SB334867 (0.1, 1 and 10 nM) and TCS OX2 29 (1, 7 and 20 nM) as OX1 and OX2 receptor antagonists, respectively, 10 min before subcutaneous administration of morphine (5 mg/kg) during 3-day sensitization period. After a 5-day drug-free period, the conditioned place preference (CPP) paradigm induced by subthreshold doses of morphine (0.5 mg/kg), and CPP scores were measured by EthoVision software. The results revealed that the blockade of both OX1 and OX2 receptors within the VTA reduced the expression of morphine-induced CPP in the sensitized rats. It is plausible that VTAs orexin receptors are involved in the development/acquisition of sensitization to morphine-induced CPP in the rats.


Assuntos
Comportamento de Procura de Droga/fisiologia , Receptores de Orexina/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Benzoxazóis/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Carbacol/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Operante/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Região Hipotalâmica Lateral/metabolismo , Isoquinolinas/farmacologia , Masculino , Morfina/metabolismo , Morfina/farmacologia , Naftiridinas/farmacologia , Núcleo Accumbens/metabolismo , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/efeitos dos fármacos , Orexinas/metabolismo , Piridinas/farmacologia , Ratos , Ratos Wistar , Recompensa , Ureia/análogos & derivados , Ureia/farmacologia , Área Tegmentar Ventral/efeitos dos fármacos
20.
Int J Dev Biol ; 64(7-8-9): 445-452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32930354

RESUMO

Planarians are traditional model invertebrates in regeneration and developmental biology research that also display a variety of quantifiable behaviors useful to screen for pharmacologically active compounds. One such behavior is the expression of seizure-like movements (pSLMs) induced by a variety of substances. Previous work from our laboratory showed that cocaine, but not nicotine, induced pSLMs in intact but not decapitated planarians. Interestingly, as decapitated planarians regenerated their heads, they gradually recovered their sensitivity to cocaine. These results suggested a method to assess planarian brain regeneration and a possible way of identifying compounds that could enhance or hold back brain regeneration. In the present work, we demonstrate that the cholinergic agent cytisine is a suitable reference compound to apply our method. Cytisine induces pSLMs in a concentration-dependent manner in intact (but not decapitated) planarians of the species Girardia tigrina. Based on our data, we developed a behavioral protocol to assess planarian brain regeneration over time. We tested this method to measure the effect of ethanol on G. tigrina's brain regeneration. We found that ethanol slows down the rate of planarian brain regeneration in a concentration-dependent manner, consistently with data from other research groups that tested ethanol effects on planarian brain regeneration using different behavioral protocols. Thus, here we establish a general method using cytisine-induced pSLMs as an indicator of brain regeneration in planarians, a method that shows potential for assessing the effect of pharmacologically active compounds in this process.


Assuntos
Alcaloides/farmacologia , Encéfalo/efeitos dos fármacos , Colinérgicos/farmacologia , Planárias/fisiologia , Regeneração/efeitos dos fármacos , Anestésicos Locais/farmacologia , Animais , Azocinas/farmacologia , Encéfalo/fisiologia , Carbacol/farmacologia , Cocaína/farmacologia , Cotinina/farmacologia , Relação Dose-Resposta a Droga , Etanol/farmacologia , Nicotina/farmacologia , Quinolizinas/farmacologia , Regeneração/fisiologia , Convulsões/fisiopatologia
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