Comparison of the Binding and Functional Actions of Angiotensin Agonists in Clone 9 Cells: Additional Evidence for Angiotensin II Receptor Heterogeneity

Abstract

The effects of the angiotensin-II (All) agonists and antagonists on both ¹²⁵I-SARILE binding and phosphoinositol (PI) accumulation in clone 9 cells were examined. Clone 9 cells, which are derived from rat liver, have been shown to respond to All agonists with an increase in PI accumulation which is inhibitable by Sar₁, Ile₈-AII (SARILE) and DUP-753 but not PD-123319, suggesting that they possess the AT₁ subtype of All receptor. The present results confirmed these properties. The order of potency of AII agonists was AII> AIII> AI. Clone 9 cells also possessed binding sites for ¹²⁵I-SARILE. The majority of these were AT₁ type receptors, although a small number of AT₂ receptors may also have been present. The order of potency of All agonists in inhibiting ¹²⁵-SARILE binding was All » AIII> = AI. The difference in rank order of potency between the functional and binding assays was due to AIII being much less potent in the binding assay than the functional assay. Since the potency of AIII relative to AII was lower than that at either AT₁ or AT₂ subtypes of AII receptor, these data suggest that an additional subtype, with selectively low affinity for AIII, exists.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Analysis of the mechanisms underlying the vasorelaxant action of angiotensin II in the isolated rat carotid

It has been suggested that low concentrations of angiotensin II cause vasoconstriction whereas high concentrations evoke vasodilation. Thus, this work aimed to functionally characterize the mechanisms underlying the relaxation induced by angiotensin II at high concentrations in isolated rat carotid rings. Experiments using standard muscle bath procedures showed that angiotensin II (0.01-3 μM) concentration dependently induces relaxation of phenylephrine-pre-contracted rings. No differences between intact or denuded endothelium were found. The angiotensin II-induced relaxation was strongly inhibited by saralasin, the non-selective antagonist of angiotensin II receptors but not by the selective antagonists of AT₁ and AT₂ receptors, losartan and PD123319, respectively. However, A-779, a selective angiotensin-(1-7) receptor antagonist, reduced the relaxation induced by angiotensin II. Administration of exogenous angiotensin-(1-7) on pre-contracted tissues produced concentration-dependent relaxation, which was also inhibited by A-779. HOE-140, the selective antagonist of the bradykinin in B₂ receptor did not produce any significant effect on angiotensin II-induced relaxation. Pre-incubation of denuded-rings with N G-nitro-l-arginine methyl ester (l-NAME) or 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced angiotensin II-induced relaxation. On the other hand, neither indomethacin nor tetraethylammonium (TEA) produced any significant effect. The major new finding of this work is that high concentrations of angiotensin II induce relaxation of the rat carotid via activation of the NO-cGMP pathway through a mechanism that seems to be partially dependent on activation of angiotensin-(1-7) receptors.     

Intracerebroventricular Administration of AT1 Receptor Antisense Oligonucleotides Inhibits the Behavioral Actions of Angiotensin II

Abstract: Antisense Oligonucleotides were developed to study the expression and function of angiotensin type 1 (AT₁) receptors in cultured cells and brain. In both liver epithelial WB and neuro-blastoma N1E-115 cells AT₁ antisense oligomers substantially decreased AT₁ receptor density, whereas angiotensin type 2 (AT₂) receptors remained unchanged. Similarly, repeated intracerebroventricular injections of AT₁ antisense oligomers in rats decreased AT₁ receptor density in hypothalamic-thalamic-septal tissue, and AT₂ receptors were unaffected. Intracerebroventricular antisense oligomers also attenuated drinking elicited by intra-cerebroventricular angiotensin II but not the cholinomimetic carbachol. Collectively, these results demonstrate that antisense Oligonucleotides attenuate angiotensin receptor expression and function in behaving animals.     

Novel O-[11C]methylated derivatives of candesartan as angiotensin II AT1 receptor imaging ligands: Radiosynthesis and ex vivo evaluation in rats

[¹¹C]Methyl-candesartan and its desethyl derivative ([¹¹C]TH4) were developed as potential radiotracers for imaging angiotensin II (Ang II) type 1 (AT₁) receptors. These compounds were synthesized via methylation of tetrazole-protected candesartan using [¹¹C]methyl iodide followed by deprotection through HCl hydrolysis at 65 °C to produce [¹¹C]methyl-candesartan, and 90 °C for [¹¹C]TH4. Ex vivo biodistribution and competition studies were carried out for both [¹¹C]methyl-candesartan and [¹¹C]TH4 to assess tissue retention time course and binding selectivity. Besides the liver, [¹¹C]methyl-candesartan and [¹¹C]TH4 displayed highest tissue retention in the AT₁ receptor-rich renal cortex and outer medulla. At tracer doses 15 min post-injection, [¹¹C]methyl-candesartan demonstrated higher specific binding proportion for AT₁ receptors, and selectivity for AT₁ over Ang II AT₂, Mas, β-adrenergic, and α₂-adrenergic receptors in rat kidneys compared to [¹¹C]TH4. This study indicates that [¹¹C]methyl-candesartan has potential for in vivo imaging renal AT₁ receptors selectively using positron emission tomography.     

Affinity Purification of Angiotensin Type 2 Receptors from N1E-115 cells: Evidence for Agonist-Induced Formation of Multimeric Complexes

Abstract: The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (Angll) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT₂-receptor subtype, whereas the density of the AT₁ receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) selectively solubilized AT₂receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (Angll) during affinity purification of AT₂ receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar¹, lle⁸-Angll was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT₂-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar¹, lle⁸-Angll, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT₁-receptor antagonist, was completely ineffective in eluting any Angll receptors from the affinity column, further confirming their AT₂ identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions. The 110-kDa and 66-kDa proteins, but not smaller, affinity-purified proteins, specifically bound ¹²⁵l-Angll as determined by covalent cross-linking of ¹²⁵l-Angll to the receptors with the homobifunctional cross-linker disuccinimidyl suberate, or by size exclusion chromatography on a TSK 3000 SW column. Lastly, immunoblot analysis of affinity- purified material with antibodies selective for AT₂ receptors revealed major immunoreactive proteins of 110 kDa and 66 kDa in the presence of an agonist, whereas the same 66-kDa protein, as well as a smaller (54-kDa) immunoreactive protein, was detected under reducing conditions. Collectively, these data suggest that CHAPS-solubilized AT₂ receptors from N1E-115 cells may consist of a binding protein of approximately 66 kDa, which in the presence of an agonist readily associates with other smaller proteins to form larger multimeric complexes.     

The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT₁ receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B₁ and B₂ bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT₁-B₂ receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT₁ agonist, [Sar¹,Ile⁴,Ile⁸]-AngII, but not the neutral AT₁ antagonist, losartan, inhibits endogenous B₂ receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar¹,Ile⁴, Ile⁸]-AngII require the AT₁ receptor and result from arrestin-dependent co-internalization of AT₁-B₂ heterodimers. BRET₅₀ measurements indicate that AT₁ and B₂ receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar¹,Ile⁴,Ile⁸]-AngII blunts B₂ receptor activation of G_q/11-dependent intracellular calcium influx and G_i/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar¹,Ile⁴,Ile⁸]-AngII has no effect on B₂ receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar¹,Ile⁴,Ile⁸]-AngII promotes internalization of AT₁-B₂ heterodimers. Thus, [Sar¹,Ile⁴,Ile⁸]-AngII exerts lateral allosteric modulation of B₂ receptor signaling by binding to the orthosteric ligand binding site of the AT₁ receptor and promoting co-sequestration of AT₁-B₂ heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT₁ receptor ligands may translate into different pharmacologic actions.     

Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E-115 Cells

Abstract: The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT₂ subtype. However, displacement of ¹²⁵I-Angll with the AT₂ nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT₂ receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound ¹²⁵I-Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ～80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT₂ receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT₁-selective antagonist Losartan. However, whereas the nonpeptidic AT₂-selective antagonist PD-123319 completely displaced the binding of ¹²⁶I-Angll from peak I in a monophasic fashion (IC₅₀= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD-123319 was much less effective in displacing ¹²⁵I-Angll from peak III (IC₅₀= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in ¹²⁵I-Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT₂ receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated ¹²⁵I-Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT₂ receptors.     

Alpha-adrenergic receptors in liver membranes: delineation with subtype selective radioligands

Alpha₁ and alpha₂ adrenergic receptors have previously been demonstrated in rat liver membranes by competition curves of [³H]dihydroergocryptine ([³H]DHE) with the alpha₁ selective antagonist prazosin (B.B. Hoffman, D. Mullikin-Kilpatrick and R.J. Lefkowitz, J. Biol. Chem. $\text{255}$:4645–4652, 1980). The present studies have utilized the radioligands [³H]prazosin and [³H]yohimbine to further define alpha receptors in rat liver membranes. [³H]Prazosin was found to label alpha₁ receptors whereas [³H]yohimbine labelled alpha₂ receptors. The proportion of alpha₁ and alpha₂ receptors determined directly with these radioligands (79% and 20% respectively) was in good agreement with the proportions determined previously with [³H]DHE. Guanine nucleotides were found to reduce the affinity of the agonist epinephrine at the alpha₂ sites labelled by [³H]yohimbine but not at the alpha₁ sites labelled by [³H]prazosin. These results have implications for the interpretation of agonist interactions with alpha receptors in liver membranes.     

Perinatal Nicotine Exposure Increases Angiotensin II Receptor-Mediated Vascular Contractility in Adult Offspring

Previous studies have reported that perinatal nicotine exposure causes development of hypertensive phenotype in adult offspring.

Aims

The present study was to determine whether perinatal nicotine exposure causes an epigenetic programming of vascular Angiotensin II receptors (ATRs) and their-mediated signaling pathway leading to heightened vascular contraction in adult offspring.

Main methods

Nicotine was administered to pregnant rats via subcutaneous osmotic minipumps from day 4 of gestation to day 10 after birth. The experiments were conducted at 5 months of age of male offspring.

Key Findings

Nicotine treatment enhanced Angitension II (Ang II)-induced vasoconstriction and 20-kDa myosin light chain phosphorylation (MLC₂₀-P) levels. In addition, the ratio of Ang II-induced tension/MLC-P was also significantly increased in nicotine-treated group compared with the saline group. Nicotine-mediated enhanced constrictions were not directly dependent on the changes of [Ca²⁺]_i concentrations but dependent on Ca²⁺ sensitivity. Perinatal nicotine treatment significantly enhanced vascular ATR type 1a (AT_1aR) but not AT_1bR mRNA levels in adult rat offspring, which was associated with selective decreases in DNA methylation at AT_1aR promoter. Contrast to the effect on AT_1aR, nicotine decreased the mRNA levels of vascular AT₂R gene, which was associated with selective increases in DNA methylation at AT₂R promoter.

Significance

Our results indicated that perinatal nicotine exposure caused an epigenetic programming of vascular ATRs and their-mediated signaling pathways, and suggested that differential regulation of AT₁R/AT₂R gene expression through DNA methylation mechanism may be involved in nicotine-induced heightened vasoconstriction and development of hypertensive phenotype in adulthood.     

AT1 receptors mediate pressor responses induced by angiotensin II in the periaqueductal gray area of rats

Microinjection of angiotensin II (ANGII) (0.01 to 1 nmol) into the periaqueductal gray area (PAG) of anaesthetised rats caused dose-dependent increases in blood pressure. Preinjection (10 min before) of losartan (a selective non-peptide AT₁ receptor antagonist; 50 nmol) to the PAG reduced the pressor response to ANGII whereas PD123319 (a selective non-peptide AT₂ receptor antagonist; 50 nmol) did not affect the ANGII-induced hypertension. Thus, our data suggest that the activation of AT₁ but not AT₂ receptors mediates ANGII-induced blood pressure changes in the PAG area.     

Involvement of the AT1 receptor in the venoconstriction induced by angiotensin II in both the inferior vena cava and femoral vein

Although angiotensin II-induced venoconstriction has been demonstrated in the rat vena cava and femoral vein, the angiotensin II receptor subtypes (AT₁ or AT₂) that mediate this phenomenon have not been precisely characterized. Therefore, the present study aimed to characterize the pharmacological receptors involved in the angiotensin II-induced constriction of rat venae cavae and femoral veins, as well as the opposing effects exerted by locally produced prostanoids and NO upon induction of these vasomotor responses. The obtained results suggest that both AT₁ and AT₂ angiotensin II receptors are expressed in both veins. Angiotensin II concentration-response curves were shifted toward the right by losartan but not by PD 123319 in both the vena cava and femoral vein. Moreover, it was observed that both 10⁻⁵ M indomethacin and 10⁻⁴ M L-NAME improve the angiotensin II responses in the vena cava and femoral vein. In conclusion, in the rat vena cava and femoral vein, angiotensin II stimulates AT₁ but not AT₂ to induce venoconstriction, which is blunted by vasodilator prostanoids and NO.     

Angiotensin II receptors: Localization of type I and type II in rat epididymides of different developmental stages

Correspondence to: H.C. Chan--> Abstract. Previous studies from our laboratory have provided evidence for the existence of a local renin-angiotensin system in the rat epididymis. Evidence has also accumulated, indicating that locally formed angiotensin II from the rat epididymis may play a paracrine and/or autocrine role in regulating epididymal electrolyte and fluid transport. In the present study, specific anti-peptide antibodies against the second extracellular loops of angiotensin II type I (AT₁) and type II (AT₂) receptors were used to localize immunocytochemically these receptors in the rat cauda epididymides of three developmental stages, namely, immature (2-week), early mature (6-week) and fully mature (10-week). The immunostaining intensity for AT₁ receptors was found to be stronger than that for AT₂ receptors throughout rat epididymides of all stages. However, the immunostaining for both AT₁ and AT₂ receptors observed in the fully mature rat epididymis was much more intense than that observed in the epididymides of the two younger stages. While the immunostaining for both AT₁ and AT₂ receptors in the younger rat epididymides appeared to be distributed in both basal and apical regions, the immunostaining in the fully mature epididymis was predominantly localized in the basal region. The present finding of the differential patterns of angiotensin II receptor immunoreactivity in three different stages of the rat epididymis may reflect the fine tuning of rat epididymal function by angiotensin II, acting as a paracrine or autocrine agent, during the course of development. Received: 18 December 1995/Revised: 19 December 1996     

[3H]A-69024: A non-benzazepine ligand for in vitro and in vivo studies of dopamine d1 receptors

[³H]A-69024 has been prepared as a radioligand for studying the dopamine D₁ receptor. [³H]A-69024 binds to rat striatal membranes with a K_D = 14.3 ± 3.2 nM (mean ± SEM; n = 3) and B_max = 63.5 ± 12.8 fmol/mg wet tissue (1.8 ± 0.3 pmol/mg protein). This ligand binds to only one site with a Hill coefficient close to unity. The in vivo biodistribution of [³H]A-69024 showed a high uptake in the striatum (5.9 %ID/g) at 5 min followed by clearance. As a measure of specificity, the striatum/cerebellar ratio reached a maximum of 6.7 at 30 min post-injection. Pre-treatment with the D₁ antagonist R(+)SCH 23390 (1 mg/kg) reduced this ratio to unity. The dopamine antagonist (+)butaclamol and unlabeled A-69024 inhibited striatal uptake by 70 and 51%, respectively. Spiperone (D₂/5-HT_2A) and ketanserin (5-HT_2A/5-HT_2C) at doses of 1 mg/kg had no inhibitory effect on [³H]A-69024 uptake in the striatum; however, increased uptake of [³H]A-69024 by > 30% in the whole brain was observed. The selectivity and affinity of [³H]A-69024 suggests that this non-benzazepine radioligand may be useful for in vitro and in vivo studies of the dopamine D₁ receptor.     

Down-Regulation of Angiotensin II Receptor Subtypes and Desensitization of Cyclic GMP Production in Neuroblastoma N1E-115 Cells

Abstract: Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either ¹²⁵I-AngII or the high-affinity antagonist ¹²⁵I-[Sarc¹,Ile⁸]-AngII. This apparent down-regulation was dose related with an ED₅₀ of 1 nM, and maximal decreases of ～90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT₁ receptor subtype during the first 5 min, followed by a decline in both AT₁ and AT₂ receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc¹, Thr⁸]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells. These results suggest that differentiated N1E-115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII receptor subtypes in the nervous system.     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.     

Increased expression of vascular endothelin type B and angiotensin type 1 receptors in patients with ischemic heart disease

Background

Endothelin-1 and angiotensin II are strong vasoconstrictors. Patients with ischemic heart disease have elevated plasma levels of endothelin-1 and angiotensin II and show increased vascular tone. The aim of the present study was to examine the endothelin and angiotensin II receptor expression in subcutaneous arteries from patients with different degrees of ischemic heart disease.

Methods

Subcutaneous arteries were obtained, by biopsy from the abdomen, from patients undergoing coronary artery bypass graft (CABG) surgery because of ischemic heart disease (n = 15), patients with angina pectoris without established myocardial infarction (n = 15) and matched cardiovascular healthy controls (n = 15). Endothelin type A (ET_A) and type B (ET_B), and angiotensin type 1 (AT₁) and type 2 (AT₂) receptors expression and function were examined using immunohistochemistry, Western blot and in vitro pharmacology.

Results

ET_A and, to a lesser extent, ET_B receptor staining was observed in the healthy vascular smooth muscle cells. The level of ET_B receptor expression was higher in patients undergoing CABG surgery (250% ± 23%; P < 0.05) and in the patients with angina pectoris (199% ± 6%; P < 0.05), than in the healthy controls (100% ± 28%). The data was confirmed by Western blotting. Arteries from CABG patients showed increased vasoconstriction upon administration of the selective ET_B receptor agonist sarafotoxin S6c, compared to healthy controls (P < 0.05). No such difference was found for the ET_A receptors. AT₁ and, to a lesser extent, AT₂ receptor immunostaining was seen in the vascular smooth muscle cells. The level of AT₁ receptor expression was higher in both the angina pectoris (128% ± 25%; P < 0.05) and in the CABG patients (203% ± 41%; P < 0.05), as compared to the healthy controls (100% ± 25%). The increased AT₁ receptor expression was confirmed by Western blotting. Myograph experiment did however not show any change in vasoconstriction to angiotensin II in CABG patients compared to healthy controls (P = n.s).

Conclusion

The results demonstrate, for the first time, upregulation of ET_B and AT₁ receptors in vascular smooth muscle cells in ischemic heart disease. These receptors may play a role in the pathophysiology of ischemic heart disease and could provide important targets for pharmaceutical interventions.     

CARDIOVASCULAR RHYTHMS AND CARDIAC BAROREFLEX SENSITIVITY IN AT1A RECEPTOR GAIN-OF-FUNCTION MUTANT MICE

A mutant mouse expressing a gain-of-function of the AT_1A angiotensin II receptor was engineered to study the consequences of a constitutive activation of this receptor on blood pressure (BP). Cardiovascular rhythms and spontaneous cardiac baroreflex sensitivity (BRS) were evaluated using telemetric BP recordings of five transgenic (AT_1AMUT) and five wild (AT_1AWT) mice. The circadian rhythms were described with the Chronos-Fit program. The gain of the transfer function between systolic BP (SBP) and pulse intervals used to estimate the spontaneous BRS (ms/mmHg) was calculated in the low frequency (0.15–0.60?Hz) band. Transgenic AT_1AMUT exhibited higher BP and heart rate (HR) levels compared to controls (SBP AT_1AMUT 134.6?±?5.9?mmHg vs. AT_1AWT 110.5?±?5.9; p?<?0.05; HR AT_1AMUT 531.0?±?14.9 vs. AT_1AWT 454.8?±?5.4 beats/min; p?=?0.001). Spontaneous BRS was diminished in transgenic mice (AT_1AMUT 1.23?±?0.17?ms/mmHg vs. AT_1AWT 1.91?±?0.18?ms/mmHg; p?<?0.05). Motor activity did not differ between groups. These variables exhibited circadian changes, and the differences between the strains were maintained throughout the cycle. The highest values for BP, HR, and locomotor activity were observed at night. Spontaneous BRS varied in the opposite direction, with the lowest gain estimated when BP and HR were elevated (i.e., at night, when the animals were active). It is likely the BP elevation of the mutant mice results from the amplification of the effects of AngII at different sites. Future studies are necessary to explore whether AT_1A receptor activation at the central nervous system level effectively contributed to the observed differences. (Author correspondence: jean-luc.elghozi@nck.aphp.fr)     

II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.