RESUMEN
P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/farmacología , Riñón/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Adenosina Trifosfato , Aminoquinolinas/farmacología , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Pirimidinas/farmacología , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X2/genética , Proteína de Unión al GTP rac1/antagonistas & inhibidoresRESUMEN
Laboratory practicals in life science subjects are traditionally assessed by written reports that reflect disciplinary norms for documenting experimental activities. However, the exclusive application of this assessment has the potential to engage only a narrow range of competencies. In this study, we explored how multiple modes of laboratory assessment might affect student perceptions of learned skills in a life science module. We hypothesized that while a mixture of assessments may not impact student summative performance, it might positively influence student perceptions of different skills that varied assessments allowed them to practice. This was informed by universal design for learning and teaching for understanding frameworks. In our study, in a third-year Bioscience program, written reports were complemented with group presentations and online quizzes via Moodle. Anonymous surveys evaluated whether this expanded portfolio of assessments promoted awareness of, and engagement with, a broader range of practical competencies. Aspects that influenced student preferences in assessment mode included time limitations, time investment, ability to practice new skills, links with lecture material, and experience of assessment anxiety. In particular, presentations were highlighted as promoting collaboration and communication and the quiz as an effective means of diversifying assessment schedules. A key takeaway from students was that while reports were important, an overreliance on them was detrimental. This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.NEW & NOTEWORTHY This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.
Asunto(s)
Disciplinas de las Ciencias Biológicas , Evaluación Educacional , Humanos , Evaluación Educacional/métodos , Disciplinas de las Ciencias Biológicas/educación , Masculino , Femenino , Estudiantes/psicología , LaboratoriosRESUMEN
TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1-/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1-/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Fenómenos Fisiológicos del Sistema Urinario , Animales , Biomarcadores , Técnica del Anticuerpo Fluorescente , Expresión Génica , Espacio Intracelular/metabolismo , Masculino , Potenciales de la Membrana , Ratones , Ratones Noqueados , Contracción Muscular/genética , Transporte de Proteínas , Canales de Potencial de Receptor Transitorio/genética , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatologíaRESUMEN
Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at â¼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na+ was substituted with N-Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The V1/2s of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC50 = 1.5 nM) and the NaV1.7 subtype-selective blocker, PF-05089771 (IC50 = 8.6 nM), consistent with NaV1.7 as the underlying pore-forming α subunit. Two NaV1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-NaV antibody. RT-PCR, performed on groups of â¼15 isolated SMCs, revealed transcripts for NaV1.7 in 7/8 samples. Veratridine (30 µM), a nonselective NaV channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE2 (prostaglandin E2), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express NaV1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.
Asunto(s)
Bronquios , Miocitos del Músculo Liso , Animales , Derivados de Atropina/metabolismo , Derivados de Atropina/farmacología , Bronquios/metabolismo , Ratones , Miocitos del Músculo Liso/metabolismo , Sodio/metabolismo , Tetrodotoxina/metabolismo , Tetrodotoxina/farmacología , Veratridina/metabolismo , Veratridina/farmacologíaRESUMEN
Junctophilin proteins maintain close contacts between the endoplasmic/sarcoplasmic reticulum (ER/SR) and the plasma membrane in many types of cells, as typified by junctophilin-2 (JPH2), which is necessary for the formation of the cardiac dyad. Here, we report that JPH2 is the most abundant junctophilin isotype in native smooth muscle cells (SMCs) isolated from cerebral arteries and that acute knockdown diminishes the area of sites of interaction between the SR and plasma membrane. Superresolution microscopy revealed nanometer-scale colocalization of JPH2 clusters with type 2 ryanodine receptor (RyR2) clusters near the cell surface. Knockdown of JPH2 had no effect on the frequency, amplitude, or kinetics of spontaneous Ca2+ sparks generated by transient release of Ca2+ from the SR through RyR2s, but it did nearly abolish Ca2+ spark-activated, large-conductance, Ca2+-activated K+ (BK) channel currents. We also found that JPH2 knockdown was associated with hypercontractility of intact cerebral arteries. We conclude that JPH2 maintains functional coupling between RyR2s and BK channels and is critically important for cerebral arterial function.
Asunto(s)
Arterias Cerebrales/fisiología , Proteínas de la Membrana/fisiología , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Animales , Arterias Cerebrales/citología , Técnicas de Silenciamiento del Gen , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Nanopartículas , Canales de Potasio Calcio-Activados/fisiología , Transducción de SeñalRESUMEN
ß3-Adrenoceptor (ß3-AR) agonists are used to treat overactive bladder syndrome; however, their mechanism of action has not been determined. The aims of this study were to compare the effects of ß3-AR agonists on cholinergic versus purinergic receptor-mediated contractions of the detrusor and to examine the mechanisms underlying inhibition of the purinergic responses by ß3-AR agonists. Isometric tension recordings were made from strips of murine detrusor and whole cell current recordings were made from freshly isolated detrusor myocytes using the patch-clamp technique. Transcriptional expression of exchange protein directly activated by cAMP (EPAC) subtypes in detrusor strips was assessed using RT-PCR and real-time quantitative PCR. The ß3-AR agonists BRL37344 and CL316243 (100 nM) inhibited cholinergic nerve-mediated contractions of the detrusor by 19 and 23%, respectively, but did not reduce contractions induced by the cholinergic agonist carbachol (300 nM). In contrast, BRL37344 and CL316243 inhibited purinergic nerve-mediated responses by 55 and 56%, respectively, and decreased the amplitude of contractions induced by the P2X receptor agonist α,ß-methylene ATP by 40 and 45%, respectively. The adenylate cyclase activator forskolin inhibited purinergic responses, and these effects were mimicked by a combination of the PKA activator N6-monobutyryl-cAMP and the EPAC activator 8-pCPT-2'-O-methyl-cAMP-AM (007-AM). Application of ATP (1 µM) evoked reproducible P2X currents in isolated detrusor myocytes voltage-clamped at -60 mV. These responses were reduced in amplitude in the presence of BRL37344 and also by 007-AM. This study demonstrates that ß3-AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 3/farmacología , Contracción Muscular/efectos de los fármacos , Agonistas del Receptor Purinérgico P2X/farmacología , Receptores Adrenérgicos beta 3/efectos de los fármacos , Receptores Purinérgicos P2X/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , Urodinámica/efectos de los fármacos , Animales , Agonistas Colinérgicos/farmacología , AMP Cíclico/metabolismo , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Masculino , Ratones Endogámicos C57BL , Receptores Adrenérgicos beta 3/metabolismo , Receptores Purinérgicos P2X/metabolismo , Transducción de Señal , Vejiga Urinaria/inervación , Vejiga Urinaria/metabolismoRESUMEN
OBJECTIVE: To test if carbachol (CCh)-evoked Ca2+ oscillations in freshly isolated murine detrusor myocytes are affected by ß3-adrenoceptor (ß-AR) modulators. MATERIALS AND METHODS: Isometric tension recordings were made from strips of murine detrusor, and intracellular Ca2+ measurements were made from isolated detrusor myocytes using confocal microscopy. Transcriptional expression of ß-AR sub-types in detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR). Immunocytochemistry experiments, using a ß3-AR selective antibody, were performed to confirm that ß3-ARs were present on detrusor myocytes. RESULTS: The RT-PCR and qPCR experiments showed that ß1-, ß2- and ß3-AR were expressed in murine detrusor, but that ß3-ARs were the most abundant sub-type. The selective ß3-AR agonist BRL37344 reduced the amplitude of CCh-induced contractions of detrusor smooth muscle. These responses were unaffected by addition of the BK channel blocker iberiotoxin. BRL37344 also reduced the amplitude of CCh-induced Ca2+ oscillations in freshly isolated murine detrusor myocytes. This effect was mimicked by CL316,243, another ß3-AR agonist, and inhibited by the ß3-AR antagonist L748,337, but not by propranolol, an antagonist of ß1- and ß2-ARs. BRL37344 did not affect caffeine-evoked Ca2+ transients or L-type Ca2+ current in isolated detrusor myocytes. CONCLUSION: Inhibition of cholinergic-mediated contractions of the detrusor by ß3-AR agonists was associated with a reduction in Ca2+ oscillations in detrusor myocytes.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 3/farmacología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Células Musculares/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Calcio/metabolismo , Etanolaminas/farmacología , Femenino , Masculino , Ratones Endogámicos C57BL , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Péptidos/farmacología , Vejiga Urinaria/efectos de los fármacosRESUMEN
Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl- (IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa T16Ainh-A01 and CaCCinh-A01 (10 µM) reduced IClCa by ~85%, while 30 µM abolished it. L-type Ca2+ current was unaffected by 10 µM CaCCinh-A01 but was reduced by 50% at 30 µM CaCCinh-A01, 46% at 10 µM T16Ainh-A01, and 78% at 30 µM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 µM and >90% at 30 µM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 µM, CaCCinh-A01; 14 µM, T16Ainh-A01). CaCCinh-A01 at 10 µM had little effect on 60 mM KCl contractures, though they were reduced by 30 µM CaCCinh-A01 and T16Ainh-A01 (10 µM and 30 µM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 µM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Músculo Liso/fisiología , Pene/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Masculino , Músculo Liso/efectos de los fármacos , Técnicas de Cultivo de Órganos , Pene/efectos de los fármacos , ConejosRESUMEN
The cough reflex becomes hyperresponsive in acute and chronic respiratory diseases, but understanding the underlying mechanism is hampered by difficulty accessing human tissue containing both nerve endings and neuronal cell bodies. We refined an adult stem cell sensory neuronal model to overcome the limited availability of human neurones and applied the model to study transient receptor potential ankyrin 1 (TRPA1) channel expression and activation.Human dental pulp stem cells (hDPSCs) were differentiated towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). Using molecular and immunohistochemical techniques, together with Ca2+ microfluorimetry and whole cell patch clamping, we investigated roles for nerve growth factor (NGF) and the viral mimic poly I:C in TRPA1 activation.PNEs exhibited morphological, molecular and functional characteristics of sensory neurons and expressed functional TRPA1 channels. PNE treatment with NGF for 20â min generated significantly larger inward and outward currents compared to untreated PNEs in response to the TRPA1 agonist cinnamaldehyde (p<0.05). PNE treatment with poly I:C caused similar transient heightened responses to TRPA1 activation compared to untreated cells.Using the PNE neuronal model we observed both NGF and poly I:C mediated sensory neuronal hyperresponsiveness, representing potential neuro-inflammatory mechanisms associated with heightened nociceptive responses recognised in cough hypersensitivity syndrome.
Asunto(s)
Tos/fisiopatología , Factor de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/metabolismo , Canal Catiónico TRPA1/metabolismo , Canales de Calcio/metabolismo , Tos/tratamiento farmacológico , Pulpa Dental/citología , Humanos , Neuronas Aferentes/citología , Poli I-C/farmacología , Células Madre/efectos de los fármacos , Canal Catiónico TRPA1/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
PURPOSE: Muscarinic receptor mediated contractions of the detrusor rely on Ca2+ influx through voltage-gated Ca2+ channels but to our knowledge the mechanism linking stimulation of M3Rs to the activation of voltage dependent Ca2+ channels has not been established. TRPC4 channels are receptor operated cation channels that couple muscarinic receptor activation to depolarization of intestinal smooth muscle cells, voltage-activated Ca2+ influx and contraction. We investigated whether TRPC4 channels are involved in cholinergic mediated contractions of the detrusor. MATERIALS AND METHODS: Isometric tension recordings were made on strips of murine detrusor and intracellular Ca2+ measurements were made on isolated detrusor myocytes using confocal microscopy. Transcriptional expression of TRPC and IP3R subtypes in intact detrusor strips and isolated detrusor myocytes was assessed using reverse transcriptase-polymerase chain reaction. RESULTS: Cholinergic stimulation of the detrusor induced by electrical field stimulation or exogenous application of carbachol or neostigmine evoked contractions consisting of a transient plus a tonic response, which was blocked by ML204, an inhibitor of TRPC4 channels. A phasic oscillatory component was blocked by the IP3R inhibitor 2-APB. Carbachol evoked reproducible Ca2+ responses in isolated detrusor myocytes, consisting of an initial Ca2+ transient followed by Ca2+ oscillations. ML204 inhibited the initial Ca2+ transient whereas 2-APB inhibited the Ca2+ oscillations. Reverse transcriptase-polymerase chain reaction experiments showed that TRPC4ß, TRPC6 and IP3R1 were selectively expressed in isolated detrusor myocytes. Control experiments demonstrated that ML204 did not affect L-type Ca2+ or BK current amplitude, caffeine induced Ca2+ transients or KCl induced contractions of the detrusor. CONCLUSIONS: Muscarinic receptor mediated contractions of the detrusor involve the activation of TRPC4ß channels.
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Contracción Muscular/fisiología , Músculo Liso/fisiología , Receptores Muscarínicos/fisiología , Canales Catiónicos TRPC/fisiología , Vejiga Urinaria/fisiología , Animales , Femenino , Masculino , Ratones Endogámicos C57BLRESUMEN
Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit+ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca2+ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca2+ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca2+ signalling. ICC-LC generated Ca2+ waves spontaneously that propagated on average 40.1 ± 0.7 µm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca2+ signalling between ICC-LC was obtained. Ca2+ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca2+ channels (GSK-7975A). Phenylephrine increased Ca2+ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca2+ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.
RESUMEN
BACKGROUND AND PURPOSE: Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (NaV ) currents in mouse CCSM and investigate their function. EXPERIMENTAL APPROACH: We used electrophysiological, pharmacological and immunocytochemical methods to study the NaV currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue. KEY RESULTS: Fast, voltage-dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of NaV currents, 'TTX-sensitive' and 'TTX-insensitive', were identified. TTX-sensitive currents showed 48% block with the NaV channel subtype-specific blockers ICA-121431 (NaV 1.1-1.3), PF-05089771 (NaV 1.7) and 4,9-anhydro-TTX (NaV 1.6). TTX-insensitive currents were resistant to blockade by A803467, specific for NaV 1.8 channels. Immunocytochemistry confirmed expression of NaV 1.5 and NaV 1.4 in freshly dispersed CCSM cells. Veratridine, a NaV channel activator, reduced time-dependent inactivation of NaV currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB-R7943. CONCLUSION AND IMPLICATIONS: There are fast, voltage-dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role.
Asunto(s)
Disfunción Eréctil , Animales , Disfunción Eréctil/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/metabolismo , Veratridina/metabolismoRESUMEN
Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.
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Calcio , Músculo Liso Vascular , Animales , Calcio/metabolismo , Señalización del Calcio , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.
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Señalización del Calcio , Calcio/metabolismo , Miocitos del Músculo Liso/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Endosomas/genética , Endosomas/metabolismo , Lisosomas/genética , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/citología , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Acetylcholine contracts the bladder by binding to muscarinic M3 receptors on the detrusor, leading to Ca2+ influx via voltage-gated Ca2+ channels. The cellular mechanisms linking these events are poorly understood, but studies have suggested that activation of TRPC4 channels could be involved. The purpose of this study was to investigate if spontaneous and cholinergic-mediated contractions of the detrusor were impaired in TRPC4 deficient (TRPC4-/-) mice. Isometric tension recordings were made from strips of wild-type (WT) and TRPC4-/- detrusor. Spontaneous phasic detrusor contractions were significantly smaller in TRPC4-/- mice compared to wild-type, however no difference in response to exogenous application of 60 mM KCl was observed. Cholinergic responses, induced by electric-field stimulation (EFS), bath application of the cholinergic agonist carbachol, or the acetylcholinesterase inhibitor neostigmine were all significantly smaller in TRPC4-/- detrusor strips than wild-type. Surprisingly, the TRPC4/5 inhibitor ML204 reduced EFS and CCh-evoked contractions in TRPC4-/- detrusor strips. However, TRPC5 expression was up-regulated in these preparations and, in contrast to wild-type, EFS responses were reduced in amplitude by the TRPC5 channel inhibitor clemizole hydrochloride. This study demonstrates that TRPC4 channels are involved in spontaneous and cholinergic-mediated contractions of the murine detrusor. TRPC5 expression is up-regulated in TRPC4-/- detrusor strips, and may partially compensate for loss of TRPC4 channels.