Leu8 and Pro8 oxytocin agonism differs across human, macaque, and marmoset vasopressin 1a receptors

Oxytocin (OXT) is an important neuromodulator of social behaviors via activation of both oxytocin receptors (OXTR) and vasopressin (AVP) 1a receptors (AVPR1a). Marmosets are neotropical primates with a modified OXT ligand (Pro8-OXT), and this ligand shows significant coevolution with traits including social monogamy and litter size. Pro8-OXT produces more potent and efficacious responses at primate OXTR and stronger behavioral effects than the consensus mammalian OXT ligand (Leu8-OXT). Here, we tested whether OXT/AVP ligands show differential levels of crosstalk at primate AVPR1a. We measured binding affinities and Ca2+ signaling responses of AVP, Pro8-OXT and Leu8-OXT at human, macaque, and marmoset AVPR1a. We found that AVP binds with higher affinity than OXT across AVPR1a, and marmoset AVPR1a show a 10-fold lower OXT binding affinity compared to human and macaque AVPR1a. Both Leu8-OXT and Pro8-OXT produce a less efficacious response than AVP at human AVPR1a and higher efficacious response than AVP at marmoset AVPR1a. These data suggest that OXT might partially antagonize endogenous human AVPR1a signaling and enhance marmoset AVPR1a signaling. These findings aid in further understanding inconsistencies observed following systemic intranasal administration of OXT and provide important insights into taxon-specific differences in nonapeptide ligand/receptor coevolution and behavior

Agonist-Specific Desensitization of PGE2-Stimulated cAMP Signaling due to upregulated Phosphodiesterase Expression in Human Lung Fibroblasts

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3′,5′-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2- mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family

Lysophosphatidic acid augments fibroblast-mediated contraction of released collagen gels.

Lysophosphatidic acid (LPA) is a glycerophospholipid released from platelets that has multiple biologic effects. The present study evaluated the potential of LPA to modulate tissue repair and remodeling by modifying human lung fibro-blast-mediated contraction of three-dimensional collagen gels. The contraction of native collagen gels caused by human fetal lung fibroblasts was augmented by LPA in a concentration-dependent manner. The estimated median effective concentration was 3 x 10(-7) mol/L, which was well below the concentrations likely released by platelets in tissues. LPA-augmented contraction was not blocked by pertussis toxin or cholera toxin but was inhibited by inhibition of phospholipase C. Neither calcium mobilization nor protein kinase C appeared to play a role. In contrast, the effect of LPA appeared to depend on a kinase inhibited by staurosporine but not by genistein or GF109203X, suggesting a process that depends on phospholipase C and may involve a novel protein kinase. By modulating fibroblast-mediated remodeling, LPA could play a role in the tissue remodeling that characterizes wound repair

Specificity and Mechanisms for Induction of PDE4 Up-Regulation by PGE2 and Related Agents in Lung Fibroblasts

Introduction Prostaglandin E2 (PGE2) is an important modulator of the fibrotic changes that occur in pulmonary fibrosis and COPD. It is important to understand the mechanisms that regulate the initiation and desensitization of lung fibroblast responses to PGE2. Our previous studies (Am J Respir Crit Care Med 191:A4945, 2015) showed that pretreating lung fibroblasts with PGE2 induced a desensitization of PGE2 stimulation of cyclic AMP (cAMP) accumulation that could be reversed by the phosphodiesterase 4 (PDE4) inhibitor roflumilast. PGE2 did not reduce the cAMP response to the beta-adrenergic receptor agonist isoproterenol (Iso), and Iso pretreatment reduced the response to Iso but not to PGE2. The current study more directly examined the specificity and mechanisms of PGE2 desensitization with direct assays of PDE enzyme activity. Methods Human fetal lung (HFL-1) fibroblasts were incubated with or without 100 nM PGE2, 10 uM Iso, or 30 uM forskolin (Fsk) for 24 hr. Cells were washed to remove pretreatment drug and then lysed for PDE4 enzyme activity assays with cell lysates. Results Pretreatment of cells with PGE2 induced a 2- to 3-fold increase in PDE enzyme activity in cell lysates, and this increased cAMP breakdown was largely reduced by including the PDE4 inhibitor roflumilast. With 24-hr pretreatments, half-maximal increases in PDE activity occurred between 1 and 10 nM PGE2. With 100 nM PGE2 pretreatments, half-maximal increases occurred between 3 and 6 hrs. In contrast to PGE2 pretreatment, pretreatment with Iso induced little or no increase in PDE activity. Although Iso did not mimic the PGE2-induced increase in PDE activity, pretreatment with the direct adenylyl cyclase activator Fsk did induce an increase in PDE activity. The mRNA synthesis inhibitor actinomycin D (2 ug/mL) and the protein synthesis inhibitor cycloheximide (5 uM) nearly completely eliminated the PGE2-induced increase in PDE activity. Conclusions Pretreatment of HFL-1 fibroblasts with PGE2, but not with Iso, induces an increase in PDE enzyme activity that is inhibited by roflumilast, implicating PDE4 as the up-regulated isozyme. The ability of receptor-independent elevation of cAMP with Fsk to up-regulate PDE activity implicates cAMP as the likely mediator of the PDE up-regulation; the failure of cAMP elevation by Iso to induce the same response is likely related to differential localization and signaling by PGE receptors vs. beta-2 adrenergic receptors (Am J Physiol - Cell Physiol 298:L819, 2010). The inhibition by actinomycin D and cycloheximide suggests that transcriptional and translational processes mediate an up-regulation of PDE enzyme expression. These adaptive changes in PDE activity due to cAMP elevation will be important considerations for future studies to explore either PGE- or PDE-targeted therapies for fibrotic diseases of the lung

Regulatory Properties of ␣ 1B -Adrenergic Receptors Defective in Coupling to Phosphoinositide Hydrolysis

ABSTRACT Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for ␣ 1B -adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and downregulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The ⌬12 and ⌬5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (⌬[246 -261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by ␣ 1B -adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes