RESUMO
Vertebrate oocytes are naturally arrested at prophase of meiosis I for sustained periods of time before resuming meiosis in a process called oocyte maturation that prepares the egg for fertilization. Members of the constitutively active GPR3/6/12 family of G-protein coupled receptors represent important mediators of meiotic arrest. In the frog oocyte the GPR3/12 homolog GPRx (renamed GPR185) has been shown to sustain meiotic arrest by increasing intracellular cAMP levels through GαSßγ. Here we show that GPRx is enriched at the cell membrane (~80%), recycles through an endosomal compartment at steady state, and loses its ability to signal once trapped intracellularly. Progesterone-mediated oocyte maturation is associated with significant internalization of both endogenous and overexpressed GPRx. Furthermore, a GPRx mutant that does not internalize in response to progesterone is significantly more efficient than wild-type GPRx at blocking oocyte maturation. Collectively our results argue that internalization of the constitutively active GPRx is important to release oocyte meiotic arrest.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Endocitose/fisiologia , Meiose/fisiologia , Modelos Biológicos , Oócitos/crescimento & desenvolvimento , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Animais , Western Blotting , AMP Cíclico/metabolismo , Primers do DNA/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteína Vermelha FluorescenteRESUMO
Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca(2+) signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca(2+) signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca(2+) release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca(2+) influx through L-type Ca(2+) channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca(2+) release, resulting in increased VSM contraction in response to agonist stimulation.
Assuntos
Sinalização do Cálcio , Hipertensão/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Contração Muscular , Proteínas Musculares/biossíntese , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Regulação para Cima , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Hipertensão/patologia , Potenciais da Membrana , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Camundongos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fatores de Transcrição NFATC/metabolismo , Ratos , Transcrição GênicaRESUMO
The TRP gene family encodes primarily cation non-selective, Ca2+ permeant channels that are involved in a dizzying array of sensory mechanisms. Two channels in this large family TRPV5 and TRPV6 are highly Ca2+ selective and are expressed in epithelia where they are important in Ca2+ uptake. TRPV5/6 are constitutively active, yet the mechanisms regulating their activation in native tissue remains elusive. Here we functionally characterize the Xenopus TRPV6 homolog. xTRPV6 is expressed in the oocyte and encodes a channel that is permeant to divalents including Ca2+ , and displays a high permeability to Mg2+ . The oocyte does not exhibit functional TRPV6-like current at rest, showing that the endogenous channel is somehow maintained in an inactive state. We show that endogenous as well as overexpressed xTRPV6 interacts with xTRPC1 and that this interaction inhibits xTRPV6 currents. As such TRPC1 is likely to regulate the activity of TRPV6 under physiological conditions.
Assuntos
Magnésio/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPV/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Mutação , Canais de Cátion TRPC/genética , Canais de Cátion TRPV/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
The activation of vertebrate development at fertilization relies on IP3-dependent Ca²âº release, a pathway that is sensitized during oocyte maturation. This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP3 receptors within ER patches have a higher sensitivity to IP3 than those in the neighboring reticular ER. The lateral diffusion rate of IP3 receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP3 receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP3 receptors through ER remodeling is sufficient to sensitize IP3-dependent Ca²âº release. Mathematical modeling supports this concept of 'geometric sensitization' of IP3 receptors as a population, and argues that it depends on enhanced Ca²âº-dependent cooperativity at sub-threshold IP3 concentrations. This represents a novel mechanism of tuning the sensitivity of IP3 receptors through ER remodeling during meiosis.