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BACKGROUND: For millennia, drug-type cannabis strains were extensively used for various medicinal, ritual, and inebriant applications. However, cannabis prohibition during the last century led to cultivation and breeding activities being conducted under clandestine conditions, while scientific development of the crop ceased. Recently, the potential of medicinal cannabis has been reacknowledged and the now expanding industry requires optimal and scientifically characterized varieties. However, scientific knowledge that can propel this advancement is sorely lacking. To address this issue, the current study aims to provide a better understanding of key physiological and phenological traits that can facilitate the breeding of advanced cultivars. RESULTS: A diverse population of 121 genotypes of high-THC or balanced THC-CBD ratio was cultivated under a controlled environment facility and 13 plant parameters were measured. No physiological association across genotypes attributed to the same vernacular classification was observed. Floral bud dry weight was found to be positively associated with plant height and stem diameter but not with days to maturation. Furthermore, the heritability of both plant height and days to maturation was relatively high, but for plant height it decreased during the vegetative growth phase. To advance breeding efficacy, a prediction equation for forecasting floral bud dry weight was generated, driven by parameters that can be detected during the vegetative growth phase solely. CONCLUSIONS: Our findings suggest that selection for taller and fast-growing genotypes is likely to lead to an increase in floral bud productivity. It was also found that the final plant height and stem diameter are determined by 5 independent factors that can be used to maximize productivity through cultivation adjustments. The proposed prediction equation can facilitate the selection of prolific genotypes without the completion of a full cultivation cycle. Future studies that will associate genome-wide variation with plants morphological traits and cannabinoid profile will enable precise and accelerated breeding through genomic selection approaches.
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Cannabis/genética , Fitomejoramiento , Carácter Cuantitativo Heredable , Cannabis/crecimiento & desarrollo , Cannabis/fisiología , Variación Genética , Fenotipo , Fitomejoramiento/métodosRESUMEN
AIMS: The present study aims to investigate the role of Akt in the regulation of urinary bladder organ hypertrophy caused by partial bladder outlet obstruction (pBOO). MAIN METHODS: Male rats were surgically induced for pBOO. Real-time PCR and western blot were used to examine the levels of mRNA and protein. A phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was used to inhibit the activity of endogenous Akt. KEY FINDINGS: The urinary bladder developed hypertrophy at 2â¯weeks of pBOO. The protein but not mRNA levels of type I collagen and α-smooth muscle actin (αSMA) were increased in pBOO bladder when compared to sham control. The phosphorylation (activation) levels of Akt1 (p-Ser473), mammalian target of rapamycin (mTOR), p70S6 kinase (p70S6K), and 4E-BP1 were also increased in pBOO bladder. LY294002 treatment reduced the phosphorylation levels of Akt1 and 4E-BP1, and the protein levels of type I collagen and αSMA in pBOO bladder. The mRNA and protein levels of proliferating cell nuclear antigen (PCNA) were increased in pBOO bladder, and PCNA up-regulation occurred in urothelial not muscular layer. LY294002 treatment had no effect on the mRNA and protein levels of PCNA in pBOO bladder. LY294002 treatment partially reduced the bladder weight caused by pBOO. SIGNIFICANCE: pBOO-induced urinary bladder hypertrophy is attributable to fibrosis, smooth muscle cellular hypertrophy, and urothelium cell hyper-proliferation. Akt1-mediated protein synthesis in pBOO bladder contributes to type I collagen and αSMA but not PCNA up-regulation. Target of Akt1 is necessary but not sufficient in treatment of urinary bladder hypertrophy following pBOO.
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Proteínas Proto-Oncogénicas c-akt/biosíntesis , Proteínas Proto-Oncogénicas c-akt/genética , Vejiga Urinaria/patología , Animales , Vías Biosintéticas/genética , Cromonas/farmacología , Inhibidores Enzimáticos , Fibrosis , Hipertrofia , Masculino , Morfolinas/farmacología , Tamaño de los Órganos/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Obstrucción del Cuello de la Vejiga Urinaria/patología , Urotelio/patologíaRESUMEN
Although recent studies reveal that activation of the metabolic and Ca2+ sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM) contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s) is(are) affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC). A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca2+- and GTPγS-induced contractions in ß-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca2+]i), inhibited a stimulus-induced increase in [Ca2+]i, and inhibited a cyclopiazonic acid (CPA)-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca2+]i. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca2+ mobilization pathway, possibly involving CPA-dependent Ca2+ entry via an AMPK-independent pathway. That MET activated AMPK without causing arterial relaxation suggests that AMPK activation does not necessarily cause VSM relaxation.
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Metabolic stress diminishes smooth muscle contractile strength by a poorly defined mechanism. To test the hypothesis that metabolic stress activates a compensatory cell signaling program to reversibly downregulate contraction, arterial rings and bladder muscle strips in vitro were deprived of O2 and glucose for 30 and 60 min ("starvation") to induce metabolic stress, and the phosphorylation status of proteins involved in regulation of contraction and metabolic stress were assessed in tissues under basal and stimulated conditions. A 15-30 min recovery period (O2 and glucose repletion) tested whether changes induced by starvation were reversible. Starvation decreased basal phosphorylation of myosin regulatory light chain (MLC-pS19) and of the rho kinase (ROCK) downstream substrates cofilin (cofilin-pS3) and myosin phosphatase targeting subunit MYPT1 (MYPT1-pT696 and MYPT1-pT853), and abolished the ability of contractile stimuli to cause a strong, sustained contraction. Starvation increased basal phosphorylation of AMPK (AMPK-pT172) and 3 downstream AMPK substrates, acetyl-CoA carboxylase (ACC-pS79), rhoA (rhoA-pS188), and phospholamban (PLB-pS16). Increases in rhoA-pS188 and PLB-pS16 would be expected to inhibit contraction. Recovery restored basal AMPK-pT172 and MLC-pS19 to control levels, and restored contraction. In AMPKα2 deficient mice (AMPK[Formula: see text]), the basal level of AMPK-pT172 was reduced by 50%, and MLC-pS19 was elevated by 50%, but AMPK[Formula: see text] did not prevent starvation-induced contraction inhibition nor enhance recovery from starvation. These results indicate that constitutive AMPK activity participates in constitutive regulation of contractile proteins, and suggest that AMPK activation is necessary, but may not be sufficient, to cause smooth muscle contraction inhibition during metabolic stress.
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Background: Many strategies have been utilized to treat traumatic shock via improved oxygen delivery (DO2), while fewer have been used to in an attempt to reduce oxygen demand (VO2). The cellular energy sensor 5' adenosine monophosphate-activated protein kinase (AMPK) has the potential to modulate both whole-body DO2 and VO2. Therefore, we determined the effect of the AMPK activator AICAR (5-aminoimidazole-4-carboxamide 1-ß-D-ribonucleoside) given acutely or chronically on key metabolites, hemodynamics, and oxygen consumption/delivery before and during hemorrhage in anesthetized male rabbits. Methods: Chronically treated animals received AICAR (40 mg/kg/day, IV) for 10 days prior to hemorrhage, while rabbits in the acute study were infused with AICAR (7.5 mg/kg bolus, 2 mg/kg/min infusion) or vehicle (0.3 ml/kg saline bolus, 0.03 ml/kg/min infusion) IV for 2 h prior to severe hemorrhage. Both acutely and chronically treated animals were sedated (ketamine/xylazine cocktail) the morning of the terminal experiment and surgically prepared for hemorrhage, including the implantation of arterial and venous catheters (for blood removal/sampling and drug/vehicle administration) and thoracotomy for implantation of transit-time flow transducers (for cardiac output determination). Results: AICAR given acutely lowered arterial blood glucose and increased blood lactate levels before hemorrhage, and abolished the well-documented hemorrhage-induced hyperglycemia seen in vehicle treated animals. Animals given AICAR chronically had blunted hemorrhage-induced hyperglycemia without prior baseline changes. Chronically treated AICAR animals showed significantly lower lactate levels during hemorrhage. Rabbits receiving AICAR both acutely and chronically experienced similar falls in mean arterial pressure, cardiac output and hence DO2 to their vehicle counterparts throughout the hemorrhage period. However, rabbits treated either acutely or chronically with AICAR accumulated lower oxygen deficits and debt during hemorrhage compared to vehicle-infused controls. Conclusions: The oxygen debt data suggest that AMPK activation could decrease trauma associated morbidity and mortality, perhaps by mechanisms related to increased glucose utilization. Additional studies are needed to investigate the effects of AICAR and associated mechanisms of action when given during resuscitation from hemorrhage.
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Biological soft tissues are viscoelastic because they display time-independent pseudoelasticity and time-dependent viscosity. However, there is evidence that the bladder may also display plasticity, defined as an increase in strain that is unrecoverable unless work is done by the muscle. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether rabbit detrusor smooth muscle (rDSM) is best described as viscoelastic or viscoelastic plastic. Using sequential ramp loading and unloading cycles, stress-strain and stiffness-stress analyses revealed that rDSM displayed reversible viscoelasticity, and that the viscous component was responsible for establishing a high stiffness at low stresses that increased only modestly with increasing stress compared with the large increase produced when the viscosity was absent and only pseudoelasticity governed tissue behavior. The study also revealed that rDSM underwent softening correlating with plastic deformation and creep that was reversed slowly when tissues were incubated in a Ca2+-containing solution. Together, the data support a model of DSM as a viscoelastic-plastic material, with the plasticity resulting from motor protein activation. This model explains the mechanism of intrinsic bladder compliance as "slipping" cross bridges, predicts that wall tension is dependent not only on vesicle pressure and radius but also on actomyosin cross-bridge activity, and identifies a novel molecular target for compliance regulation, both physiologically and therapeutically.
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Actomiosina/metabolismo , Contracción Muscular , Músculo Liso/enzimología , Vejiga Urinaria/enzimología , Quinasas Asociadas a rho/metabolismo , Animales , Fenómenos Biomecánicos , Adaptabilidad , Masculino , Modelos Biológicos , Conejos , Transducción de Señal , Estrés Mecánico , Factores de Tiempo , ViscosidadRESUMEN
Teaching large numbers of students can be a challenge for both teachers and students. Implementing new teaching strategies may be 1 way to address the problem. This article presents the impact of using Gagne's 9 events of instruction on student learning and course evaluations over a 3-semester period. Student evaluations indicated enhanced teacher mastery, effectiveness, and enthusiasm. Overall student final grades increased.
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Actitud del Personal de Salud , Curriculum , Bachillerato en Enfermería/organización & administración , Evaluación Educacional/estadística & datos numéricos , Aprendizaje , Estudiantes de Enfermería/psicología , Enseñanza/métodos , Humanos , Investigación en Educación de Enfermería , Investigación en Evaluación de Enfermería , Investigación Metodológica en EnfermeríaRESUMEN
In rabbit bladder wall (detrusor) muscle, the degree of tone induced during physiological filling (filling tone) is the sum of adjustable preload tension and autonomous contractile tension. The present study was designed to determine whether the level of filling tone is dependent on detrusor muscle length. Maximum active tension induced by KCl was parabolic in relation to length [tension increased from 70% to 100% of a reference length (L(ref)) and decreased at longer muscle lengths]. Filling tone, however, increased in a linear fashion from 70% to 120% L(ref). In the presence of ibuprofen to abolish autonomous contraction and retain adjustable preload tension, tension was reduced in strength but remained linearly dependent on length from 70% to 120% L(ref). In the absence of autonomous contraction, stretching detrusor muscle from 80% to 120% L(ref) still caused an increase in tone during PGE(2)-induced rhythmic contraction, suggesting that muscle stretch caused increases in detrusor muscle contractile sensitivity rather than in prostaglandin release. In the absence of autonomous contraction, the degree of adjustable preload tension and myosin phosphorylation increased when detrusor was stretched from 80% to 120% L(ref), but also displayed length-hysteresis, indicating that detrusor muscle senses preload rather than muscle length. Together, these data support the hypothesis that detrusor muscle acts as a preload tension sensor. Because detrusor muscle is in-series with neuronal mechanosensors responsible for urinary urgency, a more thorough understanding of detrusor muscle filling tone may reveal unique targets for therapeutic intervention of contractile disorders such as overactive bladder.
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Tono Muscular/fisiología , Músculo Liso/fisiología , Cadenas Ligeras de Miosina/metabolismo , Vejiga Urinaria/fisiología , Animales , Dinoprostona/farmacología , Femenino , Técnicas In Vitro , Masculino , Modelos Animales , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Tono Muscular/efectos de los fármacos , Músculo Liso/anatomía & histología , Fosforilación , Cloruro de Potasio/farmacología , ConejosRESUMEN
K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, ß-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 µM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in ß-escin permeabilized tissue. GF-109203X at 1 µM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 µM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and ß-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that ß-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Calcio/farmacología , Fármacos Cardiovasculares/farmacología , Inhibidores Enzimáticos/farmacología , Escina/farmacología , Arteria Femoral/enzimología , Indoles/farmacología , Maleimidas/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Octoxinol/farmacología , Tensoactivos/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Técnicas de Cultivo de Órganos , Permeabilidad , Cloruro de Potasio/metabolismo , Cloruro de Potasio/farmacología , Conejos , Quinasas Asociadas a rho/metabolismoRESUMEN
INTRODUCTION: The purpose of this investigation was to determine if prostaglandin E2(PGE2) is produced by rabbit detrusor free of urothelium and demonstrate that PGE2 is responsible for the generation of spontaneous rhythmic contraction (SRC). METHODS: A bioassay was performed in which contraction frequency in strips of rabbit detrusor was compared before and after addition of superfusate from incubating sections of rabbit detrusor. Specificity was determined by testing the effects of SC-51089, a PGE2(EP1) antagonist. Effects on development of tension were determined in artery segments after treatment with increasing doses of PGE2, PGF2α, and TXA2, and a section of femoral artery was used as a negative control. Confirmation of PGE2 production was then determined using EIA kits. RESULTS: Increased rhythmic frequency was identified after superfusate from a section of rabbit detrusor free of urothelium was added to strips of detrusor from the same animal. Additional experiments demonstrated that rhythmic frequency generated after treatment with PGE2 was significantly reduced after treatment with SC-51089. In artery smooth muscle, prostaglandin dose response experiments demonstrated that only TXA2 induced contraction at physiologic doses (<10â»7M). As a negative control, subsequent treatment of a section of femoral artery with detrusor superfusate failed to increase tension, confirming a lack of TXA2 production. EIA confirmed that PGE2 production increased by 4.8-fold in strips of detrusor free of urothelium after 15 minutes of incubation and that this production was blocked by ibuprofen and a COX-1 inhibitor. CONCLUSIONS: Rabbit detrusor produces PGE2 which is the most likely mediator of SRC.
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Dinoprostona/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Animales , Dinoprostona/metabolismo , Músculo Liso/metabolismo , Músculo Liso/fisiología , Conejos , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/fisiologíaRESUMEN
Unlike the static length-tension curve of striated muscle, airway and urinary bladder smooth muscles display a dynamic length-tension curve. Much less is known about the plasticity of the length-tension curve of vascular smooth muscle. The present study demonstrates that there were significant increases of â¼15% in the phasic phase and â¼10% in the tonic phase of a third KCl-induced contraction of a rabbit femoral artery ring relative to the first contraction after a 20% decrease in length from an optimal muscle length (L(0)) to 0.8-fold L(0). Typically, three repeated contractions were necessary for full length adaptation to occur. The tonic phase of a third KCl-induced contraction was increased by â¼50% after the release of tissues from 1.25-fold to 0.75-fold L(o). The mechanism for this phenomenon did not appear to lie in thick filament regulation because there was no increase in myosin light chain (MLC) phosphorylation to support the increase in tension nor was length adaptation abolished when Ca(2+) entry was limited by nifedipine and when Rho kinase (ROCK) was blocked by H-1152. However, length adaptation of both the phasic and tonic phases was abolished when actin polymerization was inhibited through blockade of the plus end of actin by cytochalasin-D. Interestingly, inhibition of actin polymerization when G-actin monomers were sequestered by latrunculin-B increased the phasic phase and had no effect on the tonic phase of contraction during length adaptation. These data suggest that for a given level of cytosolic free Ca(2+), active tension in the femoral artery can be sensitized not only by regulation of MLC phosphatase via ROCK and protein kinase C, as has been reported by others, but also by a nonmyosin regulatory mechanism involving actin polymerization. Dysregulation of this form of active tension modulation may provide insight into alterations of large artery stiffness in hypertension.
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Citocalasina D/farmacología , Arteria Femoral/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Actinas/efectos de los fármacos , Actinas/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Contracción Muscular/fisiología , Tono Muscular/efectos de los fármacos , Tono Muscular/fisiología , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina/metabolismo , Cadenas Ligeras de Miosina/fisiología , Nifedipino/farmacología , Fosforilación , Cloruro de Potasio/farmacología , Conejos , Tiazolidinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
This study examined the role of ethnicity in untrained observers' ratings of videotaped mother-child interactions. Participants were Black, White, and Latino undergraduates (N = 109), who rated videotapes of 4 Black, 4 White, and 4 Latino mother-child dyads. Overall, participants of different ethnicities showed more similarities than differences in their ratings of parent-child behavior. There was, however, evidence that participant ethnicity and parent-child ethnicity interacted for ratings of child defiance/negative emotion. Black and White participants differed in their ratings of Black and White children's defiance/negative emotion, with members of each ethnic group favoring children of their own ethnic group. Intergroup contact appeared to play a role in ratings of parent behavior among Black observers. Black observers who reported low intergroup contact tended to rate Black mothers high on strictness and low on permissiveness. More research is needed to better understand the role of ethnicity in observers' ratings of parent and child behavior.
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Relaciones Madre-Hijo , Variaciones Dependientes del Observador , Grupos Raciales , Adolescente , Niño , Conducta Infantil , Cultura , Femenino , Humanos , Masculino , Conducta Materna , Prejuicio , Factores Socioeconómicos , Estereotipo , Grabación en Video , Adulto JovenRESUMEN
In vascular smooth muscle, KCl not only elevates intracellular free Ca(2+) ([Ca(2+)](i)), myosin light chain kinase activity and tension (T), but also can inhibit myosin light chain phosphatase activity by activation of rhoA kinase (ROCK), resulting in Ca(2+) sensitization (increased T/[Ca(2+)](i) ratio). Precisely how KCl causes ROCK-dependent Ca(2+) sensitization remains to be determined. Using Fura-2-loaded isometric rings of rabbit artery, we found that the Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, bromoenol lactone (BEL), reduced the KCl-induced tonic but not early phasic phase of T and potentiated [Ca(2+)](i), reducing Ca(2+) sensitization. The PKC inhibitor, GF-109203X (> or =3 microM) and the pseudo-substrate inhibitor of PKCzeta produced a response similar to BEL. BEL reduced basal and KCl-stimulated myosin phosphatase phosphorylation. Whereas BEL and H-1152 produced strong inhibition of KCl-induced tonic T (approximately 50%), H-1152 did not induce additional inhibition of tissues already inhibited by BEL, suggesting that iPLA(2) links KCl stimulation with ROCK activation. The cPLA(2) inhibitor, pyrrolidine-1, inhibited KCl-induced tonic increases in [Ca(2+)](i) but not T, whereas the inhibitor of 20-HETE production, HET0016, acted like the ROCK inhibitor H-1152 by causing Ca(2+) desensitization. These data support a model in which iPLA(2) activity regulates Ca(2+) sensitivity.
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Calcio/metabolismo , Músculo Liso Vascular/enzimología , Fosfolipasas A2 Calcio-Independiente/metabolismo , Cloruro de Potasio/farmacología , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Animales , Ácido Araquidónico/antagonistas & inhibidores , Cinética , Contracción Muscular/efectos de los fármacos , Tono Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Naftalenos/farmacología , Fenilefrina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Pironas/farmacología , Pirrolidinas/farmacología , Conejos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
In strips of rabbit bladder free of urothelium, the beta-adrenoceptor agonist, isoproterenol, significantly reduced basal detrusor smooth muscle tone and inhibited contractions produced by low concentrations of the muscarinic receptor agonist, carbachol. During a carbachol concentration-response curve, instead of inhibiting, isoproterenol strengthened contractions produced by high carbachol concentrations. Thus, the carbachol concentration-response curve was shifted by isoproterenol from a shallow, graded relationship, to a steep, switch-like relationship. The tyrosine kinase inhibitor, genistein, inhibited carbachol-induced contractions only in the presence of isoproterenol. Contraction produced by a single high carbachol concentration (1 microM) displayed 1 fast and 1 slow peak. In the presence of isoproterenol, the slow peak was not strengthened, but was delayed, and U-0126 (mitogen-activated protein kinase kinase inhibitor) selectively inhibited this delay concomitantly with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. Isoproterenol reduced ERK phosphorylation only in the absence of carbachol. These data support the concept that, by inhibiting weak contractions, potentiating strong contractions, and producing a more switch-like concentration-response curve, beta-adrenoceptor stimulation enhanced the effectiveness of muscarinic receptor-induced detrusor smooth muscle contraction. Moreover, beta-adrenoceptor stimulation changed the cellular mechanism by which carbachol produced contraction. The potential significance of multi-receptor and multi-cell crosstalk is discussed.
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Carbacol/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Receptores Adrenérgicos/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Técnicas In Vitro , Isoproterenol/farmacología , Proteínas de Microfilamentos/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Norepinefrina/farmacología , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Conejos , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Interstitial cells of Cajal (ICCs) have been identified as pacemaker cells in the upper urinary tract and urethra, but the role of ICCs in the bladder remains to be determined. We tested the hypotheses that ICCs express cyclooxygenase (COX), and that COX products (prostaglandins), are the cause of spontaneous rhythmic contraction (SRC) of isolated strips of rabbit bladder free of urothelium. SRC was abolished by 10 microM indomethacin and ibuprofen (non-selective COX inhibitors). SRC was concentration-dependently inhibited by selective COX-1 (SC-560 and FR-122047) and COX-2 inhibitors (NS-398 and LM-1685), and by SC-51089, a selective antagonist for the PGE-2 receptor (EP) and ICI-192,605 and SQ-29,548, selective antagonists for thromboxane receptors (TP). The partial agonist/antagonist of the PGF-2alpha receptor (FP), AL-8810, inhibited SRC by approximately 50%. Maximum inhibition was approximately 90% by SC-51089, approximately 80-85% by the COX inhibitors and approximately 70% by TP receptor antagonists. In the presence of ibuprofen to abolish SRC, PGE-2, sulprostone, misoprostol, PGF-2alpha and U-46619 (thromboxane mimetic) caused rhythmic contractions that mimicked SRC. Fluorescence immunohistochemistry coupled with confocal laser scanning microscopy revealed that c-Kit and vimentin co-localized to interstitial cells surrounding detrusor smooth muscle bundles, indicating the presence of extensive ICCs in rabbit bladder. Co-localization of COX-1 and vimentin, and COX-2 and vimentin by ICCs supports the hypothesis that ICCs were the predominant cell type in rabbit bladder expressing both COX isoforms. These data together suggest that ICCs appear to be an important source of prostaglandins that likely play a role in regulation of SRC. Additional studies on prostaglandin-dependent SRC may generate opportunities for the application of novel treatments for disorders leading to overactive bladder.
Asunto(s)
Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Vejiga Urinaria/metabolismo , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Femenino , Hidrazinas/farmacología , Ibuprofeno/farmacología , Indometacina/farmacología , Células Intersticiales de Cajal , Oxazepinas/farmacología , Prostaglandinas/metabolismo , Conejos , Vimentina/metabolismoRESUMEN
The degree of tonic force (F) maintenance induced in vascular smooth muscle upon K(+) depolarization with 110 mM KCl can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a protein kinase C (PKC) isotype may also play a role in causing KCl-induced Ca(2+) sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go-6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF-109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), and a cell-permeable (myristoylated) pseudosubstrate inhibitor of PKCzeta (PI(PKCzeta)) inhibited KCl-induced tonic F. A myristoylated pseudosubstrate inhibitor of PKCalpha/beta that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F and attenuated 30 mM KCl-induced F. Although the ROCK inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)-sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride], reduced basal phosphorylation of myosin light-chain phosphatase-targeting subunit at Thr853 (MYPT1-pT853), 3 and 10 muM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PI(PKCzeta) elevated basal [Ca(2+)](i) (calcium) and potentiated KCl-induced tonic increases in calcium while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca(2+) entry through voltage-operated Ca(2+) channels reduced KCl-induced Ca(2+) sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKCzeta are constitutively active and function in "resting" muscle to regulate the basal levels of MYPT1-pT853 and calcium, respectively. In this model, KCl-induced increases in calcium activate PKCzeta to feed forward and cause additional MYPT1-pT853 above that induced by constitutive ROCK, permitting Ca(2+) sensitization and strong F maintenance. Active PKCzeta also feeds back to attenuate the degree of KCl-induced increases in calcium.
Asunto(s)
Calcio/metabolismo , Retroalimentación/fisiología , Músculo Liso Vascular/metabolismo , Proteína Quinasa C/fisiología , Animales , Células Cultivadas , Retroalimentación/efectos de los fármacos , Indoles/farmacología , Maleimidas/farmacología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Nifedipino/farmacología , Fosforilación , Cloruro de Potasio/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/farmacología , Conejos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Urinary bladder wall muscle (i.e., detrusor smooth muscle; DSM) contracts in response to a quick-stretch, but this response is neither fully characterized, nor completely understood at the subcellular level. Strips of rabbit DSM were quick-stretched (5 ms) and held isometric for 10 s to measure the resulting peak quick-stretch contractile response (PQSR). The ability of selective Ca(2+) channel blockers and kinase inhibitors to alter the PQSR was measured, and the phosphorylation levels of myosin light chain (MLC) and myosin phosphatase targeting regulatory subunit (MYPT1) were recorded. DSM responded to a quick-stretch with a biphasic response consisting of an initial contraction peaking at 0.24+/-0.02-fold the maximum KCl-induced contraction (F(o)) by 1.48+/-0.17 s (PQSR) before falling to a weaker tonic (10 s) level (0.12+/-0.03-fold F(o)). The PQSR was dependent on the rate and degree of muscle stretch, displayed a refractory period, and was converted to a sustained response in the presence of muscarinic receptor stimulation. The PQSR was inhibited by nifedipine, 2-aminoethoxydiphenyl borate (2-APB), 100 microM gadolinium and Y-27632, but not by atropine, 10 microM gadolinium, LOE-908, cyclopiazonic acid, or GF-109203X. Y-27632 and nifedipine abolished the increase in MLC phosphorylation induced by a quick-stretch. Y-27632, but not nifedipine, inhibited basal MYPT1 phosphorylation, and a quick-stretch failed to increase phosphorylation of this rhoA kinase (ROCK) substrate above the basal level. These data support the hypothesis that constitutive ROCK activity is required for a quick-stretch to activate Ca(2+) entry and cause a myogenic contraction of DSM.
Asunto(s)
Calcio/metabolismo , Contracción Muscular/fisiología , Músculo Liso/fisiología , Quinasas Asociadas a rho/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Femenino , Técnicas In Vitro , Contracción Isométrica/fisiología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Cadenas Ligeras de Miosina/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Cloruro de Potasio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Conejos , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Quinasas Asociadas a rho/efectos de los fármacosRESUMEN
Overactive bladder syndrome (OBS) results from disturbances of bladder function. Bladder smooth muscle (detrusor) exhibits spontaneous rhythmic activity (tone) independent of neurogenic control, which is enhanced in patients with OBS. We have now uncovered a prominent role for the bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), in regulating rabbit detrusor smooth muscle tone and contraction. S1P-induced contraction of detrusor muscle was dependent on stretch and intracellular calcium. Although detrusor expresses the S1P receptors S1P1 and S1P2, only S1P2 appeared to be involved in S1P-induced contraction, since SEW2871 (S1P1 agonist) and dihydro-S1P (potent agonist for all S1P receptors except S1P2) were poor contractile agents. In agreement, the S1P2 antagonist JTE013 inhibited S1P-induced contraction. The fast, transient muscle contraction (phasic) mediated by S1P was dependent on phospholipase C (PLC) whereas the slower, sustained contraction (tonic) was not. Surprisingly, the immunosuppressant FTY720-phosphate, an agonist for all S1P receptors except S1P2, had distinct contractile properties and also induced slow, sustained contraction. Thus, FTY720-phosphate and/or S1P may regulate calcium channels in an S1P receptor-independent manner. Collectively, our results demonstrate that S1P may regulate detrusor smooth muscle tone and suggest that dysregulation of complex S1P signaling might contribute to OBS.
Asunto(s)
Inmunosupresores/farmacología , Lisofosfolípidos/farmacología , Tono Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Animales , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Femenino , Clorhidrato de Fingolimod , Immunoblotting , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Conejos , Receptores de Lisoesfingolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/farmacología , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC(50) = 0.5-5 microM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC(50) approximately 80 microM). We found that blebbistatin potently (IC(50) approximately 3 microM) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC(50) approximately 5 microM). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited beta-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC(50) approximately 20 microM). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.