Submandibular Gland Acinar Cells Express Multiple α<sub>1</sub>-Adrenoceptor Subtypes

Bockman, Charles S.; Bruchas, Michael R.; Zeng, Wanyun; O’Connell, Kelly A.; Abel, Peter W.; Scofield, Margaret A.; Dowd, Frank J.

doi:10.1124/jpet.104.066399

Cited by 12 publications

(17 citation statements)

References 18 publications

(20 reference statements)

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“…For example, in a recent study, mRNA for all three ␣ 1 -AR subtypes was detectable in rat submandibular gland cells. However, BMY 7378 detected only a single population of low-affinity binding sites in radioligand binding experiments (Bockman et al, 2004), which is consistent with our findings suggesting that coexpression of ␣ 1B -and ␣ 1D -ARs results in the masking of high-affinity ␣ 1D -AR binding sites. In addition, the affinity of BMY 7378 in inhibiting phenylephrine-mediated contraction was found to be significantly increased in isolated carotid arteries from ␣ 1B -AR knockout mice (Deighan et al, 2005), and phenylephrinestimulated increases in left ventricular-developed pressure were only inhibited by BMY 7378 in ␣ 1A -/␣ 1B -AR double knockout mice (Turnbull et al, 2003).…”

Section: Discussionsupporting

confidence: 81%

Heterodimers of α_1B- and α_1D-Adrenergic Receptors Form a Single Functional Entity

et al. 2005

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Heterologous expression of ␣ 1D -adrenergic receptors (␣ 1D -ARs) in most cell types results in intracellular retention and little or no functionality. We showed previously that heterodimerization with ␣ 1B -ARs promotes surface localization of ␣ 1D -ARs. Here, we report that the ␣ 1B -/␣ 1D -AR interaction has significant effects on the pharmacology and signaling of the receptors, in addition to the effects on trafficking described previously. Upon coexpression of ␣ 1B -ARs and epitope-tagged ␣ 1D -ARs in both human embryonic kidney 293 and DDT 1 MF-2 cells, ␣ 1D -AR binding sites were not detectable with the ␣ 1D -AR selective antagonist 8-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]-8-azaspiro [4,5]decane-7,9-dione (BMY 7378), despite the ability to detect ␣ 1D -AR protein using confocal microscopy, immunoprecipitation, and a luminometer cell-surface assay. However, the ␣ 1B -AR-selective mutant F18A conotoxin showed a striking biphasic inhibition in ␣ 1B /␣ 1D -AR-expressing cells, revealing that ␣ 1D -ARs were expressed but did not bind BMY 7378 with high affinity. Studies of norepinephrine-stimulated inositol phosphate formation showed that maximal responses were greatest in ␣ 1B /␣ 1D -AR-coexpressing cells. Stable coexpression of an uncoupled mutant ␣ 1B -AR (⌬12) with ␣ 1D -ARs resulted in increased responses to norepinephrine. However, Schild plots for inhibition of norepinephrine-stimulated inositol phosphate formation showed a single low-affinity site for BMY 7378. Thus, our findings suggest that ␣ 1B /␣ 1D -AR heterodimers form a single functional entity with enhanced functional activity relative to either subtype alone and a novel pharmacological profile. These data may help to explain why ␣ 1D -ARs are often pharmacologically undetectable in native tissues when they are coexpressed with ␣ 1B -ARs.

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Section: Discussionsupporting

confidence: 81%

Heterodimers of α_1B- and α_1D-Adrenergic Receptors Form a Single Functional Entity

et al. 2005

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“…Similarly, an α 1D -adrenoceptor-selective concentration of BMY 7378 (10 nM) had little effect on phenylephrinestimulated ERK1/2 activation (8% inhibition). In contrast, 30 μM chloroethylclonidine for 12 min, a treatment that we previously showed to inactivate only the α 1B -adrenoceptor subtype in SMG-C10 cells (Bockman et al, 2004), significantly inhibited (p < 0.05 ) phenylephrinestimulated ERK1/2 activation (Fig. 4A).…”

Section: Functional Characterization Of the α 1 -Adrenoceptor Subtypementioning

confidence: 73%

“…Where it was appropriate, cells or gland slices were incubated with a competitive receptor antagonist or an inhibitor for 30 min before agonist addition. In some experiments, cells were pretreated with the irreversible α 1B -adrenoceptor antagonist, chloroethylclonidine, as previously described (Bockman et al, 2004). Briefly, cells were incubated with 30 μM chloroethylclonidine for 12 min at 37 °C.…”

Section: Erk1/2 Assaymentioning

confidence: 99%

“…Recently, Huang et al (2007) reported in cardiomyocytes that α 1 -adrenoceptors activate an anti-apoptotic, cell survival pathway by stimulation of ERK 1/2. We have reported that both the native submandibular gland and an immortalized acinar cell line (SMG-C10) cloned from submandibular glands similarly express both the α 1A -and α 1B -adrenoceptor subtypes (Bockman et al, 2004). Like native acinar cells, SMG-C10 cells are polarized epithelia, express functional adrenergic, muscarinic and purinergic receptors that couple to an elevation in intracellular calcium, and share other morphological characteristics unique to secretory epithelia (Quissell et al, 1997;Liu et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the α1-adrenoceptor subtype activating extracellular signal-regulated kinase in submandibular gland acinar cells

Bruchas

Toews

Bockman

et al. 2008

European Journal of Pharmacology

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Abstractα 1 -Adrenoceptors and extracellular signal-regulated kinases 1 and 2 (ERK1/2) regulate salivary secretion. However, whether α 1 -adrenoceptors couple to ERK1/2 activation and the specific α 1 -adrenoceptor subtypes involved in salivary glands is unknown. Western blotting of ERK1/2 phosphorylation showed phenylephrine activated ERK1/2 by 2-3-fold in submandibular gland slices and 3-4-fold in submandibular acinar (SMG-C10) cells with an EC 50 of 2.7 ± 2 μM. ERK1/2 activation was blocked by either prazosin or HEAT, indicating α 1 -adrenoceptors stimulate ERK1/2 in native glands and SMG-C10 cells. Inhibition of [ 125 I]HEAT binding by 5-methylurapidil (selective for α 1A over α 1B/ α 1D ), but not BMY 7378 (selective for α 1D over α 1A/ α 1B ), was biphasic and bestfit by a two-site binding model with K iH and K iL values for 5-methylurapidil of 0.64 ± 0.3 and 91 ± 7 nM, respectively, in SMG-C10 membranes. From these binding data, we obtained subtype-selective concentrations of 5-methylurapidil to determine the α 1 -adrenoceptor subtype/s activating ERK1/2 in SMG-C10 cells. 5-methylurapidil (20 nM) did not affect phenylephrine-or A-61603-(α 1A -selective agonist) induced ERK1/2 activation; whereas, 30 μM chloroethylclonidine (α 1B -selective antagonist) inhibited ERK1/2 activation by phenylephrine, indicating α 1B -adrenoceptors, but not α 1A -adrenoceptors, activate ERK1/2 in submandibular cells. We also examined α 1 -adrenoceptor location and dependence on cholesterol-rich microdomains for activating ERK1/2. Sucrose density gradient centrifugation showed 71 ± 3% of α 1 -adrenoceptor binding sites were in plasma membranes. Cholesterol-disrupting agents filipin and methyl-β-cyclodextrin inhibited phenylephrine-stimulated ERK1/2. These results show only α 1B -adrenoceptors activate ERK1/2 and suggest subtype-specific ERK1/2 signaling by α 1B -adrenoceptors may be determined by localization to cholesterol-rich microdomains in submandibular cells.

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“…This suggests that the ␣ 1D -AR is indeed expressed in the vasculature and contributes to maintenance of blood pressure-a contention that is also evident from the reduced blood pressure and impaired vasoconstrictor responses to norepeinephrine of ␣ 1D -knockout mice (Tanoue et al, 2002)-but that its expression is masked by the coincident expression of the ␣ 1B -AR. Of course, this interpretation, and the increasingly robust evidence suggesting that endogenously expressed ␣ 1 -ARs heterodimerize, is predicated on more than one ␣ 1 -AR subtype being expressed in a single cell, a contention supported by studies of various cell lines (Esbenshade et al, 1993;Bockman et al, 2004), albeit one that has not yet been definitively demonstrated using a single isolated cell.…”

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confidence: 91%

The α_1D-Adrenergic Receptor: Cinderella or Ugly Stepsister

Finch

Graham

2005

Mol Pharmacol

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This Perspective focuses on the ␣ 1D -adrenergic receptor (AR), the often neglected sibling of the ␣ 1 -AR family. This neglect is due in part to its poor cell-surface expression. However, it has recently been shown that dimerization of the ␣ 1D -AR with either the ␣ 1B -AR or the ␤ 2 -AR increases ␣ 1D -AR cell-surface expression, and in this issue of Molecular Pharmacology, Hague et al. (p. 45) demonstrate that dimerization of the ␣ 1D -AR with the ␣ 1B -AR not only leads to increased cell-surface expression but also results in the formation of a novel functional entity.

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Submandibular Gland Acinar Cells Express Multiple α₁-Adrenoceptor Subtypes

Cited by 12 publications

References 18 publications

Heterodimers of α_1B- and α_1D-Adrenergic Receptors Form a Single Functional Entity

Heterodimers of α_1B- and α_1D-Adrenergic Receptors Form a Single Functional Entity

Characterization of the α1-adrenoceptor subtype activating extracellular signal-regulated kinase in submandibular gland acinar cells

The α_1D-Adrenergic Receptor: Cinderella or Ugly Stepsister

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Submandibular Gland Acinar Cells Express Multiple α1-Adrenoceptor Subtypes

Cited by 12 publications

References 18 publications

Heterodimers of α1B- and α1D-Adrenergic Receptors Form a Single Functional Entity

Heterodimers of α1B- and α1D-Adrenergic Receptors Form a Single Functional Entity

Characterization of the α1-adrenoceptor subtype activating extracellular signal-regulated kinase in submandibular gland acinar cells

The α1D-Adrenergic Receptor: Cinderella or Ugly Stepsister

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Product

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Submandibular Gland Acinar Cells Express Multiple α₁-Adrenoceptor Subtypes

Heterodimers of α_1B- and α_1D-Adrenergic Receptors Form a Single Functional Entity

Heterodimers of α_1B- and α_1D-Adrenergic Receptors Form a Single Functional Entity

The α_1D-Adrenergic Receptor: Cinderella or Ugly Stepsister