L-748,337

The new radioligand [(3)H]-L 748,337 differentially labels human and rat beta3-adrenoceptors.[Pubmed:24183974]

Eur J Pharmacol. 2013 Nov 15;720(1-3):124-30.

As no suitable radioligand exists for the detection of beta3-adrenoceptors, we have explored the radioligand binding properties of a tritiated version of the selective beta3-adrenoceptor antagonist L 748,337. Kinetic and equilibrium saturation and competition binding experiments were performed with [(3)H]-L 748,337 on membrane fractions of HEK and CHO cells stably transfected with human and rat beta-adrenoceptor subtypes. Based on both association/dissociation kinetic and equilibrium saturation binding studies in transfected HEK cells, [(3)H]-L 748,337 exhibited an affinity of approximately 2 nM for human beta3-adrenoceptors. Competition studies with agonists and subtype-selective antagonists validated its binding to beta3-adrenoceptors. In CHO cells transfected with human beta3-adrenoceptors similar saturable high-affinity of [(3)H]-L 748,337 was observed. While some isoprenaline-sensitive [(3)H]-L 748,337 binding was also observed in CHO cells transfected with human beta1- or beta2-adrenoceptors, this was not saturable in a similar concentration range and/or not sensitive to the antagonists propranolol and SR 59,230, indicating that it did not primarily involve beta-adrenoceptors. In CHO cells transfected with rat beta3-adrenoceptors [(3)H]-L 748,337 exhibited a considerably lower affinity than with the human subtype (12-95 nM). Low affinity for the rat beta3-adrenoceptor was also found with unlabelled L 748,337 in rat bladder strip relaxation experiments. We conclude that L 748,337 apparently has lower affinity for the rat than the human beta3-adrenoceptors and that [(3)H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of beta-adrenoceptors. Nevertheless, [(3)H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human beta3-adrenoceptors reported to date.

beta3-adrenergic receptor activity modulates melanoma cell proliferation and survival through nitric oxide signaling.[Pubmed:24599317]

Naunyn Schmiedebergs Arch Pharmacol. 2014 Jun;387(6):533-43.

We have recently shown in B16F10 melanoma cells that blockade of beta3-adrenergic receptors (beta3-ARs) reduces cell proliferation and induces apoptosis, likely through the involvement of nitric oxide (NO) signaling. Here, we tested the hypothesis that the effects of beta3-AR blockade on melanoma cells are mainly mediated by a decrease in the activity of the NO pathway, possibly due to reduced expression of inducible NO synthase (iNOS). B16F10 cells were used. Nitrite production, iNOS expression, cell proliferation, and apoptosis were evaluated. beta3-AR blockade with L-748,337 reduced basal nitrite production, while beta3-AR stimulation with BRL37344 increased it. The effects of beta3-AR blockade were prevented by NOS activation, while the effects of beta3-AR activation were prevented by NOS inhibition. Treatments increasing nitrite production also increased iNOS expression, while treatments decreasing nitrite production reduced iNOS expression. Among the different NOS isoforms, experiments using L-748,337 or BRL37344 with activators or inhibitors targeting specific NOS isoforms demonstrated a prominent role of iNOS in nitrite production. beta3-AR blockade decreased cell proliferation and induced apoptosis, while beta3-AR activation had the opposite effects. The effects of beta3-AR blockade/activation were prevented by iNOS activation/inhibition, respectively. Taken together, these results demonstrate that iNOS-produced NO is a downstream effector of beta3-ARs and that the beneficial effects of beta3-AR blockade on melanoma B16F10 cell proliferation and apoptosis are functionally linked to reduced iNOS expression and NO production. Although it is difficult to extrapolate these data to the clinical setting, the targeted inhibition of the beta3-AR-NO axis may offer a new therapeutic perspective to treat melanomas.

The function of alpha- and beta-adrenoceptors of the saphenous artery in caveolin-1 knockout and wild-type mice.[Pubmed:17179950]

Br J Pharmacol. 2007 Feb;150(3):261-70.

BACKGROUND AND PURPOSE: Adrenoceptors can associate with cardiac caveolae. To investigate the function of vascular caveolae, adrenoceptor-mediated effects were compared in the saphenous artery of caveolin-1 knockout (cav-1KO) and wild-type (WT) mice. EXPERIMENTAL APPROACH: Electronmicroscopy was used to detect caveolae. Real-Time quantitative PCR was used for adrenoceptor subtypes. Catecholamine-evoked contractions and relaxations were studied in arterial segments. KEY RESULTS: Caveolae were found in arterial smooth muscle from WT but not from cav-1KO mice. Arterial mRNA levels for the adrenoceptors alpha1A, alpha1B, alpha1D, beta1, beta2 and beta3 were similar in cav-1KO and WT. (-)-Noradrenaline contracted cav-1KO (-log EC50M=7.1) and WT (-log EC50M=7.3) arteries through prazosin-sensitive receptors. Maximum (-)-noradrenaline-evoked contractions were greater in cav-1KO than WT arteries. (-)-Isoprenaline relaxed WT arteries (-log EC50M=7.3) more potently than cav-1KO arteries (-log EC50M=6.8); the effects were antagonized partially and similarly by the beta2-selective antagonist ICI118551 (50 nM). The (-)-isoprenaline-evoked relaxation was partially antagonized by the beta1-adrenoceptor-selective antagonist CGP20712 (300 nM) in WT but not cav-1KO arteries. The beta3-adrenoceptor-selective antagonist L748337 (100 nM) partially antagonized the relaxant effects of (-)-isoprenaline in cav-1KO but not in WT arteries. BRL37344 partially relaxed arteries through beta3-adrenoceptors in cav-1KO but not WT. The relaxant effects of BRL37344 were decreased by the NO synthase inhibitor OmegaL-nitroarginine. CONCLUSIONS AND IMPLICATIONS: The function of arterial alpha1- and beta2-adrenoceptors is similar in cav-1KO and WT mice. beta1-adrenoceptor-mediated relaxation in WT is lost in cav-1KO and replaced by the appearance of beta3-adrenoceptors.

Potent and selective human beta(3)-adrenergic receptor antagonists.[Pubmed:10411574]

J Pharmacol Exp Ther. 1999 Aug;290(2):649-55.

Although the functional presence of beta(3)-adrenergic receptors (beta(3)-AR) in rodents is well established, its significance in human adipose tissue has been controversial. One of the issues confounding the experimental data has been the lack of potent and selective human beta(3)-AR ligands analogous to the rodent-specific agonist BRL37344. Recently, we described a new class of aryloxypropanolamine beta(3)-AR agonists that potently and selectively activate lipolysis in rhesus isolated adipocytes and stimulate the metabolic rate in rhesus monkeys in vivo. In this article, we describe novel and selective beta(3)-AR antagonists with high affinity for the human receptor. L-748,328 and L-748,337 bind the human cloned beta(3)-AR expressed in Chinese hamster ovary (CHO) cells with an affinity of 3.7 +/- 1.4 and 4.0 +/- 0.4 nM, respectively. They display an affinity of 467 +/- 89 and 390 +/- 154 nM for the human beta(1)-AR. Their selectivity for human beta(3)-AR versus beta(2)-AR is greater than 20-fold (99 +/- 43 nM) and 45-fold (204 +/- 75 nM), respectively. These compounds are competitive antagonists capable of inhibiting the functional activation of agonists in a dose-dependent manner in cells expressing human cloned beta(3)-AR. Moreover, both L-748,328 and L-748,337 inhibit the lipolytic response elicited by the beta(3)-AR agonist L-742,791 in isolated nonhuman primate adipocytes. The aryloxypropanolamine benzenesulfonamide ligands illustrated here and elsewhere demonstrate high-affinity human beta(3)-AR binding. In addition, we describe specific 3'-phenoxy substitutions that transform these compounds from potent agonists into selective antagonists.