Beta Subtypes Research Articles

β-adrenoceptor signaling and its control of cell replication in MDA-MB-231 human breast cancer cells

MDA-MB-231 human breast cancer cells express high beta-adrenoceptor levels, predominantly the beta2 subtype. Receptor stimulation by isoproterenol evoked immediate reductions in DNA synthesis which were blocked completely by propranolol and were of the same magnitude as effects elicited by high concentrations of 8-Br-cAMP. Isoproterenol-induced inhibition of DNA synthesis was maintained throughout several days of exposure, resulting in a decrement in total cell number, and the effects were augmented by cotreatment with dexamethasone; an even greater effect was seen when cAMP breakdown was inhibited by theophylline, with or without addition of isoproterenol. Despite the persistent effect of isoproterenol, receptor downregulation was evident with as little as 1 h of treatment, and over 90% of the receptors were lost within 24 h. Receptor downregulation was paralleled by homologous desensitization of the adenylyl cyclase response to beta-adrenoceptor stimulation. Dexamethasone augmented the effects of isoproterenol on DNA synthesis but did not prevent receptor downregulation or desensitization. These results indicate that beta-adrenoceptors are effectively linked, through cAMP, to the termination of cell replication in MDA-MB-231 human breast cancer cells, and that activation of only a small number of receptors is sufficient for a maximal effect. Novel pharmacologic strategies that focus on cell surface receptors operating through adenylyl cyclase may offer opportunities to combat cancers that are unresponsive to hormonal agents, or that have developed multidrug resistance.

Co-expression of calcium signaling components in vertebrate taste bud cells.

In order to investigate the molecular mechanism of calcium signaling pathways common to the vertebrate gustatory systems, we have analyzed the expression of their molecular components. We first identified a phospholipase C (PLC) beta subtype expressed in the taste buds of pond loach (Misgurnus anguillicaudatus), designated DPLCbeta2, which is closely related to mammalian PLCbeta2 shown recently to be expressed in rat taste buds. The taste bud-specific expression of PLCbeta2 in a fish species as well as rat strongly suggests that PLCbeta2 mediates the tastant-induced second messenger response in taste buds, which is common to vertebrates. Next, we examined the correlation of gene expression of the candidate components leading to PLCbeta2 activation in rat circumvallate papillae, including G proteins, G(i2) and gustducin, and a G protein-coupled receptor, TR2. As a result, it was shown that the mRNAs for PLCbeta2 and G(i2) co-exist in the same cells, and PLCbeta2- and G(i2)-positive cells include both gustducin-positive cells and TR2-positive cells. However, no correlation was found between the expressions of TR2 and gustducin as reported previously. Our results thus indicate that a taste transduction pathway comprising TR2, G(i2) and PLCbeta2 occurs in a subset of taste cells.

Structure, function, and control of phosphoinositide-specific phospholipase C.

Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function.

Characterization of the endothelin receptor subtypes in human prostate.

Endothelin (ET) receptor subtypes in human prostate with benign prostatic hyperplasia were investigated by binding and functional studies. In the displacement experiment, LU224332 [endothelin-A/-B (ET(A)/ET(B)) nonselective antagonist] competed for [125I]ET-1 binding with a monophasic curve. On the other hand, LU135252 (ET(A)-selective antagonist) and sarafotoxin S6c (S6c, ET(B)-selective agonist) competed for [125I]ET-1 binding with shallow and biphasic curves. The analysis of the displacement curves for LU135252 and S6c showed that both ET(A) and ET(B) subtypes coexist but that ET(A) is the dominantly expressed receptor. In human prostate strips, 10 microM of both LU135252 and LU224332 strongly inhibited the contractile response to ET-1 with equal potency. However, 10 microM of BQ788 (ET(B)-selective antagonist) did not show a clear inhibition. S6c also produced a contractile response, which was potently inhibited by LU224332 or BQ788, and slightly suppressed by LU135252. These results suggest that in human prostate both ET(A) and ET(B) subtypes are functional receptors mediating contraction, but that ET-1-mediated contractions are predominantly mediated by activation of dominant receptor subtype, ET(A).

Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [(3)H]inositol phosphate ([(3)H]IP(x)) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alphaT3-1). Here we show that when alphaT3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [(3)H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by trans-activation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of alpha and beta (but not beta2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for alphaT3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K(d) for (125)I-buserelin 4.33 nM) and that their activation by GnRH caused a concentration-dependent increase in [(3)H]IP(x) (in cells labelled with [(3)H]inositol) and inositol 1,4,5 trisphophate (in unlabelled cells). Addition of 10 nM oestradiol (to overcome receptor blockade by raloxifene) reduced GnRH receptor number by 31% but increased maximal effects on [(3)H]IP(x) and Ins(1,4,5)P(3) approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2- to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRH-stimulated [(3)H]IP(x) accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in alphaT3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.

Subtype specific downregulation of thyroid hormone receptor mRNA by beta-adrenoreceptor blockade in the myocardium.

The beta-adrenergic system is very important in cardiovascular medicine. Thyroid hormone (T3) affects beta-adrenergic receptors. In cell culture, isoproterenol, a beta-adrenergic agonist, has been shown to upregulate thyroid hormone receptor (TR) mRNA, thus indicating a bi-directional regulation. The aim of this study was to evaluate if beta-adrenoreceptor blockade may affect subtype TR mRNA expression in vivo. Propranolol or vehicle was delivered by implanting an Alzet osmotic pump subcutaneously in mice for 14d. The concentration of TRalpha1, alpha2, beta1 and beta2 subtype mRNA concentrations were quantified by reverse transcription-polymerase chain reaction and ELISA. Propranolol downregulated the levels of TRalpha1 by 44% (p < 0.0005) and beta1 mRNA by 39% (p < 0.0005) in mouse heart, in comparison to the control, while no difference in the TRalpha2 or beta2 mRNA levels occurred. The heart rate was reduced by 10% (p < 0.05) in the propranolol group, whereas no reduction was detected in the control group. In mouse treated with propranolol serum, T3 levels were 21% lower, (p < 0.05) while serum T4 levels were 23% higher (p < 0.05) in comparison to the control. This is the first study suggesting that a beta-adrenoreceptor blockade subtype selectively regulates TR mRNA subtypes, thus giving us further knowledge about the interaction between the beta-adrenergic system and the thyroid hormone sytem.

Venous endothelin receptor function in patients with chronic heart failure

Cardiac preload reduction through venodilatation is beneficial in chronic heart failure. The recent development of endothelin receptor antagonists for possible therapeutic use in heart failure has hastened the need for a clearer understanding of the venoconstrictor actions of endothelin-1 in this disease. Two main subtypes of endothelin receptor, ETA and ETB, exist in human blood vessels. We studied the venoconstrictor effects of endothelin-1 (a non-selective ETA and ETB agonist) and sarafotoxin S6c (a selective ETB agonist) in vivo in patients with chronic heart failure and in age-matched healthy controls. On separate days at least 1 week apart, locally active doses of endothelin-1 or sarafotoxin S6c were infused into a suitable dorsal hand vein for 1 h, and the venous internal diameter was measured using a displacement technique. Venoconstriction in response to endothelin-1 was significantly blunted in heart failure patients compared with controls (26±7% and 51±6% peak reduction in vein calibre respectively; P = 0.013). Venoconstriction to sarafotoxin S6c was similar in heart failure patients and controls (17±5% and 17±4% peak reduction in vein calibre respectively). Both ETA and ETB receptors mediate venoconstriction in healthy subjects and in patients with chronic heart failure. Optimal inhibition of the venoconstrictor effects of endothelin-1 in chronic heart failure may therefore require administration of an antagonist with ETA- and ETB-receptor-blocking properties. Chronic heart failure may be associated with a selective decrease in venous ETA receptor sensitivity, but further studies are required to clarify the functional significance of this observation.

Mechanisms and receptor subtypes involved in the stimulatory action of endothelin-1 on rat adrenal zona glomerulosa.

Endothelin (ET)-1 is the prototype of a family of 21-amino acid residue hypertensive peptides, acting through two subtypes of receptors, named ETA and ETB. ETs and their receptors are expressed in the adrenal cortex and medulla, and ET-1 enhances both corticosteroid and catecholamine release. ET-1 concentration-dependently (from 10(-11) to 10(-8) M) increased aldosterone secretion of both dispersed rat zona glomerulosa (ZG) cells and adrenal slices containing a core of medullary chromaffin tissue, but the response of the latter preparations was significantly more intense than that of the formers. The stimulatory effect of 10(-8) M ET-1 on dispersed ZG cells was blocked by the ETB-receptor antagonist BQ-788 (10(-7) M), but not by the ETA-receptor antagonist BQ-123 (10(-7) M); conversely, both ET-receptors antagonists counteracted aldosterone response of adrenal slices to ET-1. The -adrenoceptor antagonist l-alprenolol (10(-6) M) did not affect aldosterone response of dispersed ZG cells to ET-1 (10(-8) M), but it significantly lowered that of adrenal slices. l-Alprenolol also counteracted the aldosterone response of adrenal slices to the pure activation of ETB or ETA receptors, as obtained by using the selective ETB-receptor agonist BQ-3020 (10(-8) M) or ET-1 (10(-8) M) plus BQ-788 (10(-7) M). ET-1 concentration-dependently (from 10(-9) to 10(-8)/10(-7) M) stimulated catecholamine release by adrenal slices, and the effect was counteracted by both BQ-123 and BQ-788 (10(-7) M). Collectively, our findings suggest that, when the integrity of adrenal tissue is preserved, a two-fold mechanism underlies the aldosterone secretagogue action of ET-1 in the rat: i) a direct mechanism mediated by ETB receptors located on ZG cells; and ii) an indirect mechanism involving the ETA and ETB receptor-mediated local release of catecholamines, which in turn stimulate ZG cells in a paracrine manner.

Modeling study of three-dimensional structure of human beta adrenoceptors and receptor-antagonists interactions

We deduced the three dimensional structures of transmembrane regions in human beta adrenoceptors by computer simulating using the 2D electron density maps of bacteriorhodopsin and frog rhodopsin as templates. The deduced membrane-spanning helical regions, their arrangements and axes to the membrane plain were different between two models. Typical beta adrenergic antagonist, propranolol, and long-lasting beta antagonist, bopindolol, were docked in these models. The receptor-antagonist interactions were different between both models, in which the 3, 4, 5 and 6th transmembrane domains might participate in the antagonists-binding in both models, however, the amino acid residues in the domains contributing to the binding might be different between models. Propranolol and bopindolol have high affinity to beta 1 and beta 2 subtypes, but low affinity to beta 3 subtype. The simulated receptor-antagonist relationships could account, in part, for the selectivity of the antagonists to the beta subtypes.

Identification of a phospholipase C β subtype in rat taste cells

From rat circumvallate papillae a novel phospholipase C (PLC) subtype has been cloned and identified as most closely related to human PLC beta2. The corresponding mRNA was only detected in sensory lingual tissue but not in non-taste lingual tissue or any other tissues examined by Northern blot analysis. In situ hybridization revealed that a subset of taste receptor cells of circumvallate papillae was specifically labeled. A functional involvement of this PLC beta subtype in taste signal transduction emerged from biochemical analysis monitoring the second messenger response in circumvallate preparations induced by denatonium benzoate. This bitter agent elicited a rapid and transient increase of the inositol 1,4,5-trisphosphate level; this response was blocked by U73122, a potent inhibitor of PLC, and by PLC beta2-specific antibodies. These data indicate that a phospholipase C beta2 isoform mediates a denatonium benzoate-induced second messenger response of taste sensory cells in the circumvallate papillae.

Transcription activation by the human estrogen receptor subtype beta (ER beta) studied with ER beta and ER alpha receptor chimeras.

We have studied the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and chimeric constructs with ER alpha and ER beta to examine the bioactivities of these receptors and their responses to estrogen and antiestrogen ligands. Transcriptional activity of ER beta is highly dependent on cell/promoter context and on the nature of the ligand. ER beta activated significant levels of transcription in response to estrogens in certain cell types, but showed only moderate activity compared with ER alpha in others. Antiestrogens such as tamoxifen and 2-phenylbenzofuran, which show some agonistic activity with ER alpha, exhibit no agonistic activity with ER beta. Alteration of the amino-terminal A/B receptor domain can result in a dramatic change in cell type- and ligand-specific transcriptional activity of ER beta. Upon replacing the A/B domain of ER beta with the A/B domain of ER alpha, this receptor chimera not only exhibits an improved transcriptional response to estrogens, but also is now able to activate transcription upon treatment with these antiestrogens. As antiestrogen agonism was lacking in ER beta and the ER beta/alpha chimera containing the amino-terminal A/B domain of ER beta fused to domains C through F of ER alpha, but was restored in an ER alpha/beta chimera containing the A/B domain of ER alpha, antiestrogen agonism was shown to depend on the A/B domain (activation function-1-containing region) of ER alpha. Together, these results indicate that the differences in the amino-terminal regions of ER alpha and ER beta contribute to the cell- and promoter-specific differences in transcriptional activity of these receptors, and their ability to respond to different ligands, thus providing a mechanism for differentially regulated transcription by these two ERs.

Effect of deletion of the major brain G-protein alpha subunit (alpha(o)) on coordination of G-protein subunits and on adenylyl cyclase activity.

Heterotrimeric G-proteins, composed of alpha and betagamma subunits, transmit signals from cell-surface receptors to cellular effectors and ion channels. Cellular responses to receptor agonists depend on not only the type and amount of G-protein subunits expressed but also the ratio of alpha and betagamma subunits. Thus far, little is known about how the amounts of alpha and betagamma subunits are coordinated. Targeted disruption of the alpha(o) gene leads to loss of both isoforms of alpha(o), the most abundant alpha subunit in the brain. We demonstrate that loss of alpha(o) protein in the brain is accompanied by a reduction of beta protein to 32+/-2% (n = 4) of wild type. Sucrose density gradient experiments show that all of the betagamma remaining in the brains of alpha(o)-/- mice sediments as a heterotrimer (s20,w = 4.4 S, n = 2), with no detectable free alpha or betagamma subunits. Thus, the level of the remaining betagamma subunits matches that of the remaining alpha subunits. Protein levels of alpha subunits other than alpha(o) are unchanged, suggesting that they are controlled independently. Coordination of betagamma to alpha occurs posttranscriptionally because the mRNA level of the predominant beta1 subtype in the brains of alpha(o)-/- mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by betagamma. Because the level of other alpha subunits is unchanged and alpha(o) itself has little or no effect on adenylyl cyclase, we could examine how a large change in the level of betagamma affects this enzyme. Surprisingly, we could not detect any difference in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice. We propose that alpha(o) and its associated betagamma are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase.

Endothelin-1 enhances thymocyte proliferation in monolaterally adrenalectomized rats with contralateral adrenocortical regeneration.

Endothelins (ETs) are a family of vasoactive peptides widely distributed in the body systems, where they exert pleiotropic biological effects, acting through two main subtypes of receptors, named ETA and ETB. Evidence indicates that ET-1 plays a permissive role in the development of neural crest-derived structures, among which are the epithelial cells of the thymus. These cells are known to control proliferation and differentiation of thymocytes, a process requiring adequate levels of glucocorticoids. Therefore, we have investigated the effects of ET-1, that binds both ETA and ETB receptors, on thymocyte proliferation in monolaterally adrenalectomized rats with contralateral enucleated adrenal at day 4 and 8 of regeneration, when glucocorticoid production is very low and, respectively, rather normal. Metaphase index (percentage of metaphase arrested cells) of thymocytes is the lowest at day 4 of regeneration, and markedly rose at day 8, thereby confirming the need of sizable levels of circulating glucocorticoid for the maintenance of a normal rate of thymocyte proliferation. ET-1 markedly increased the mitotic index of thymocytes at both times of adrenal regeneration. At day 8 of regeneration, the ETA-receptor antagonist BQ-123 markedly lowered mitotic index of thymocytes, and annulled its ET-1-evoked raise. Conversely, the ETB-receptor antagonist BQ-788 was ineffective. Collectively, these findings clearly indicate that endogenous ETs, through the activation of ETA receptors, are involved in the maintenance and stimulation of thymocyte proliferation in the adult rat, thereby playing a possibly important role in the modulation of the immune-system functions.

Identification of Endothelin Receptor Subtypes in Rat Ciliary Body Using Subtype-Selective Ligands

The endothelins are important vasoactive ocular peptides and there is some evidence that they may modulate intraocular pressure. We investigated the existence and localization of endothelin receptor subtypes using subtype selective ligands in rat ciliary body. Scatchard transformation of saturation binding experiments revealed that the KDand Bmaxfor [125I]ET-1 and [125I]ET-3 to membranes from ciliary body were 41.7±9 pM and 236±20 fmol mg−1protein and 37.8±0.4 pM and 160±2.0 fmol mg−1protein, respectively. Competitive experiments in the presence of cyclic pentapeptide BQ123 (selective for ETA receptors) and BQ3020 (selective for ETB receptors), demonstrated the existence of ETA and ETB receptors in a ratio of 35:65. Cross-linking of [125I]ET-1 and [125I]ET-3 to ciliary body membranes resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa, suggesting that ETA and ETB receptors have similar molecular mass. The 34 Kda band is a proteolytic degradation product of the 52 Kda band. Autoradiographic results show that specific [125I]ET-1 binding sites, displaced by BQ123 and BQ3020, are localized to the ciliary epithelium, supporting the idea that ETA and ETB subtype receptors exist in this tissue.

An epitope-tagging system for studying regulation of the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a group of compounds which cause peroxisome proliferation and hepatocellular carcinomas in rodents, and form a class of non-genotoxic carcinogens. It is thought that PPs act via a receptor similar to members of the nuclear hormone superfamily termed the peroxisome proliferator activated receptor (PPAR). Multiple subtypes (alpha, beta, delta and gamma) of the receptor exist and are differentially expressed between tissues and species. PPAR alpha has been shown to activate transcription by binding to response elements upstream of peroxisome proliferator responsive genes. However, despite the isolation of transcriptionally active human subtypes of the receptor, hPPAR alpha and hNUC1, humans are thought to be non-responsive to PPs. This is possibly due to regulation of PPAR, and it has been recently reported that PPAR alpha is a phosphoprotein in vivo and insulin regulates its phosphorylation. A system employing epitope-tagged receptors has been developed to study this further, with the aim of establishing stably transfected cell lines expressing high levels of epitope-tagged mouse and human PPAR alpha. Our experiments clearly demonstrate that an epitope-tagged mPPAR alpha receptor has an equal ability to modulate transcription as the native receptor in transactivation assays and will be further used to examine the molecular mechanisms of peroxisome proliferation.

Subunit combinations defined for K+ channel Kv1 subtypes in synaptic membranes from bovine brain.

Voltage-dependent Shaker-related (Kv1) K+ channels are composed of transmembrane alpha subunits and peripheral Kv beta proteins that exist as octomers with (alpha)4(beta)4 stoichiometry. Although several alpha (designated Kv1.X) and three Kv beta subunits are known to be expressed in brain, their oligomeric combinations in neurons have yet to be deciphered. Herein, the subunits comprising a number of neuronal K+ channels from bovine brain cortex were deduced by immunoprecipitation and Western blotting, using antibodies specific for Kv1.X and Kv beta subtypes. Only a subset of the theoretically possible oligomers was detected, showing that the synthesis and/or assembly of these multisubunit K+ proteins is controlled to yield a limited variety of K+ channels. Except for a small population of Kv1.4 containing K+ channels, all the recognizable species contained Kv1.2 and beta2 subunits. Furthermore, several subpopulations were identified including a fully defined complex of Kv1.2/1.3/1.4/1.6 and Kv beta2, plus oligomers containing three or two assigned alpha subunits. Kv1.2 was also shown to occur in the absence of these other subunits as a putative homo-oligomer. Thus, for the first time, the complete subunit combination of an authentic K+ channel has been elucidated; also, the strategy employed to establish this can now be applied to closely related members of other K+ channel families.

Identification of a short sequence highly divergent between beta-adrenergic-receptor kinases 1 and 2 that determines the affinity of binding to betagamma subunits of heterotrimeric guanine-nucleotide-binding regulatory proteins.

A 28-residue peptide (peptide G), derived from the C-terminal (W643-S670) of the beta-adrenergic receptor kinase (betaARK), was previously identified as the critical domain for binding to the betagamma subunits of the heterotrimeric guanine-nucleotide-binding regulatory protein (G betagamma). We observed that the 18-amino-acid core of this domain is poorly conserved between betaARK1 and betaARK2 and so may provide the basis for differences in G betagamma-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for betaARK1 and betaARK2, respectively) competitively inhibited G betagamma-activation of the related betaARK subtype, confirming the involvement of this region in binding to G betagamma. Peptides P1 and P2 inhibited G betagamma-stimulated activity of both betaARK1 and betaARK2, with P2 being significantly more potent than P1 (IC50 of 179+/-5 microM for P2 and >500 microM for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC50 = 48+/-5 microM for G2 and 146+/-8 microM for G1). This relative order of potency G2 > G1 approximately P2 > P1 was confirmed in a direct G betagamma-binding assay. No binding selectivity for the beta1, beta2, beta3 and beta4 G beta subtypes was observed. The EC50 value for G betagamma-activation of betaARK1 was about double of that for betaARK2, indicating a higher affinity between G betagamma and betaARK2, which is the expected result based on the findings with the peptides. These findings show that the 18-residue peptides P represent the shortest sequence of betaARK that can bind to G betagamma and provide a demonstration of a functional difference between the G betagamma binding domains of betaARK1 and betaARK2.

Distribution and functional significance of the endothelin receptor subtypes in the rat adrenal gland.

Endothelins (ET) are a family of vasoactive peptides that act via two subtypes of receptors, named ETA and ETB. ET-1 binds to both ETA and ETB, whereas the isopeptide ET-3 preferentially binds to ETB. The localization of ETA and ETB receptors in the rat adrenal gland and their involvement in the adrenal secretagogue effect of ETs has been studied in vitro. Autoradiographic assessment of the selective displacement of [125I]ET-1, [125I]ET-3 and [125I]BQ-3020 (an ETB agonist) by BQ-123 or BQ-788 (specific antagonists of ETA and ETB, respectively) indicates that the zona glomerulosa and adrenal medulla possess both ETA and ETB, whereas the zona fasciculata/reticularis is exclusively provided with ETB. ET-1, ET-3 and BQ-3020 enhance aldosterone and corticosterone secretion by dispersed cells of the zona glomerulosa and zona fasciculata/reticularis, respectively. BQ-123 does not affect the secretagogue action of these three agonists, whereas BQ-788 completely annuls it. ET-1 induces a marked rise in catecholamine release by fragments of the adrenal medulla, and both BQ-123 and BQ-788 partially reverse this effect. ET-3 and BQ-3020 elicit a catecholamine release that is less intense than that produced by ET-1; this response is unaffected by BQ-123 and abolished by BQ-788. Thus, in the rat, the corticosteroid secretagogue effect of ETs seems to be exclusively mediated by the ETB receptor subtype, and the catecholamine secretagogue action by both ETA and ETB. The functional relevance of ETA receptors present in the zona glomerulosa remains to be investigated.

β‐Adrenoceptor‐mediated effects in rat cultured thymic epithelial cells

1. Sympathetic nerves were visualized in sections from rat thymus by immunostaining of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, and by glyoxylic acid-induced fluorescence of catecholamines. Catecholaminergic nerve fibres were detected in close connection to thymic epithelial cells which therefore might be preferred target cells. To evaluate this, rat immunocytochemically defined, cultured thymic epithelial cells were investigated for adrenoceptors and adrenergic effects. 2. In rat cultured thymic epithelial cells mRNA for beta 1- and beta 2-adrenoceptors was detected by reverse transcription-polymerase chain reaction by use of sequence-specific primers. Specific, saturable binding to the cultivated cells was observed with the beta-adrenoceptor agonist CGP 12177. 3. Adrenaline, noradrenaline or the beta-adrenoceptor agonist, isoprenaline, increased intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultivated thymic epithelial cells dose-dependently about 25 fold. The pharmacological properties revealed that this response was mediated by receptors of the beta 1- and the beta 2-subtypes. The selective beta 3-adrenoceptor agonist BRL 37344 had no effect on cyclic AMP levels. The increase in cyclic AMP was downregulated by preincubation with glucocorticoids like dexamethasone or cortisol which also changed the relative importance of beta 1-/beta 2-adrenoceptors to the response. 4. Incubation with isoprenaline or the adenylate cyclase activator forskolin decreased basal and serum-stimulated proliferation of thymic epithelial cells. However, adrenergic stimulation of thymic epithelial cells did not induce interleukin 1 production. Since thymic epithelial cells create a microenvironment which influences the maturation and differentiation of thymocytes to T-lymphocytes, their observed capacity to respond to catecholamines provides novel evidence for the suggestion that adrenergic stimulation may interfere with the regulation of immune functions.

Beta-adrenoceptor subtype expression and function in rat white adipocytes.

1. The pharmacological features of rat white adipocyte beta-adrenoceptor subtypes were investigated by saturation and beta-agonist competition studies with [3H]-CGP 12177 and by lipolysis induced by beta-agonists as well as their inhibition by CGP 20712A (selective beta 1-antagonist) and ICI 118551 (selective beta 2-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of beta-adrenoceptor subtypes. 2. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22 +/- 0.07 nM) and the other with low affinity (23 +/- 7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3. The competition curves, with 15 nM [3H]-CGP 12177, for the beta-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective beta 3-agonist, BRL 37344 (BRL), were clearly biphasic (P < 0.001). The rank orders of agonist potency (pKi) in competing for [3H]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28 +/- 0.24) > NA (8.90 +/- 0.12) > Ad (8.65 +/- 0.12) > > BRL (4.53 +/- 0.17) and BRL (7.38 +/- 0.19) > > Iso (2.96 +/- 0.26) > or = NA (2.80 +/- 0.17) > Ad (2.10 +/- 0.11) indicating the expression of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective beta 1-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81 +/- 9 fmol mg-1) and high pKi value (7.23 +/- 0.26) confirming the presence of beta 1-adrenoceptors. 4. To assess a possible contribution of the beta 2-subtype, procaterol (Proc), a selective beta 2-agonist, was used to compete with 2 nM [3H]-CGP 12177. A single low affinity (4.61 +/- 0.07) population of binding sites was identified. The density of these sites (71 +/- 12 fmol mg-1) was similar to the one obtained with Xam, suggesting that Proc displaced [3H]-CGP 12177 from the beta 1-subtype. 5. The functional potency (pD2) order with BRL (9.07 +/- 0.20) and catecholamines (Iso: 7.26 +/- 0.06, NA: 6.89 +/- 0.02 and Ad: 6.32 +/- 0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31 +/- 0.06 and 5.66 +/- 0.10, respectively. Proc stimulated lipolysis with a low potency (5.59 +/- 0.21). 6. The lipolytic response to 0.001 microM BRL was inhibited by both, selective beta 1- and beta 2-antagonist, in a monophasic manner with low potencies (CGP 20712A pKi: < 4.5 and ICI 118551 pKi: 5.57 +/- 0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (> 10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 microM Iso or 0.13 microM NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pKi values for the first sites were 8.41 +/- 0.09 and 8.58 +/- 0.17, respectively, and for the second population of sites 4.73 +/- 0.22 and 4.27 +/- 0.27, respectively. The proportion of the first sites was low: 19 +/- 4 and 22 +/- 5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 microM Proc (CGP 20712A: pKi1: 8.17 +/- 0.08, site1: 23 +/- 6%, pKi2: 4.77 +/- 0.14; ICI 118551: pKi1: 7.78 +/- 0.03, site1: 37 +/- 2%, pKi2: 5.35 +/- 0.25). 7. Our results show that the radioligand [3H]-CGP 12177 allows the characterization of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on beta 1- and beta 3-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the beta 3-subtype.