Expression Of Beta3-adrenoceptors Research Articles

Beta‐3 adrenoceptor expression in the uterosacral ligament in the postmenopausal women with pelvic organ prolapse

β3-adrenoceptor (β3-AR) agonist is known to relax the detrusor muscles. Considering that both pelvic organ prolapse (POP) and overactive-bladder (OAB) are frequently seen in the elderly population, the effect of β3-AR agonist on the pelvic floor tissues should be investigated. As a first step, this pilot, experimental, and descriptive study was designed to investigate the presence of β3-AR expression in the uterosacral-ligaments (USLs) in postmenopausal (PMP) women with POP. After informed consent, PMP women undergoing hysterectomy for POP were recruited for the intraoperative collection of distal USL between 5/2016 and 8/2016. H&E analysis for the USLs and immunohistochemistry (IHC) analysis for β3-AR and α-actin smooth muscle (SM) were carried out. Tissue composition and percentage of β3-AR expression in the USLs were subjectively measured and the staining results were expressed as descriptive analysis. Total 11 USL specimens were collected and nine specimens were included for the final analysis (one specimen: used for antibody concentration adjustment and one specimen: failed proper staining). Under high-power-field, the USLs were composed of SM cells (81.5 ± 7.47%) and connective tissues (16.5 ± 7.9%). On IHC analysis, six out of nine specimens expressed β3-AR with a different level of expression in SM cells in the USLs. The majority of distal USLs were composed of SM cells. β3-ARs are expressed in 67% of the study specimens. Considering that both POP and OAB are frequently seen in the elderly population, the effect of β3-AR agonist on the pelvic floor tissues should be investigated further.

OP.8C.02] NORADRENERGIC RECEPTOR FUNCTION IN HEALTHY AND OBESE PERIVASCULAR ADIPOSE TISSUE

Objective: Healthy perivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating blood pressure, and evidence suggests that sympathetic nervous stimulation of PVAT triggers the release of anti-contractile factors via activation of beta3-adrenoceptors. In obesity there is sympathetic over-activity, which may result in a loss of PVAT function and subsequent hypertension. Therefore we examined beta3-adrenoceptor function in obesity. Design and method: Electrical field stimulation (EFS) profiles of healthy and obese mouse mesenteric arteries (<200um, +/−PVAT) were characterised using wire myography (0.1–30 Hz, 20 V, 0.2ms pulse duration, 4 s train duration). To demonstrate the release of an anti-contractile factor in health, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT denuded vessel. Beta3-adrenoceptor function was investigated using the agonist CL-316,243 (10uM) and antagonist SR59203A (100 nM). The role of the vasodilator nitric oxide (NO) was studied using nitric oxide synthase (NOS) inhibitor L-NMMA (100uM), and NOS activator histamine (100uM). Results: During EFS healthy PVAT elicited an anti-contractile effect; which was lost in obesity. Solution transfer from stimulated healthy exogenous PVAT to a –PVAT vessel significantly reduced contraction, confirming that stimulated PVAT releases a transferable anti-contractile factor. Solution transfer from obese PVAT had no effect on contraction. Inhibition of beta3-adrenoceptors in healthy PVAT using SR59230A significantly reduced the anti-contractile effect, whereas activation of beta3-adrenoceptors in obese PVAT using CL-316,243 did not restore function. Using immunohistochemistry, expression of beta3-adrenoceptors is reduced in obesity. In healthy PVAT, inhibition of NOS using L-NMMA abolished the anti-contractile effect. In obese PVAT, activation of NOS using histamine was able to restore the anti-contractile function. Conclusions: These results demonstrate that in health PVAT releases an anti-contractile factor via activation of beta3-adrenoreceptors, which downstream triggers the release of NO. In obesity, the anti-contractile effect is lost and cannot be restored by beta3-activation, but is restored by activation of NOS. This reveals that in obesity beta3-adrenoreceptors are downregulated, leading to a loss of anti-contractile function, which may contribute to the development of hypertension. Targeting the NOS pathway may restore anti-contractile function, and treat the vascular complications of obesity.

Evaluation of beta3-adrenoceptor-mediated relaxation in intact and endotoxin-treated equine digital veins.

To investigate the functional expression of beta3-adrenoceptors (beta3-ARs) in equine digital veins (EDVs) and to examine whether beta3-AR relaxation was altered in EDVs incubated with endotoxin. Forelimbs obtained from 30 horses. Forelimbs were obtained from horses in an abattoir. Equine digital veins were carefully removed from distal portions of the forelimbs. Rings of dissected EDVs were mounted in 5-mL organ baths to record isometric tension in the presence of various beta3-AR agonists (SR 58611A, ZD 2079, and ZM 215001). In intact EDVs, isoprenaline, SR 58611A, ZD 2079, and ZM 215001 induced concentration-dependent relaxation. Isoprenaline and SR 58611A-induced relaxations were reduced or unaffected by nadolol, respectively. In intact EDVs, SR 58611A-induced relaxation was significantly reduced in the presence of 2 microM ZM 215001 (used as a beta3-AR antagonist). In endothelium-denuded EDVs or intact EDVs in the presence of a nitric oxide synthase inhibitor, isoprenaline and SR 58611A-induced relaxations were significantly decreased. The endothelium-independent relaxation to SR 58611A was significantly inhibited in the presence of ZM 215001. In endotoxin-treated EDV, isoprenaline- and SR 58611A-induced relaxations were significantly reduced. In these conditions, cycloheximide (a protein synthesis inhibitor) and ibuprofen (a cyclooxygenase inhibitor) restored the relaxant response to SR 58611A. Beta3-adrenoceptors are functionally expressed in EDVs. Incubation in the presence of endotoxin, used as an in vitro model of laminitis, induced an alteration of beta-AR-mediated relaxations in EDVs, which could be the consequence of cyclooxygenase induction and subsequent prostanoid production.

Pro-inflammatory cytokines and adipose tissue.

Cytokines appear to be major regulators of adipose tissue metabolism. Therapeutic modulation of cytokine systems offers the possibility of major changes in adipose tissue behaviour. Cytokines within adipose tissue originate from adipocyte, preadipocyte and other cell types. mRNA expression studies show that adipocytes can synthesise both tumour necrosis factor alpha (TNF-alpha) and several interleukins (IL), notably IL-1beta and IL-6. Other adipocyte products with 'immunological' actions include complement system products and macrophage colony-stimulating factor. Cytokine secretion within adipocytes appears similar to that of other cells. There is general agreement that circulating TNF-alpha and IL-6 concentrations are mildly elevated in obesity. Most studies suggest increased TNF-alpha mRNA expression or secretion in vitro in adipose tissue from obese subjects. The factors regulating cytokine release within adipose tissue appear to include usual 'inflammatory' stimuli such as lipopolysaccaride, but also the size of the fat cells per se and catecholamines. There is conflicting data about whether insulin and cortisol regulate TNF-alpha. The effects of cytokines within adipose tissue include some actions that might be characterised as metabolic. TNF-alpha and IL-6 inhibit lipoprotein lipase, and TNF-alpha additionally stimulates hormone-sensitive lipase and induces uncoupling protein expression. TNF-alpha also down regulates insulin-stimulated glucose uptake via effects on glucose transporter 4, insulin receptor autophosphorylation and insulin receptor substrate-1. All these effects will tend to reduce lipid accumulation within adipose tissue. Other effects appear more 'trophic', and include the induction of apoptosis, regulation of cell size and induction of de-differentiation (the latter involving reduced peroxisome proliferator-activated receptor gamma). Cytokines are important stimulators and repressors of other cytokines. In addition, cytokines appear to modulate other regulatory systems. Examples of the latter include effects on leptin secretion (probably stimulation followed by inhibition) and reduction of beta3-adrenoceptor expression. There seems to be no clear agreement as to which cytokines derived from adipose tissue act as remote regulators, i.e. hormones. Leptin, which is structurally a cytokine, is also a hormone. IL-6 appears to be released systemically by adipose tissue, but TNF-alpha is probably not. Both leptin and IL-6 appear to act on the hypothalamus, IL-6 acts on the liver, while leptin may have actions on the pancreas. The importance of the immune system in whole-body energy balance provides a rationale for the links between cytokines and adipose tissue. It seems clear that TNF-alpha is a powerful autocrine and paracrine regulator of adipose tissue. Other cytokines, notably leptin, and possibly IL-6, have lesser actions on adipose tissue. These cytokines act as hormones, reporting the state of adipose tissue stores throughout the body.

The tissue distribution of the human beta3-adrenoceptor studied using a monoclonal antibody: direct evidence of the beta3-adrenoceptor in human adipose tissue, atrium and skeletal muscle.

To develop a monoclonal antibody that recognises an epitope of the native beta3-adrenoceptor expressed on the extracellular surface of human cells and tissues. A high affinity monoclonal antibody, Mab72c, was raised against the human beta3-adrenoceptor expressed on a transfected mammalian cell line. In CHO (Chinese hamster ovary) cells transfected with beta3-adrenoceptor cDNA, antibody labelling was found to be proportional to receptor density measured by the binding of the radiolabelled beta-adrenoceptor antagonist, [125I]-iodocyanopindolol. The use of Mab 72c has demonstrated the expression of the beta3-adrenoceptor in a variety of human tissues, including gall bladder, prostate and colon, where a mRNA signal had been detected previously. This study also provides the first direct demonstration of the expression of beta3-adrenoceptors in human skeletal muscle, atrium and adipose tissue. The development of this antibody represents an important addition to the armentarium of reagents that are available to study the localisation of beta3-adrenoceptors in human tissues.

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle.

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction.

Beta-3 adrenoceptor (beta-3AR) expression in leptin treated Ob/Ob mice

Leptin-deficient Ob/Ob mice are hypometabolic and have reduced fat cell expression of beta-3 adrenoceptors (ARs). To determine whether leptin repletion restores beta-3 AR number, C57BL/6J Ob/Ob mice were given exogenous leptin (5 mg/kg IP daily) for 21 days. Leptin administration reduced body weight from 43.1 ± 3.7 to 34.1 ± 3.7g in Ob/Ob animals but had no effect on weight in wildtype animals. Body weight increased by 12% in Ob/Ob mice receiving saline. Beta-3 AR mRNA concentrations were markedly reduced in Ob/Ob animals at baseline. Leptin increased beta-3 AR mRNA to control levels in Ob/Ob mice, but had no effect in wildtype animals. Adipocyte leptin mRNA was increased by 400% in Ob/Ob mice and did not suppress with exogenous leptin administration, suggesting no direct feedback regulation of leptin synthesis. We speculate that restoration of beta-3 AR expression by repleting leptin may be important in correcting hypometabolism in Ob/Ob animals.

Beta 3-adrenoceptor in guinea pig brown and white adipocytes: low expression and lack of function.

In the guinea pig, cold acclimation induced a conversion of unilocular to multilocular adipocytes in interscapular (IS) and retroperitoneal (RP) fat depots but not in the epididymal (EP) fat pad. The conversion was associated with an increase in mitochondriogenesis and the appearance of the uncoupling protein. The maximal lipolytic responses to norepinephrine and dibutyryl adenosine 3',5'-cyclic monophosphate were decreased in IS cells, unchanged in RP cells, and increased in EP cells, suggesting a site-specific regulation of lipolysis at the postreceptor level. beta 3-Adrenergic agonists were not lipolytic regardless of the depot and the thermal environment of the animal. These agents did not inhibit glucose transport and lipogenesis, as was previously reported for rodents. Cloning and sequencing of the guinea pig beta 3-adrenoceptor gene revealed a slightly higher amino acid sequence similarity with the human than with the rodent beta 3-adrenoceptors. beta 3-Adrenoceptor transcripts were present at a very low level in guinea pig adipocytes, and mRNA levels did not increase to a significant extent after cold acclimation. The guinea pig thus differs from rodents by an absence of beta 3-adrenergic effects and by low beta 3-adrenoceptor expression in brown and white adipose tissues.

Triiodothyronine regulates beta 3-adrenoceptor expression in 3T3-F442A differentiating adipocytes.

The effect of thyroid hormones on the beta 3-adrenoceptor expression was studied in the preadipose 3T3-F442A cell line. As assessed by molecular and pharmacological analyses, triiodothyronine addition to differentiating 3T3-F442A cells caused a 2.3-fold increase in beta 3-adrenoceptor mRNA levels, which was correlated with a parallel induction of beta 3-adrenoceptor number and of beta 3-adrenoceptor coupling to the adenylate cyclase system. Nuclear transcription experiments showed that triiodothyronine did not significantly alter the transcription rate of the beta 3-adrenoceptor gene. By contrast, the hormone increased by 36% the half-life of beta 3-adrenoceptor mRNA. Triiodothyronine exhibited a discrete effect on beta 3-adrenoceptor expression when added to mature 3T3-F442A adipocytes. This study indicates that thyroid hormones exert a differentiation-dependent and post-transcriptional regulation of beta 3-adrenoceptor expression in adipocytes.

Expression of beta3-adrenoceptors with low lipolytic action in human subcutaneous white adipocytes.

Beta3-Adrenoceptors are involved in the control of catecholamine-induced lipolysis in rodent adipose tissues. The expression and function of human beta3-adrenoceptors were investigated in subcutaneous white adipocytes of young healthy women. In these cells, beta3-adrenoceptor mRNAs represent 20% of total amount of beta-adrenoceptor transcripts and less than half of beta1-adrenoceptor transcripts. Among beta3-agonists known to stimulate beta3-adrenoceptor-mediated lipolysis in rodent fat cells, only CGP12177 was able to mediate such activity in human fat cells. In in vitro lipolysis experiments and in situ microdialysis studies, CGP12177 had a 4- to 5-times lower lipolytic efficacy than isoprenaline, a nonselective beta-adrenoceptor agonist. CGP12177-induced lipolysis was antagonized in vitro by bupranolol, a beta-adrenergic antagonist potent on rodent beta3-adrenoceptors but not by nadolol, a beta1- and beta2-adrenoceptor antagonist. The in vitro blockade of isoprenaline-stimulated lipolysis by nadolol showed that the agonist acted solely via beta1- and beta2-adrenoceptors. Isoprenaline and CGP12177 were able to increase the nutritive blood flow suggesting an interaction of these molecules with receptors present in adipose tissue vessels. In conclusion, beta3-adrenoceptors are expressed in human subcutaneous white adipocytes but do not significantly contribute to isoprenaline-induced lipolysis.

Regulation of beta 3-adrenoceptor expression in white fat cells.

Catecholamines (adrenaline and noradrenaline) stimulate adipocyte lipolysis via three beta-adrenoceptor subtypes beta 1, beta 2 and beta 3. beta 3-adrenoceptor-mediated lipolysis varies according to the species. Rodent adipocytes exhibit the strongest response to beta 3 agonists while human fat cells are poorly responsive. The species-related differences can partly be explained by lower beta 3-adrenoceptor mRNA levels in human adipocytes compared to rat adipocytes. Poor coupling efficiency of human adipocyte beta 3-adrenoceptors cannot, however, be ruled out. The regulation of beta 3-adrenoceptor gene expression has been studied in the adipocytes of the murine cell line 3T3-F442A which express high levels of beta 3-adrenoceptors. Insulin and glucocorticoids down-regulate beta 3-adrenoceptor expression through a transcriptional effect. The impairment of beta 3-adrenoceptor gene expression in adipocytes of congenitally obese ob/ob mice could be related to the higher glucocorticoid plasma levels when compared to lean littermates although the direct involvement of glucocorticoids remains to be demonstrated. In the rat and the rabbit, the beta 3-adrenergic responsiveness varies according to the anatomical location of the fat pad. There is a marked decrease in beta 3-adrenergic response in rabbit retroperitoneal fat cells during ageing. cAMP modulates the beta 3-adrenergic response in white adipocytes at different levels. Human beta 3-adrenoceptor expression seems to be up-regulated by cAMP through an interaction with the promoter of the gene. It has been shown in cells transfected with cDNAs for the different beta-adrenoceptors that the beta 3-adrenoceptor is less prone to desensitization than the beta 1 and beta 2-subtypes. This observation is in agreement with the absence of desensitization of the beta 3-adrenoceptor response in isolated rat fat cells. Continuous infusion of noradrenaline for six days into hamsters does not lead to an alteration of the beta-adrenergic response. A similar treatment undertaken in the guinea pig, a species, unlike the hamster, devoid of beta 3-adrenoceptor responsiveness, promoted strong desensitization of the beta-adrenergic response through down-regulation of beta 1- and beta 2-adrenoceptors. From these observations, it could be hypothesized that the beta 3-adrenoceptor, that shows a low affinity for catecholamines, is the "emergency" beta-adrenoceptor which is essential under conditions of strong and sustained sympathetic nervous system activation.