Mechanisms Underlying Activation of ??-Adrenergic Receptor-Induced Trafficking of AQP5 in Rat Parotid Acinar Cells under Isotonic or Hypotonic Conditions.
ABSTRACT: Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of ??-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the ?1A-AR subtype, but not the ?1B- and ?1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La(3+), implying the participation of store-operated Ca(2+) channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La(3+), suggesting the involvement of extracellular Ca(2+) entry. Thus, ?1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca(2+)/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca(2+) entry.
Project description:Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown.Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo.Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro.These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.
Project description:Aquaporins (AQPs) are important mediators of water permeability and are closely associated with tumor cell proliferation, migration, angiogenesis and chemoresistance. Moreover, the chemosensitivity of tumor cells to cisplatin (CDDP) is potentially affected by osmotic pressure. The present study was undertaken to determine whether hyperosmosis regulates ovarian cancer cell sensitivity to CDDP in vitro and to explore whether this is associated with AQP expression. The hyperosmotic stress was induced by D-sorbitol. 3AO ovarian cancer cells were treated with different concentrations of hypertonic medium and/or CDDP for various times, followed by measuring the inhibition rate of cell proliferation using an MTT assay. In addition, AQP expression in response to osmotic pressure and/or CDDP was measured by reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation in response to hypertonic stress was also measured when AQP5 was knocked down by small interfering (si)RNA. 3AO cell proliferation was inhibited by hyperosmotic stress, while the expression of AQP5, but not that of AQP1, AQP3 or AQP9, was increased in a dose- and time-dependent manner in hypertonic sorbitol-containing medium. When AQP5 was silenced by siRNA, cells were susceptible to hypertonic stress. MTT analyses showed that the inhibition of cell proliferation by a low dose of CDDP increased significantly with exposure to a hyperosmotic stimulus, and this effect was reduced when a high dose of CDDP was used. AQP5 expression was induced by a low dose of CDDP, but was reduced by a high dose of CDDP. However, hyperosmosis enhanced AQP5 mRNA expression at every dose of CDDP tested, compared with isotonic medium. With prolonged treatment time, AQP5 expression was reduced by CDDP in hypertonic and isotonic culture medium. Thus, the effects of hyperosmosis on cell sensitivity to CDDP were associated with AQP5 expression. These results suggest that AQP5 expression in ovarian cancer cells is induced by hypertonic medium, and that the sensitivity of ovarian cancer cells to CDDP can be regulated by hyperosmosis associated with AQP5 expression.
Project description:Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with ?1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that ?1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, ?1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between ?1A/B-AR and CXCR4, and inhibited Ca(2+) mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon ?1-AR activation. CXCR4 silencing reduced ?1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca(2+) fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of ?1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit ?1-AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of ?1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating ?1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the ?1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.
Project description:Small cholangiocytes proliferate via activation of calcium (Ca(2+) )-dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate-dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca(2+) -dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an ?(1) -adrenergic agonist stimulating intracellular D-myo-inositol-1,4,5-triphosphate (IP(3) )/Ca(2+) levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP(3) /Ca(2+) -dependent signaling. Phenylephrine stimulated the production of intracellular IP(3) . The Ca(2+) -dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca(2+) -dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca(2+) -dependent activation of NFAT2 and Sp1.Selective stimulation of Ca(2+) -dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins.
Project description:Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²? from intracellular stores to activate stage-specific Ca²?-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²? required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²? signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²? effectors, PKG emerges as a unifying factor to control multiple cellular Ca²? signals essential for malaria parasite development and transmission.
Project description:Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.
Project description:Recent evidence suggests that C-X-C chemokine receptor type 4 (CXCR4) heteromerizes with α1A/B-adrenoceptors (AR) and atypical chemokine receptor 3 (ACKR3) and that CXCR4:α1A/B-AR heteromers are important for α₁-AR function in vascular smooth muscle cells (VSMC). Structural determinants for CXCR4 heteromerization and functional consequences of CXCR4:α1A/B-AR heteromerization in intact arteries, however, remain unknown. Utilizing proximity ligation assays (PLA) to visualize receptor interactions in VSMC, we show that peptide analogs of transmembrane-domain (TM) 2 and TM4 of CXCR4 selectively reduce PLA signals for CXCR4:α1A-AR and CXCR4:ACKR3 interactions, respectively. While both peptides inhibit CXCL12-induced chemotaxis, only the TM2 peptide inhibits phenylephrine-induced Ca(2+)-fluxes, contraction of VSMC and reduces efficacy of phenylephrine to constrict isolated arteries. In a Cre-loxP mouse model to delete CXCR4 in VSMC, we observed 60% knockdown of CXCR4. PLA signals for CXCR4:α1A/B-AR and CXCR4:ACKR3 interactions in VSMC, however, remained constant. Our observations point towards TM2/4 of CXCR4 as possible contact sites for heteromerization and suggest that TM-derived peptide analogs permit selective targeting of CXCR4 heteromers. A molecular dynamics simulation of a receptor complex in which the CXCR4 homodimer interacts with α1A-AR via TM2 and with ACKR3 via TM4 is presented. Our findings further imply that CXCR4:α1A-AR heteromers are important for intrinsic α₁-AR function in intact arteries and provide initial and unexpected insights into the regulation of CXCR4 heteromerization in VSMC.
Project description:In ureteric microvessels the antagonistic relationship between Ca(2+) signalling in endothelium and Ca(2+) oscillations in myocytes and pericytes of arterioles and venules involves nitric oxide (NO), but the underlying mechanisms are not well understood. In the present study we investigated the effects of carbachol and NO donor SNAP on Ca(2+) signalling and vasomotor responses of arterioles and venules in intact urteric microvascular network in situ using confocal microscopy. Vasomotor responses of arterioles and venules induced by AVP correlated with the occurrence of Ca(2+) oscillations in the myocytes and pericytes and were not abolished by the removal of Ca(2+) from extracellular fluid. Carbachol-induced rise of intracellular Ca(2+) in endothelium was accompanied by the termination of the Ca(2+) oscillations in myocytes and pericytes. This carbachol-induced inhibitory effect on Ca(2+) oscillations in myocytes and pericytes was reversed by ODQ, an inhibitor of soluble guanylyl cyclase (sGC) and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Ca(2+) oscillations in myocytes and pericytes were also effectively blocked by NO donor SNAP. An Inhibitory effect of SNAP was markedly enhanced by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase-5, and reversed by sGC inhibitor, ODQ and PKG inhibitor, Rp-8-pCPT-cGMPS. The cGMP analogue and selective PKG activator 8pCPT-cGMP also induced inhibition of the AVP-induced Ca(2+) oscillations in myocytes and pericytes. SNAP had no effects on Ca(2+) oscillations induced by caffeine in distributing arcade arterioles. Consequently, we conclude that NO- mediated inhibition of Ca(2+) oscillations in myocytes and pericytes predominantly recruits the cGMP/PKG dependent pathway. The inhibitory effect of NO/cGMP/PKG cascade is associated with suppressed Ca(2+) release from the SR of myocytes and pericytes selectively via the inositol triphosphate receptor (IP3R) channels.
Project description:BACKGROUND:Recently, we provided evidence that ?1-adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls ?1-ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and ?1-ARs is unknown. METHODS AND RESULTS:We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co-immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with ?1A/B/D-AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced ?1B/D-AR:ACKR3, CXCR4:ACKR3, and ?1B/D-AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced ?1B/D-AR:ACKR3 heteromers. Phenylephrine-induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, ?1A/B/D-AR, and CXCR4. While the transmembrane domain 2 peptide interfered with ?1B/D-AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and ?1A/B-AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect ?1b-AR in ?-arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine-induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine-induced constriction of mesenteric arteries. CONCLUSIONS:?1-ARs form hetero-oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for ?1B/D-AR function, and activation of ACKR3 negatively regulates ?1-ARs. G protein-coupled receptor hetero-oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous ?1-ARs function within a network of hetero-oligomeric receptor complexes.
Project description:Hydrogen peroxide (H(2)O(2)) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H(2)O(2) elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H(2)O(2) involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-I?, thereby modulating their activities.Here we hypothesize that H(2)O(2) dilates HCAs through direct oxidation and activation of PKG-I? leading to the opening of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and subsequent smooth muscle hyperpolarization.Flow and H(2)O(2) induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK(Ca) channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H(2)O(2)-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H(2)O(2) elicited dose-dependent, reversible dimerization of PKG-I?, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K(+) current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H(2)O(2) into the bath solution significantly increased the probability of BK(Ca) single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-I? inhibitor DT-2. H(2)O(2) exhibited an attenuated stimulatory effect on BK(Ca) channel open probability in inside-out membrane patches.H(2)O(2) dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-I? and subsequent opening of smooth muscle BK(Ca) channels.