Variability in the regulation of beta-adrenoceptor subtypes.
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Animals↗
Biomedical subjects
F Nantel
Publications and source records attributed to F Nantel.
Functional effects of long-term activation on human beta 2- and beta 3-adrenoceptor signalling.
The functional effects of long-term activation of beta-adrenoceptors were investigated by measuring adenylyl cyclase activity, cyclic AMP accumulation and cyclic AMP-dependent protein kinase activity in CHW and L cells expressing either human beta 2- or beta 3-adrenoceptors. Pre-incubation of CHW and L cells expressing beta 2-adrenoceptors with 10 microM isoprenaline for 24 h produced a marked reduction in the total receptor number and dramatically reduced the capacity of the receptor to stimulate adenylyl cyclase maximally. In contrast, the ability of beta 3-adrenoceptors number was observed in L but not in CHW cells. Maximal levels of intracellular cyclic AMP concentrations were reached during the first hour of receptor activation with isoprenaline in all four cell lines. In the absence of phosphodiesterase inhibitors, cyclic AMP decreased to basal levels within 24 h of continuous stimulation. This phenomenon occurred more rapidly in cells expressing the beta 2- than the beta 3-adrenoceptors. These results confirm that, at the level of adenylyl cyclase stimulation and cyclic AMP accumulation, the beta 3-adrenoceptor is more resistant than the beta 2-adrenoceptor to long-term desensitization. However, when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and when cyclic AMP-dependent protein kinase activity was considered, a 24 h stimulation of beta 2- and beta 3-adrenoceptor expressing cells led to the desensitization of the kinase in L but not in CHW cells. In conclusion, long-term desensitization may have distinct functional effects on cell signalling depending on the receptor subtype and the cell type considered. These findings might have practical implications for future strategies involving long-term therapies with receptor agonists.
Adenylyl Cyclases↗
Characterization of the vasorelaxant activity of tyramine and other phenylethylamines in rat aorta.
We recently reported that tyramine caused concentration-dependent relaxation of rat aorta, which was endothelium independent and was not exerted via alpha 1-adrenoceptors (AR), alpha 2AR, beta 1AR, beta 2AR, or receptors for 5-hydroxytryptamine, histamine, and adenosine. The present studies were done on endothelium-denuded strips to determine structure-vasorelaxant activity after blockade of beta AR by propranolol plus irreversible blockade of alpha 1AR with benextramine. Vasorelaxation under these conditions was limited to noncatecholamines, and their vasorelaxant potencies were methoxyphenamine > tyramine > p-hydroxyephedrine > L-amphetamine > L-ephedrine > phenylethylamine > synephrine > methoxamine > octopamine. beta 3AR agonists (BRL 37344 and CGP 12177A) did not produce vasorelaxation, although tyramine could compete for cyanopindolol binding to murine L cells expressing human beta 2AR or beta 3AR. There was no significant specific binding of [3H]tyramine to aortic membrane preparations after the inhibition of monamine oxidase. Yohimbine, which has a high affinity for Drosophila tyramine receptors, also caused dose-dependent vasorelaxation like tyramine. It is concluded that tyramine and several other phenylethylamines produce relaxation of rat aorta, which does not involve any of the known adrenoceptors but may be exerted via novel tyramine receptors.
Animals↗
Cell-specific down-regulation of the beta 3-adrenergic receptor.
Previous studies have shown that beta 3-adrenergic receptors, in contrast to the beta 1 and beta 2 subtypes, do not undergo desensitization following short term activation (minutes) with agonists. Longer activation (hours) has been shown to induce desensitization of the beta 3-adrenergic receptor in some, but not all, cases, suggesting that cell- or species-specific mechanisms may be involved. We investigated the contribution of the cell type to the pattern of beta 3-adrenergic receptor long term desensitization by studying, in parallel, two cell lines (Chinese hamster fibroblasts and murine Ltk- cells) expressing the human beta 3-adrenergic receptor. Sustained agonist-promoted down-regulation of the beta 3-adrenergic receptor could be induced in Ltk- cells, whereas only a transient and weak reduction of receptor number was observed in Chinese hamster fibroblasts. The half-life of the beta 3-adrenergic receptor was not affected by the agonist activation in either cell line, indicating that in contrast to the beta 2-adrenergic receptor, degradation of preexisting receptor protein does not contribute to down-regulation. Sustained reduction of receptor RNA levels, monitored by reverse transcriptase polymerase chain reaction, was exclusively shown in Ltk- cells and probably accounted for most of the observed down-regulation. Differences in the ability of the receptor to stimulate adenylyl cyclase activity in the two cell lines may be responsible for the distinct patterns of beta 3-adrenergic receptor down-regulation.
Adenylyl Cyclases↗
Cellular characterization of the pharmacological selectivity and tachyphylactic properties of denopamine for the human beta adrenergic receptors.
The pharmacological properties of the cardiotonic agent denopamine toward human beta-1 and beta-2 adrenergic receptors (AR) were assessed in a heterologous expression system. In whole cells radiolabeled with the nonselective beta antagonist [125I]iodopindolol, denopamine displayed a 7-fold lower inhibition constant for the beta-1 than for the beta-2 AR. Similarly, denopamine exhibited a 7-fold greater potency to stimulate the adenylyl cyclase activity in cells expressing the beta-1 AR than in cells expressing the beta-2 subtype, which confirmed the subtype selectivity of this drug. The maximal stimulation of adenylyl cyclase activity by denopamine was less than 10% of that produced by isoproterenol at both receptor subtypes, which indicated that denopamine is a weak partial agonist. The extent of beta-1 AR desensitization induced by denopamine was then compared with that induced by the full agonist isoproterenol. By contrast with isoproterenol, preincubation with denopamine for 20 min did not induce any decrease in the responsiveness of the receptor to subsequent stimulation nor did it promote any sequestration of the receptor. A 24-hr pretreatment with denopamine produced a time-dependent reduction in the number of beta-1 AR with a rate of down-regulation slower than that produced by isoproterenol. These data therefore indicate that denopamine is a beta-1 adrenergic selective agonist with low intrinsic activity that is less prone than full agonists to cause desensitization. This may explain the long-lasting cardiotonic effects of denopamine compared with those of full agonists.
Adenylyl Cyclases↗
The human beta 3-adrenergic receptor is resistant to short term agonist-promoted desensitization.
The human beta 3-adrenergic receptor (beta 3AR) lacks most of the structural determinants that, in the beta 2AR, contribute to agonist-induced receptor desensitization. To evaluate the effect of these structural differences on the beta 3AR desensitization profile, the human beta 2- and beta 3AR were stably expressed in Chinese hamster fibroblasts (CHW) and murine Ltk- cells (L cells). Incubation of CHW-beta 2 or L-beta 2 cells with 10 microM isoproterenol for 30 min induced a decrease in the maximal agonist-stimulated adenylyl cyclase activity and a cAMP-dependent reduction in the potency of isoproterenol to stimulate the receptor. In addition, this pretreatment impaired the formation of the high affinity heterotrimeric agonist-receptor-guanine nucleotide-binding protein complex and induced the sequestration of approximately 30% of the beta 2AR away from the cell surface. In contrast, similar treatment of CHW-beta 3 and L-beta 3 cells did not affect the maximal receptor-stimulated adenylyl cyclase activity, nor did it induce any significant sequestration of the beta 3AR. In fact, only a modest cAMP-independent decrease in the potency of isoproterenol to stimulate the receptor could be observed after isoproterenol treatment. The rapid desensitization pattern of a chimeric beta 3AR, in which the third cytoplasmic loop and the carboxyl-terminal tail were exchanged with those of the beta 2AR (which include potential phosphorylation sites and other possible molecular determinants of desensitization), was found to be intermediate between those of the two original receptor subtypes. These results demonstrate that (i) the beta 3AR is less prone than the beta 2AR to undergo rapid agonist-promoted desensitization and, (ii) in addition to the phosphorylation sites located in the third cytoplasmic loop and the carboxyl-terminal tail of the beta 2AR, other molecular determinants contribute to short term desensitization.
Adenylyl Cyclases↗
Distinct regulation of beta 1- and beta 2-adrenergic receptors in Chinese hamster fibroblasts.
The agonist-induced reduction of beta-adrenergic receptor (beta AR) cell surface density is a well documented phenomenon. The mechanisms responsible for this regulation have been well characterized for the beta 2AR. They include a rapid sequestration of the receptor away from the cell surface in a vesicular compartment and a longer term down-regulation of the total beta 2AR number. In contrast, very little is known about the cell surface regulation of the beta 1AR. In the present study, we have compared the agonist-mediated regulation of beta 1- and beta 2AR in Chinese hamster fibroblasts transfected with the cDNA encoding either beta AR subtype. Cells expressing similar numbers of the two beta AR subtypes were selected for the study. The expressed receptors exhibit typical beta 1- and beta 2AR selectivity for agonists and antagonists, as assessed by radioligand binding. Both receptors were found to be positively coupled to the adenylyl cyclase stimulatory pathway, but marked differences in the receptor regulation profiles were observed. Treatment of the cells expressing the beta 2AR with the agonist isoproterenol leads to a rapid sequestration of greater than 30% of the receptors away from the cell surface into a light vesicular fraction, where they are inaccessible to the hydrophilic ligand CGP-12177. In contrast, virtually no agonist-induced sequestration is observed in the cells expressing the beta 1AR. Longer exposure of the cells to isoproterenol leads to a time-dependent reduction in the total number of beta ARs in both beta 1- and beta 2AR-expressing cell lines. However, this down-regulation is significantly slower in the cells expressing the beta 1AR. In fact, no appreciable down-regulation of the beta 1ARs is detected in the first 4 hr of agonist treatment, compared with a down-regulation of greater than 50% of the beta 2ARs for the same period. After a 24-hr treatment with isoproterenol, less than 20% of the original number of beta 2ARs remain, whereas 60% of the beta 1ARs are still present after the same treatment. These results, therefore, suggest that, when expressed in an identical cell line, beta 1AR and beta 2AR follow distinct patterns of regulation. In fact, both agonist-induced sequestration and down-regulation are considerably blunted for the beta 1AR, compared with the beta 2AR.
Adenylyl Cyclases↗
Pharmacology of neurokinin receptors.
The neurokinins are a group of naturally occurring peptides with the common C-terminal sequence Phe-X-Gly-Leu-Met.NH2. They include substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). SP and NKA are coded on the same gene, the PPT-A, while NKB is coded on a separate gene, the PPT-B. Neurokinins are present in the central nervous system and in peripheral organs where they exert various actions. They act on three receptors--NK-1, NK-2, and NK-3--characterized through pharmacological, biochemical, and histochemical studies. Selective agonists for each neurokinin receptor were developed and evaluated on isolated smooth muscle preparations containing only one neurokinin receptor type. All three neurokinin receptors were cloned and expressed in Xenopus oocytes. Relative affinities of those receptors to neurokinins are the same as in their respective smooth muscle preparation. Finally, the mechanism of action of SP on histamine release from rat peritoneal mast cell has been studied and a direct activation of G proteins by peptides with basic amino acids is proposed as a working hypothesis.
Amino Acid Sequence↗
New highly potent bradykinin B2 receptor antagonists.
pA2 values of new B2 receptor antagonists ranging from 7.51 to 8.86 were measured on the rabbit jugular vein, while lower values were observed in the other preparations (for instance, the hamster urinary bladder). The most potent antagonists were those containing a hydroxyproline (Hyp) in position 3, a D-Arg at the N-terminal and a Leu instead of a Phe in position 8, with or without other chemical changes. D-Arg[Hyp3,D-Phe7,Leu8]-BK was found to be competitive, selective for B2 receptors and specific for kinins since it was without effect against substance P and angiotensin II in the rabbit jugular vein. The essential feature for obtaining B2 receptor antagonists appears to be the replacement or reorientation of Phe8 of bradykinin. The rabbit jugular vein provides a sensitive bioassay in which the potency and specificity of B2 receptor antagonists can be adequately evaluated.
Amino Acid Sequence↗
Pharmacological evaluation of the angiotensin, kinin, and neurokinin receptors on the rabbit vena cava.
Angiotensin II, bradykinin, and substance P are powerful vasoconstrictors of venous smooth muscle. In this report, we have characterized the receptors and the cellular mechanisms of these vasoactive peptides on a new isolated smooth muscle preparation, the rabbit vena cava. Receptors were characterized using agonists and antagonists and were found to be of the AT, B2, and NK-1 types. The myotropic responses of the vein to KCl was completely abolished in calcium-free medium; in the presence of nicardipine, nifedipine, and verapamil, three calcium channel antagonists; and of trifluoperazine, a calmodulin antagonist. AT II-, BK-, and SP-induced responses were slightly attenuated in calcium-free medium and in the presence of nifedipine and trifluoperazine. Pinacidil inhibited the contractile response of KCl and the three peptides while lidocaine was active against KCl only. Staurosporine and cholera toxin strongly inhibited the contractile responses of the vein to AT II, BK, SP, and KCl, probably by a nonspecific effect. It is concluded that AT II-, BK-, and SP-induced contractions of the rabbit vena cava are mediated by specific receptors and in part by an influx of extracellular Ca2+ through dihydropyridine-insensitive channels. Opening of K+ channels and inhibition of the Ca(2+)-calmodulin complex appear to interfere with the smooth muscle response to the peptides.
Angiotensin II↗
Characterization of bombesin receptors in peripheral contractile organs.
1 Guinea-pig and rat urinary bladders, rat stomach and the guinea-pig gall bladder, four isolated organs that show high sensitivity to bombesin, were used to characterize bombesin receptors in peripheral organs. 2 The order of potency of agonists was determined with several naturally occurring peptides of the bombesin series, namely bombesin (BBS), litorin (Lit), neuromedin B (NMB), the gastrin-releasing peptide (GRP 18-27), neuromedin C (NMC) and with some bombesin fragments. It was found that bombesin, neuromedin C, litorin and two bombesin fragments, BBS (6-14) and AcBBS (6-14) had similar activities in the four preparations, while neuromedin B and [Phe6]-neuromedin C were more active on the rat urinary bladder than on the other tissues. 3 The order of potency of agonists determined in the rat urinary bladder was as follows: BBS = NMB greater than Lit greater than NMC greater than [Phe6]NMC = GRP and it was found to be different from that observed in the other preparations: BBS greater than GRP = Lit greater than or equal to NMC much greater than NMB greater than [Phe6]NMC, suggesting the existence of two different bombesin receptors, BBS1 and BBS2. 4 This interpretation was convalidated by the finding that bombesin antagonists, namely Ac.GRP(20-26)OCH3 and Ac.GRP(20-26)OC2H5 reduced or blocked the effects of bombesin-related peptides on BBS2 receptor systems while being completely inactive on the rat urinary bladder (BBS1 system).
Animals↗
Neurokinin agonists and antagonists.
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Amino Acid Sequence↗
Multiple NK2 receptor subtypes are suggested by physiological and biochemical studies with neurokinin A (NKA) analogues and antagonists.
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Animals↗
DuP 753 is a specific antagonist for the angiotensin receptor.
2-n-Butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biph enyl-4- yl)methyl]imidazol potassium salt (DuP 753) is a nonpeptide angiotensin II receptor antagonist that inhibits the contractile effects of angiotensin II competitively and shows pA2 values of 8.27 on the rabbit aorta and jugular vein, 8.66 on the rat portal vein and stomach, 8.19 on the rat urinary bladder, and 8.36 on human colon, ileum, and urinary bladder. This agent (more than 10(-5) M) exhibits no agonistic activity and does not affect the contractile effects of norepinephrine, acetylcholine, bradykinin, desArg9-bradykinin, substance P, neurokinin A, neurokinin B, or bombesin in the various tissues. The present results demonstrate that DuP 753 is a potent nonpeptide antagonist with high affinity, specificity, and selectivity for the angiotensin receptor.
Adrenergic alpha-Antagonists↗
Antagonists for the neurokinin NK-3 receptor evaluated in selective receptor systems.
Four isolated vessels that are monoreceptor systems for neurokinins, the dog carotid artery and rabbit jugular vein (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3), were used to compare the activities of selective neurokinin agonists and evaluate the affinities of new NK-3 antagonists. Chemical modifications in the partial sequences NKA (4-10) and NKB (4-10), particularly the replacement of Val7 with an aromatic residue (Tyr, MePhe or Trp) and the extension of the peptide backbone in position 8, obtained with beta-Ala, led to compounds that maintain weak agonistic activities on the NK-1 and NK-2, and some of them also on NK-3 receptors but exert potent antagonism against NKB on the NK-3 receptor of the rat portal vein. Antagonistic affinity is the highest when Trp is used in position 7 of [beta-Ala8]-NKA (4-10) and MePhe in position 7 of [beta-Ala8]-NKB (4-10). Antagonism is selective for NKB or [MePhe7]-NKB, and appears to be specific, since the most active compound [Trp7, beta-Ala8]-NKA (4-10) is inactive against bradykinin on the rabbit jugular vein (B2 receptor), against SP on the rabbit jugular vein (NK-1 receptor), against desArg9-bradykinin on the rabbit aorta (B1 receptor), and against angiotensin II and histamine (AT and H receptors, respectively) in the rabbit aorta. The new NK-3 receptor antagonists described in the present study provide useful tools for neurokinin receptor characterization and for determining the roles of neurokinins in physiopathology.
Animals↗
The rabbit jugular vein is a contractile NK-1 receptor system.
Rabbit isolated jugular veins respond to substance P and related neurokinins with concentration-dependent contractions which appears to be due to the activation of a single neurokinin receptor of the NK-1 type. This is demonstrated by the order of potency of neurokinins and some of their fragments as well as by the strong activity of NK-1-selective agonists and the weakness of NK-2- and NK-3-selective agonists. The present results indicate that the rabbit jugular vein provides a sensitive, specific and selective NK-1 preparation which responds directly to neurokinins with contractions and therefore can be useful for characterization of NK-1 agonists and antagonists.
Angiotensin II↗
Receptors for neurokinins in human bronchus and urinary bladder are of the NK-2 type.
Human tissues such as the isolated bronchus and urinary bladder respond to neurokinins with concentration-dependent contractions, which appear to be due to the activation of receptors. We characterized these receptors in the present study using agonists (the naturally occurring neurokinins and some selective agonists) as well as newly identified antagonists. The order of potency of the agonists in the two preparations was as follows: neurokinin A (NKA) greater than substance P (SP) greater than neurokinin B (NKB) (bronchus) and NKA greater than NKB greater than SP (bladder), which suggests the presence of NK-2 receptors. This was confirmed by data obtained with two antagonists, one of which was shown to be competitive and selective for NK-2 type receptors. It thus appears that receptors of the NK-2 type are present in humans along the tracheo-bronchial tree and in the urinary system.
Bronchi↗
[Leu9 psi(CH2NH)Leu10]-neurokinin A (4-10) (MDL 28,564) distinguishes tissue tachykinin peptide NK2 receptors.
The neurokinin A analogue, MDL 28,564 (Asp-Ser-Phe-Val-Gly-Leu-CH2NH-Leu-NH2), inhibited 125I-NKA binding to hamster urinary bladder NK2 receptors with a KI of 130 nM. For rat submaxillary gland NK1 receptors and cerebral cortical NK3 receptors, the KI's for MDL 28,564 were greater than 250 microM and greater than 500 microM, respectively. MDL 28,564 did not relax dog carotid artery (NK1 tissue) or contract rat portal vein (NK3 tissue). In guinea-pig trachea tissues, MDL 28,564 stimulated phosphatidylinositol turnover and induced contraction with maximum effects similar to those of neurokinin A. In hamster urinary bladder tissue, MDL 28,564 stimulated phosphatidylinositol turnover with maximum effect only 10% of that of neurokinin A, did not produce sustained contraction itself and antagonized NKA-induced contraction. MDL 28,564 also produced full contraction in rabbit pulmonary artery (NK2 tissue) but was inactive in rat vas deferens (NK2 tissue). These data with MDL 28,564 are consistent with the NK2 receptors in guinea-pig trachea and rabbit pulmonary artery being different from those in hamster urinary bladder and rat vas deferens.
Amino Acid Sequence↗