Valerie J. Horn

Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

RAR and RXR selective ligands cooperatively induce apoptosis and neuronal differentiation in P19 embryonal carcinoma cells.

Retinoids cause differentiation in embryonal carcinoma (EC) cells, thus mimicking events in mammalian development. Here, we show that retinoids also cause apoptosis in PI9 EC cells. Characteristic DNA fragmentation was observed within 36 h after addition of retinoic acid (RA). Synthetic retinoids that are selective for RA receptors (RAR) were also effective in inducing apoptosis, whereas RXR selective ligands were without effect. The combination of RAR and RXR ligands resulted in a synergistic increase in apoptotic cell death. As with apoptosis, neuronal differentiation of P19 cells was synergistic ally induced by the combination of RAR and RXR ligands. Data obtained with an RAR antagonist and with P19 cells carrying a dominant negative RXR indicate that the two processes are receptor mediated. Together, our results indicate that reti‐ noid‐induced apoptosis and neuronal differentiation are closely coupled, and that both RAR and RXR play a role in these processes as active receptors for their respective ligands.—Horn, V., Minucci, S., Ogryzko, V. V., Adamson, E. D., Howard, B. H., Levin, A. A., Ozato, K. RAR and RXR selective ligands cooperatively induce apoptosis and neuronal differentiation in P19 embryonal carcinoma cells. FASEB J. 10, 1071‐1077(1996)

Signaling mechanisms that regulate saliva formation.

The precipitating event in the formation of saliva is the binding of neurotransmitter molecules to cell surface receptor proteins. The principal neurotransmitters involved are acetylcholine and norepinephrine that bind, respectively, to muscarinic-cholinergic, and alpha- and beta-adrenergic receptors. The transduction of the extracellular signal requires an integral membrane protein capable of binding GTP, a G protein, that specifically interacts with the receptor. The components of G protein transduction systems are fairly well studied, but the pathways by which signals are routed are just being recognized. Delineation of such routing pathways is essential to understanding the regulation of saliva formation.

Dispersed salivary gland acinar cell preparations for use in studies of neuroreceptor-coupled secretory events

Publisher Summary This chapter focuses on the utilization of the rat parotid and submandibular glands because relatively less study of other salivary glands has occurred. The use of the term coupling here refers to the signal transduction mechanism linking agonist binding at the receptor site to the physiological response (secretion). Salivary gland acinar cells have two functional signal transduction mechanisms common to most cell types. This chapter present ways to measure the appropriate coupled secretory events in these cells. There are two major types of responses associated with secretion: (1) the release of prepackaged protein and (2) the activation of ion fluxes required for transepithelial water movement to occur. This chapter also describes the dispersed acinar cell preparations currently in usage as well as to provide information on how these preparations may be used in evaluating neurotransmitter coupling to exocrine secretory events.

Evidence against a role for a pertussis toxin-sensitive G protein in Ca2+ mobilization in rat parotid acinar cells

Hormone-induced Ca2+ mobilization in rat parotid acinar cells is reportedly mediated via an as yet uncharacterized G protein. We have studied the sensitivity to pertussis toxin (PTx) of this signal transduction mechanism. When rats were treated with Ptx (1.3-1.5 micrograms per animal) for 72 h, a 41 kDa membrane protein was ADP-ribosylated. This PTx treatment regimen, also, resulted in a more than 80% block of the ability of the muscarinic agonist carbachol to inhibit beta-adrenergic receptor-stimulated parotid adenylyl cyclase activity. However, cytosolic Ca2+ levels, in response to either carbachol or AIF-4, were comparable in cells prepared from both untreated or PTx-treated rats, when incubated either in the absence or presence of extracellular Ca2+. Further, both the sensitivity of the Ca2+ response to carbachol and the ability of the agonist-sensitive intracellular Ca2+ stores to be refilled by extracellular Ca2+ were unaffected by PTx treatment. Parotid membranes also contained three low-molecular-weight GTP-binding proteins (25, 22 and 18 kDa) which were unaffected by PTx. These results show that there is only one detectable substrate in parotid membranes for a PTx-catalyzed ADP-ribosylation and that hormone-induced Ca2+ mobilization events in parotid acinar cells are not mediated via PTx-sensitive components.

Chondronectin interactions with proteoglycan

We have investigated whether proteoglycans are involved in the attachment of embryonic chick chondrocytes to type II collagen. Chondroitin sulfate proteoglycan, when added exogenously, promotes the binding of chondronectin, the chondrocyte attachment factor, to type II collagen substrates and thereby stimulates chondrocyte adhesion. Blockage of endogenous proteoglycan synthesis with beta-xylosides prevents chondronectin-mediated chondrocyte attachment, confirming that proteoglycan is required. The intact proteoglycan must be present since chondroitin sulfate glycosaminoglycans did not promote chondronectin-mediated cell attachment but, rather, inhibited it in a dose-dependent manner. This inhibition, however, could be overcome with excess exogenous proteoglycan. Consequently, chondronectin interacts with proteoglycan and then the complex interacts with the collagen substrate and with the cell surface to promote cell adhesion. Further evidence for a direct interaction of chondronectin with the glycosaminoglycan portion of the proteoglycan is the selective binding of chondronectin to dextran-Sepharose, dextran having been shown to inhibit attachment to an extent similar to that of chondroitin sulfate.

Response of rat salivary glands to mastication of pelleted vitamin A-deficient diet

Interpretation of previous studies of the effects of hypovitaminosis. A on salivary glands is confounded by the atrophic effects of liquid or powdered diets. The purpose of this study was to reevaluate the effects of vitamin. A deficiency on the morphology and function of rat salivary glands using a pelleted diet that promotes physiological levels of masticatory stimulation. Profound vitamin A deficiency resulted in a marked decrease in stimulated parotid secretion. Histological evaluation demonstrated the development of squamous metaplasia in the ducts of parotid, submandibular and sublingual salivary glands; however, atrophy was observed only in serious salivary glands. In the parotid gland the degree of atrophy corresponded to the decrease in stimulated secretion. Mild hypovitaminosis A (before the development of squamous metaplasia in ducts) was associated with distinctly different effects. The parotid gland was moderately enlarged. There was also a significant increase in stimulated secretion, which was not explained by changes in gland size, muscarinic receptor number or affinity, or receptor-mediated calcium signalling.

Evidence for the convergence of β-adrenergic and muscarinic signalling systems at a post-receptor site

The β‐adrenergic agonist isoproterenol stimulates inositol trisphosphate (IP3) formation and cytosolic Ca2+ ([Ca2+]i) mobilization in rat parotid acini via a cAMP‐dependent process. Atropine, a muscarinic antagonist, inhibited these isoproterenol responses without affecting isoproterenol‐induced amylase secretion or peak [Ca2+]i and IP3 responses elicited by α1‐adrenergic stimulation with epinephrine. Atropine had no effect on isoproterenol‐induced [Ca2+]i responses in a cell line which lacked muscarinic receptors and did not alter β‐adrenoreceptor ligand binding. These results suggest that the inhibition by atropine results from a post‐receptor effect on cAMP‐mediated stimulation of phosphatidylinositol 4,5 bisphosphate (PIP2) hydrolysis.

Attenuation of inositol trisphosphate generation and cytosolic Ca2+ elevation in dispersed rat parotid acini stimulated simultaneously at muscarinic and α1-adrenergic receptors

We have examined intracellular signalling events, peak cytosolic [Ca2+] and inositol trisphosphate levels, in rat parotid acini simultaneously stimulated with two Ca2+ mobilizing agonists, carbachol (muscarinic-cholinergic) and epinephrine (alpha 1-adrenergic). When the agonists were added together, either at sub-maximal (200 nM each, i.e. 400 nM total agonist concentration) or maximal (10 uM each, i.e. 20 uM total) stimulatory concentrations, the resulting elevations in both cytosolic [Ca2+] and inositol trisphosphate levels were not greater than those achieved when each agonist was added individually. However, with 400 nM carbachol these responses were significantly greater than those seen with either 200 nM carbachol or 200 nM carbachol + 200 nM epinephrine. The data indicate that when muscarinic and alpha 1-adrenergic receptors of rat parotid acini are simultaneously stimulated a novel regulatory mechanism is induced, which attenuates inositol trisphosphate generation and, consequently, intracellular Ca2+ release.

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor G proteins interactions

The binding of the non‐selective muscarinic antagonists [3H] quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions. [3H]QNB bound to a homogenous population of muscarinic receptors (Kd . 118±19 pM; B max 572±42 fmol/mg membrane protein. n = 12). The addition of G protein activators AIF− 4 or guanosine‐5′‐O‐(3‐thiotriphospate) (GTPγS) + Mg2+ increased the K d by 77 ± 7% (n=4. P < 0.05) and 83 ± 27% (n=7, P < 0.05), respectively, without a change in the B max or homogeneity of the binding site. GTPγS added without exogenous Mg2+ did not affect [3H] QNB binding requires a muscarinic receptor/G protein interaction.