Commentary

Structural basis for functional diversity of β₁-, β₂- and β₃-adrenergic receptors

β-Adrenoceptors (β-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of β-AR subtypes in rabbit submandibular gland (SMG). Both β₁- and β₂-ARs, but not β₃-AR, were strongly expressed in rabbit SMG. β₁-AR proteins were widely expressed in acinar and ductal cells whereas β₂-AR proteins were mainly detected in ductal cells. A [³H]-dihydroalprenolol binding assay revealed that β-AR B_max was 186 ± 11.9 fmol/mg protein and K_d was 2.71 ± 0.23 nM. A competitive binding assay with CGP 20712A, a β₁-AR antagonist, indicated that the proportion of β₁-AR and β₂-AR was 71.9% and 28.1%, respectively. Gland perfusion with the β-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective β-AR antagonist propranolol. Pretreatment with β₁- or β₂-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of α-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased α-amylase protein storage in SMG and increased α-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both β₁- and β₂-ARs are expressed in rabbit SMG. β₁-AR is the predominant subtype and may play an important role in regulating saliva and α-amylase secretion.

In this study, we investigated the signal transduction pathway involved in β₃-adrenoceptor-mediated relaxations of guinea pig gastric fundus and duodenum. In the presence of β₁- and β₂-adrenoceptor blockade, the potency (pD₂ value) of catecholamines ((−)-isoprenaline, (−)-noradrenaline and (−)-adrenaline) and β₃-adrenoceptor agonists ((R*, R*)-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (±)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((±)-CGP12177A)) to induce relaxation was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22,536, 100 μM). Catecholamines induced an elevation of cyclic AMP and SQ-22,536 significantly abolished the responses of gastric fundus. However, cyclic AMP levels were unaltered by the β₃-adrenoceptor agonists in gastric fundus and by the five agonists in duodenum. Furthermore, the relaxant responses to catecholamines and to β₃-adrenoceptor agonists were unaffected by the cyclic AMP-dependent protein kinase inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89, 10 μM) in gastric fundus. These results suggest that β₃-adrenoceptor-induced relaxation is mediated through both cyclic AMP-dependent and cyclic AMP-independent pathways in gastric fundus and through a cyclic AMP-independent pathway in duodenum.

The influence of autonomic neurotransmitters on the growth and melanogenesis of cultured uveal melanocytes was studied. Uveal melanocytes were cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (complete medium). The cells were plated into multiple well plates, and various concentrations of adrenergic and cholinergic agents were added to the media (complete medium or various deleted media). After 6 days, the cells were detached for cell counting and melanin measurement and compared to controls. Epinephrine, isoproterenol, salbutamol and metaproterenol (adrenergic agonists that can activate β₂-adrenoceptors) substantially stimulated growth and melanogenesis of cultured uveal melanocytes in cAMP-deleted medium. Methoxamine, clonidine, prenalterol and D-7114 (adrenergic agonists that do not activate β₂-adrenoceptors) showed no effect under similar experimental conditions. Muscarine (a cholinergic agonist) inhibited the growth and melanogenesis of uveal melanocytes in complete medium. It indicates that adrenergic agents (β₂-adrenoceptor agonists) stimulate growth and melanogenesis in uveal melanocytes, while cholinergic agonist has an inhibitory effect. This effect appears to involve the cAMP second messenger system. These studies suggest that homeostasis of the uveal melanocytes may be maintained, in part, by regulating the autonomic nervous system in vivo.

Induction of cell and gland enlargement (growth-in-size) and induction of a group of secretory polypeptides (polypeptides C–G) seem to occur in close relationship in mouse parotid glands stimulated chronically by the nonselective β-adrenergic agonist isoproterenol. To determine whether β₁, β₂, or both subtypes of β-adrenergic receptors are involved in those responses, dose-dependency studies were carried out during a 7-day period of daily stimulations to assess the relative abilities of the selective β-adrenergic agonists dobutamine (β₁) and salbutamol (β₂) to induce polypeptides C–G and growth-in-size. The relative abilities of the selective β-adrenoceptor antagonists atenolol (β₁) and I.C.I. 118.551 (β₂) to interfere with the induction of both responses by chronic treatment with the various β-adrenergic agonists were also studied. Parotid growth-in-size was assessed by evaluating wet weight, whole protein content, and light microscopy histology. The presence of polypeptides C–G was evaluated after SDS–polyacrylamide gel electrophoresis and Coomassie blue staining. Under these experimental conditions, dobutamine was found to be at least one order of magnitude more potent than salbutamol at inducing growth-in-size. Dobutamine was also found to be clearly stronger than salbutamol as an inducer of polypeptides C–G. On the other hand, atenolol was more effective than I.C.I. 118.551 at preventing the induction of polypeptides C–G and growth-in-size by isoproterenol, dobutamine, or salbutamol. Taken together, these results suggest that in mouse parotid glands, polypeptides C–G and growth-in-size are induced preferentially via adrenergic receptors of the β₁-subtype.

• 1.

  1. Experiments were carried out to characterize the [³H]CGP12177 binding sites in the microsomal fraction from the guinea pig taenia caecum.

• 2.

  2. The Scatchard plot in the absence of butoxamine (10⁻⁴ M) or bupranolol (10⁻⁴ M) was concave, suggesting two affinity sites: high and low affinity binding sites.

• 3.

  3. The Scatchard plot in the presence of butoxamine gave a straight line, indicating only the low affinity site.

• 4.

  4. The Scatchard plot in the presence of bupranolol was undetectable.

• 5.

  5. These results suggest that the high affinity binding sites for [³H]CGP12177 correspond to β₂ adrenoceptors in the microsomal fraction from the guinea pig taenia caecum, whereas the low affinity binding sites represent β₃-adrenoceptors.