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(Hypertension. 1998;31:1171-1177.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Angiotensin AT_1B Receptor Mediates Calcium Signaling in Vascular Smooth Muscle Cells of AT_1A Receptor–Deficient Mice

Zhiming Zhu; Sunny H. Zhang; Charlotte Wagner; Armin Kurtz; Nobuyo Maeda; Thomas Coffman; ; William J. Arendshorst

From the Departments of Physiology (Z.Z., W.J.A.) and Pathology (S.H.Z., N.M.), University of North Carolina at Chapel Hill; the Department of Medicine, Duke University, Durham, NC (T.C.), and the Institute of Physiology, University of Regensburg (Germany) (C.W., A.K.).

	Abstract

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Abstract—Our studies on angiotensin II receptor subtype 1A (AT_1A) knockout mice define how endogenous receptors other than AT_1A receptors stimulate changes in cytosolic calcium concentration ([Ca²⁺]_i) in cultured aortic vascular smooth muscle cells (VSMCs). Wild-type cells have a 1.7 ratio of AT_1A/AT_1B receptor mRNA as determined by semiquantitative reverse transcriptase–polymerase chain reaction. Mutant cells express AT_1B receptor mRNA but not that for the AT_1A receptor. In wild-type cells with AT_1A present, Ang II (10^-7 mol/L) produces a characteristic rapid peak increase in [Ca²⁺]_i of 150 to 180 nmol/L, followed by a plateau phase characterized by a sustained 70 to 80 nmol/L increase in [Ca²⁺]_i. An unexpected finding was that the magnitude and time-dependent pattern of [Ca²⁺]_i changes produced by Ang II were similar in cells that lacked AT_1A receptors but possessed AT_1B receptors. The response in mutant cells indicates effective coupling of an Ang II receptor to one or more second messenger systems. The similarity of response patterns between cells with and without AT_1A receptors suggests that non-AT_1A receptors are functionally linked to similar signal transduction pathways in mutant cells. The fact that mutant and wild-type cells exhibit similar patterns of calcium mobilization and entry supports the notion that AT_1A and non-AT_1A receptors share common signal transduction pathways. The AT₂ receptor ligands PD-123319 and CGP-42112 do not alter Ang II effects in either VSMC type, suggesting a paucity of AT₂ receptors and/or an absence of their linkage to [Ca²⁺]_i pathways. The nonpeptide AT₁ receptor blocker losartan antagonizes Ang II–induced [Ca²⁺]_i increases in both cell groups, supporting mediation by native AT_1B receptors and effective coupling of this subtype to second messenger systems leading to calcium entry and mobilization. Our results demonstrate that Ang II causes calcium signaling in AT_1A–deficient VSMCs that is mediated by an endogenous losartan-sensitive AT_1B receptor.

Key Words: muscle, smooth, vascular • angiotensin II • receptors, angiotensin • calcium channels • antagonists • losartan • fura 2

	Introduction

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Angiotensin II, a potent hormone or paracrine substance, stimulates cell surface receptors to produce a variety of regulatory actions in the cardiovascular, renal, endocrine, and neural systems.^{1 2 3} The multiple actions of Ang II are mediated by different receptors and various combinations of cell-specific signaling mechanisms. Based on pharmacological criteria, Ang II exerts its actions via two classes of receptors. Radioligand receptor-binding studies using nonpeptide ligands such as losartan and CGP-42112 have defined two distinct types, with losartan having high affinity for AT₁ receptors, whereas CGP-42112 is a selective ligand for the AT₂ receptor.^{1 2 3} AT₁ receptors can be subdivided into AT_1A and AT_1B subtypes, which differ in distribution and regulation among tissues and cell types.^{1 2 3} The AT_1A receptor is the major subtype in the cardiovascular and renal systems.^{4 5} The degree to which the biological effects of Ang II are mediated by the predominant and minority receptor subtype in each cell awaits clarification. Although the current generation of nonpeptide receptor ligands allows differentiation between the major classes of AT₁ and AT₂ receptors, it has not been possible to discriminate between AT_1A and AT_1B subtypes.^{2 3 6 7} Thus, it is not known whether AT_1A and AT_1B receptors are functionally different or similar in natural cells; also unknown are the functional consequences of different relative densities of these receptors in multiple cell types.

Mouse models in which a single receptor is completely eliminated by gene targeting provide an alternative approach to investigate receptor regulation and function.⁸ Coffman, Smithies, Sugaya, and associates (Ito et al,⁹ Sugaya et al¹⁰) have developed and used such a model to investigate ligand binding and functional characterization of endogenous angiotensin receptors other than AT_1A in the AT_1A knockout mouse. Autoradiographic characterization of receptors reveals a markedly reduced density of losartan-sensitive [¹²⁵I]Ang II binding in the kidneys of AT_1A mutant mice. Furthermore, the importance of AT_1A receptors in blood pressure control is suggested by the reduction in basal arterial pressure, attenuated acute pressor responses to administered Ang II, and increased plasma renin activity.^{9 10} Thus, Ang II appears to exert most, if not all, of its effects in the cardiovascular and renal systems via the AT_1A receptor, the predominant subtype in these systems. Little is known about the function of the native AT_1B receptor in cells that normally possess this receptor subtype. AT_1B receptors are assumed to have a minor influence on the smooth muscle function and the regulation of arterial pressure. The mouse model deficient in AT_1A receptors affords a unique opportunity to investigate Ang II effects mediated by the remaining receptor subtypes, including the AT_1B.

The present studies were conducted on VSMCs obtained from wild-type control mice and mutant mice with absent AT_1A receptor. We investigated the ability of Ang II to stimulate [Ca²⁺]_i in cultured aortic VSMCs. A major finding was that Ang II produced changes in [Ca²⁺]_i in VSMCs lacking the AT_1A receptor that were similar in magnitude and overall pattern to changes in cells with AT_1A receptor present. Thus, Ang II stimulated signal transduction mechanisms by a native receptor(s) other than AT_1A in mutant cells. Losartan (DuP 753) effectively antagonized the Ang II–induced increase in [Ca²⁺]_i, reflecting mediation by AT₁ receptors in control cells and AT_1B receptors in VSMCs lacking AT_1A receptors. Possible mediation by AT₂ receptors was eliminated by observations that AT₂ receptor ligands had no effect on Ang II–induced changes in [Ca²⁺]_i in VSMCs with or without AT_1A receptors. Our studies provide new information about the mediation of calcium stimulation by Ang II acting on endogenous AT_1B receptors in mouse aortic VSMCs.

	Methods

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Culture of Aortic VSMCs
Four-month-old adult AT_1A homozygous knockout mice with C57B/6 and 129 mixed genetic background were used; age-matched wild type mice with C57B/6 and 129 F1 backgrounds served as controls.⁹ Mice were anesthetized with Avertin, and the thoracic and abdominal aorta was isolated, extirpated, and cut longitudinally. The internal surface was gently scraped with watchmaker forceps to remove endothelial cells, and the adventitia was removed by stripping with the aid of microscopy and forceps.

Aortic VSMCs were cultured by an explant method using standard methods.¹¹ Briefly, the aortic media was cut into 1- to 2-mm pieces and put into 24-well plates with 100 to 200 µL DMEM-H medium (Gibco BRL) containing 10% fetal calf serum (HyClone), 100 U/mL penicillin, 100 µg/mL streptomycin, 25 µg/mL amphotericin B, and 200 mg/mL L-glutamine incubated at 37°C, in a humidified 5% CO₂/95% air incubator. About 0.5 mL of fresh medium was gently added 3 days later to each well. Cells formed a confluent monolayer in 10 to 14 days. The medium was changed twice weekly. The growth rates of wild-type and AT_1A-deficient cells did not differ appreciably, suggesting that the AT_1A receptor was not essential for growth. Immunocytochemistry was used to verify the presence of smooth muscle–specific -actin using a monoclonal antibody (Clone 1A4; 1:200 dilution, Dako Corp) and to verify the absence of endothelial cell contamination using von Willebrand factor (Dako, 1:50 dilution) as previously described.¹² Reactions of the subcultured VSMCs with these antibodies revealed that all cells contained smooth muscle–specific -actin with an abundance of filaments. No endothelial cell contamination was evident.

Southern Blot Analysis
To determine cell genotypes, genomic DNA was purified from cultured VSMCs with and without AT_1A receptor gene mutation and analyzed by Southern blot analysis.⁹ After digestion of DNA with HindIII, size separation in a 0.8% agarose gel, and transfer to nylon membrane, previously described probes were used to identify generation of 3.3-kb wild-type and 5.0-kb mutant fragments.

RT-PCR for AT_1A and AT_1B mRNA
AT_1A and AT_1B mRNA were determined by RT-PCR, performed as described previously.¹³ Briefly, 2 µg of total RNA was reverse transcribed with an RT mixture consisting of oligo dT (12 to 18) and 200 U of M-MLV RT (Gibco/BRL). After reverse transcription, a small aliquot of RT mixture was used for PCR with sense primer (5'-CCAAAGTCACCTGCATCATC-3') and antisense primer (5'-CACAATCGCCTAATTATCCTA-3'), which are common for both AT_1A and AT_1B receptors.⁷ The PCR reaction was carried out in a total volume of 20 µL containing 3 µL RT mixture, 1 µL of each primer (10 pmol · L^-1 · µL^-1), 5 µL MgCl₂ (25 mmol/L), 2.5 µL 10x PCK buffer, 1 µL dNTP (925 mmol/L), 3 µCi ³H-dCTP (64 Ci · mmol^-1 · L^-1), and one U Taq polymerase (Boehringer Mannheim). To distinguish between AT_1A and AT_1B receptors, amplification products (1.5 µL EcoRI, 25 U/mL) were added to 20 µL of the PCR product obtained with the AT₁ primers. The digestion yielded in fragments of the expected sizes of 128 and 177 bp, as has been visualized with agarose gel electrophoresis and ethidium bromide staining. For quantitative analysis, the PCR products were separated by polyacrylamide gel electrophoresis. N,N'-methylene-bis-acrylamide was replaced by dihydroxyethylene-bis-acrylamide. The bands were excised and solubilized in 0.025 mol/L periodic acid, and radioactivity was measured in a liquid scintillation spectrophotometer.

Measurement of [Ca²⁺]_i
Measurements of [Ca²⁺]_i in cultured VSMCs were performed using the calcium-sensitive dye fura 2-AM as previously described.^{12 14} A monolayer of VSMCs was grown on 22-mm² glass coverslips as described above. Confluent cells were rendered quiescent by maintenance in a serum-free medium for 24 hours before an experiment. Calcium determinations were performed on subcultures between the second and sixth passages. On the day of study, the VSMCs were washed twice in physiological salt solution (PSS, in mmol/L: 135 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 5 D-glucose, 10 HEPES; pH 7.4) and incubated with 4 µmol/L fura 2-AM in 0.02% pluronic F-127 (Molecular Probes Inc) for 60 minutes at room temperature. After fura 2 loading, monolayers were washed twice in PSS, and the cells were centered in the optical field of a x40 oil immersion fluorescence objective of an inverted microscope (Olympus IMT-2). The cells were excited alternately with light of 340- and 380-nm wavelength from dual monochronometers of a Photon Technology International (PTI) dual-excitation wavelength Deltascan (model RMD). Fluorescence was detected with a photon-counting device after passing through a dichroic mirrored barrier filter (510 nm). Fluorescence signal intensity of 20 to 30 cells was acquired, stored, and processed by an IBM-PC–compatible computer and Felix software (PTI), with calibration of [Ca²⁺]_i based on the ratio at 340/380 nm. The [Ca²⁺]_i was calculated according to the formula described by Grynkiewicz et al¹⁴: [Ca²⁺]_i=[(R-R_min)/(R_max-R)]*(S_f/S_b)*K_d, where R is the ratio of the 340/380 nm of the fluorescence signal, R_max is the 340/380 ratio in the presence of saturating calcium, R_min is the 340/380 ratio in calcium-free media with 10 mmol/L EGTA added, and S_f/S_b is the ratio of the 380-nm fluorescence measured in a calcium-free solution to that measured in a calcium-replete solution. The K_d value for fura 2 is 224 nmol/L.¹⁴

The effects of Ang II were determined from changes in [Ca²⁺]_i in response to Ang II concentrations ranging from 10^-13 to 10^-5 mol/L. Measurements were performed on 5 to 30 cell preparations per concentration. After a control recording was made of baseline [Ca²⁺ ]_i for 50 seconds, Ang II was added to the bath chamber containing 100 µL PSS buffer. To avoid possible receptor desensitization with repeated applications, each cell preparation was tested once only. The peak 340/380 ratio recorded during the initial 10 seconds after Ang II addition was equated with the maximal response.

Ang II receptor ligands were used to define which receptor subtypes were coupled to calcium signaling in wild-type and mutant aortic VSMCs. The effects of the AT₁ receptor antagonist losartan and the AT₂ receptor ligands PD-123319 and CGP-42112 were evaluated regarding their ability to attenuate Ang II–induced increases in [Ca²⁺ ]_i.^{1 2 3}

Statistical Methods
Statistical analyses were performed using the SigmaStat software package. Comparisons between two groups were analyzed using Student's unpaired t test. Larger data sets were tested with ANOVA. Results with a value of P<.05 were considered statistically significant. All values reported are mean±SE (number of observations).

	Results

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Cell genotypes were confirmed by Southern blot analysis of cultured VSMCs. Fig 1 shows a 5.0-kb band for cells lacking the AT_1A receptor, which contrasts with the 3.3-kb band present in wild-type control cells. AT₁ receptor subtype mRNA was analyzed using RT-PCR methodology. The results in Fig 2 demonstrate the presence of the AT_1B receptor mRNA in both groups of VSMCs, whereas the AT_1A mRNA is absent in the gene-knockout mice. The ratio of mRNA for AT_1A/AT_1B receptors was 1.7 in wild-type mice. A relative ratio does not apply to cells from knockout mice with only AT_1B mRNA present. Light microscopy showed no discernible differences between the general appearance of subcultured VSMCs derived from control and mutant mice.

View larger version (85K): [in this window] [in a new window]   Figure 1. Genotype analysis of purified genomic DNA from cultured aortic VSMCs from mice with and without AT1A receptor gene mutation assayed by Southern blot analysis. After HindIII restriction enzyme digestion, the wild-type allele was detected as a 3.3-kb fragment, and the disrupted AT1A allele appeared as a 5.0-kb fragment.

View larger version (48K): [in this window] [in a new window]   Figure 2. Determination of AT1A and AT1B mRNA expression in VSMCs with and without AT1A gene using PT-PCR. Ethidium bromide–stained DNA is shown. This was generated from a total of 2 µg RNA derived from wild-type and AT1A knockout cells. The PCR was run for 35 cycles with use of AT1A- and AT1B-specific primers as described in "Methods." Lanes 1 and 2 show the expression of the AT1A and AT1B mRNA in wild-type cells; lanes 3 and 4 show the expression of the AT1B mRNA in AT1A knockout cells; lanes 4 and 6 are internal standard for wild-type cells; and lanes 7 and 8 are internal standard for AT1A knockout cells.

Functional activity of endogenous Ang II receptors was evaluated by signal transduction in aortic VSMCs. Fura 2 fluorescence was used to determine the ability of different concentrations of Ang II to produce a rapid change in [Ca²⁺]_i. Before Ang II addition, the basal [Ca²⁺]_i was greater in AT_1A mutant cells than in wild-type control cells: 96±2 (n=74) versus 74±2 (n=68) nmol/L, P<.001. The reason for this difference is not known, but it may reflect changes in the relative importance of various calcium entry versus mobilization pathways under basal conditions. As noted below, the resting control values persist when the external medium is rendered calcium free for a brief period to minimize calcium entry and suggest involvement of intracellular sources.

Stimulation of VSMCs with Ang II caused concentration-dependent increases in [Ca²⁺]_i. After the basal [Ca²⁺]_i was recorded for 50 seconds, Ang II was added to maintain stimulation for the duration of a recording period of 250 seconds. A peak [Ca²⁺]_i increase was observed within the initial 30 seconds following addition of Ang II. Fig 3 presents the summarized data for control and mutant cells. A major finding was that the maximum [Ca²⁺]_i response was as large in mutant cells as it was in wild-type cells studied under identical conditions. Each preparation was challenged only once with a given concentration of Ang II to eliminate uncertainty that may result from receptor desensitization and increased variability with repeated applications.

View larger version (19K): [in this window] [in a new window]   Figure 3. Dose-dependent increases in [Ca2+]i produced by Ang II (10-13 to 10-5 mol/L). Each mean represents 5 to 30 measurements. The changes in [Ca2+]i were similar in AT1A knockout and wild-type control VSMCs.

The time-dependent changes in [Ca²⁺]_i showed a characteristic peak response immediately after Ang II addition, followed by a decline that plateaued at a sustained level for the duration of the recording period. The averaged responses to Ang II (10^-7 mol/L) in all cell preparations are presented in Fig 4. Interestingly, the maximum change in [Ca²⁺]_i did not differ between mutant and control cells [180±24 (30) versus 155±28 (20) nmol/L, P>.1]. Likewise, the steady-state increases recorded at 200 seconds were similar in both groups of cells [70±9 (30) in mutant versus 79±11 (20) nmol/L in control cells, P>.2]. The plateau levels averaged 36±21% and 57±6% of the maximum response (P>.1). The general shape of the calcium response is similar to that previously reported for an Ang II effect on rat aortic VSMCs.¹²

View larger version (14K): [in this window] [in a new window]   Figure 4. Temporal [Ca2+]i responses to stimulation by Ang II. After a baseline recording for 50 seconds, Ang II (10-7 mol/L) was added and remained present for the duration of the recording period. The increases in [Ca2+]i were characterized by a rapid increase to a peak, followed by a decline to a level sustained throughout the recording period in wild-type cells (left, n=20) and AT1A knockout cells (right, n=30).

To determine the contribution of calcium entry versus mobilization from internal stores after activation of native AT₁ receptor subtypes in VSMCs, the [Ca²⁺]_i response was evaluated in a calcium-free medium, achieved by adding 5 mmol/L EGTA to an otherwise normal solution containing 1 mmol/L CaCl₂ at 10 seconds before starting a recording. Preliminary studies showed that short-term exposure to EGTA had no effect on basal [Ca²⁺]_i. Calcium responses to Ang II were strongly dependent on calcium entry in both control and AT_1A mutant cells (Fig 5). In the calcium-free medium, stimulation by Ang II (10^-7 mol/L) produced smaller changes in [Ca²⁺]_i, with attenuation of both the peak and plateau phases. Ang II produced a peak increase in [Ca²⁺]_i of 103±29 (7) nmol/L in cells with only AT_1B receptors compared with a 82±25 (10)–nmol/L increase in control cells with both AT₁ receptors. These peak responses in calcium-free medium (line with circles) were 57±16% and 53±16% of the changes observed in mutant and control cells, respectively, compared with responses when calcium was present in the bathing medium (line with triangles). In addition, the sustained plateau phase was abolished as the initial calcium transient returned to baseline levels. This observation indicates that calcium entry is responsible for approximately one half of the peak response and almost all of the sustained increase in [Ca²⁺]_i that follows stimulation of AT₁ receptors. On the other hand, about 50% of the initial [Ca²⁺]_i response was independent of external calcium, suggesting a major contribution of calcium mobilization.

View larger version (16K): [in this window] [in a new window]   Figure 5. Temporal effects of Ang II on [Ca2+]i in a calcium-free medium. The extracellular Ca2+ was chelated by adding 5 mmol/L EGTA 10 seconds before the control recording was started. The Ang II (10-7 mol/L)–induced maximum increase in [Ca2+]i was reduced by 50% in the calcium-free medium (line with circles) compared with the mean response observed in normal medium containing 1 mmol/L CaCl2 (line with triangles). The sustained plateau phase was abolished in the calcium-free medium. Similar responses were recorded in wild-type (left, n=10) and AT1A knockout cells (right, n=7).

In other studies we evaluated the effect of an AT₁ receptor antagonist on Ang II–induced [Ca²⁺]_i responses. In control cells (Fig 6, left panel), losartan (10^-5 or 10^-7 mol/L) markedly antagonized the response to Ang II (10^-7 mol/L). The degree of inhibition was about 80%. An important observation was that losartan likewise caused marked inhibition of Ang II–induced changes in [Ca²⁺]_i in mutant cells lacking AT_1A receptors. Furthermore, we demonstrated that the AT₂ receptor is either absent or nonfunctional in calcium signaling in mouse VSMCs. The AT₂ receptor ligand CGP-42112 had no discernible effect on the ability of Ang II to elicit [Ca²⁺]_i increases in cells with the AT_1A receptor present or absent [84±17% (6) versus 91±22% (13) of Ang II effect, respectively]. Likewise, another AT₂ receptor ligand, PD-123319, failed to influence the ability of Ang II to stimulate [Ca²⁺]_i; in the presence of the PD compound, Ang II elicited a normal response averaging 93±33% (7) in control and 94±31% (7) in mutant cells.

View larger version (50K): [in this window] [in a new window]   Figure 6. Effects of the AT1 receptor antagonist losartan and AT2 receptor ligands CGP-42112 and PD-123319 on [Ca2+]i response to Ang II (10-7 mol/L). Mean inhibitory effects are shown for wild-type cells (left) and for AT1A knockout cells (right). Each group consisted of at least 10 determinations.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study provides new information about the ability of Ang II to produce rapid changes in [Ca²⁺]_i via stimulation of endogenous Ang II receptors in cultured mouse aortic VSMCs. In control cells obtained from wild-type mice, Ang II initiates a calcium response by acting on receptors inhibited by the AT₁ receptor antagonist losartan. In contrast, the AT₂ receptor agents PD-123319 and CGP-42112 have no discernible effect on either cytosolic calcium before addition of Ang II or the calcium response to Ang II. Our observations agree with previous reports that these agents act as specific antagonists without any partial agonist effects in rat VSMCs.^{15 16} Earlier studies on rat aortic VSMCs establish that losartan effectively and almost completely attenuates the [Ca²⁺]_i response to Ang II.^{14 17 18 19}

Although cardiovascular and renal cells in rodents express both AT_1A and AT_1B receptors, the AT_1A subtype predominates. AT₂ receptors are prevalent in young animals but sparse in adult animals.^{1 2 3} We observed a ratio of 1.7 for mRNA for AT_1A/AT_1B receptors in VSMCs of wild-type mice with semiquantitative RT-PCR methodology. This value does not differ appreciably from previous reports for AT_1A/AT_1B mRNA ratios of 1 to 2 for rat aortic VSMCs.^{4 5} By means of gene targeting, VSMCs of mutant mice lack the native AT_1A receptor. The absence of mRNA for this receptor is confirmed by RT-PCR. Semiquantitative RT-PCR suggests that AT_1B receptor expression is upregulated in the absence of AT_1A receptors.

Our results demonstrate that Ang II produces increases in [Ca²⁺]_i in mouse VSMCs by stimulating two basic signal transduction pathways. About one half of the calcium response is mediated by calcium entry across the plasma membrane, as evidenced by attenuated responses to Ang II when calcium entry is prevented by short-term EGTA addition to the medium. The fact that roughly one half of the [Ca²⁺]_i change persists in a nominally calcium-free medium implicates a second major hormone-responsive calcium-regulating site involving calcium mobilization from intracellular reserves. These results contrast with previous studies on cultured rat aortic VSMCs that generally point to a predominant, if not exclusive, role for calcium mobilization.^{19 20} Recent evidence, however, supports calcium entry as a more important mechanism in renal resistance vessels examined in isolation and in vivo.^{19 20 21}

The present findings provide new evidence that a losartan-sensitive Ang II receptor can elicit rather normal changes in [Ca²⁺]_i in VSMCs lacking AT_1A receptors and demonstrate that an endogenous non-AT_1A, perhaps the AT_1B receptor, is functionally linked to several signal transduction pathways in VSMCs of AT_1A knockout mice. The surprising finding of a robust [Ca²⁺]_i response in mutant VSMCs advances new information about stimulation of cytosolic calcium mechanisms. The magnitude and shape of the overall response pattern in the knockout cells does not differ appreciably from wild-type cells. Thus, one can conclude that similar hormone-responsive calcium-regulating steps trigger calcium entry and mobilization mediating Ang II effects in VSMCs with and without native AT_1A receptors; the relative proportions of calcium mechanisms are similar.

A goal of our studies was to obtain information about the endogenous receptor(s) utilized by AT_1A knockout cells. In addition to gene targeting, we used currently available pharmacological agents that are known to act in a selective manner on either AT₁ or AT₂ receptors.^{1 2 3} In wild-type VSMCs, the major calcium response to Ang II is mediated by AT₁ receptors that are antagonized by losartan, indicating mediation by AT_1A, AT_1B, or both. The presence or effect of an AT₂ receptor is essentially nonexistent based on the relatively low abundance revealed by RT-PCR and the absence of an AT₂ receptor ligand effect on cytosolic calcium. Interestingly, losartan blocks Ang II effects on [Ca²⁺]_i as effectively in AT_1A knockout cells as in wild-type VSMCs. Thus, AT₁ receptors are responsible for Ang II–induced calcium changes in both cell types. One can make a reasonable prediction based on known receptor distribution as indicated by mRNA expression, assuming each has a similar coupling efficiency and assuming that AT₁ receptors are the only mediator of Ang II effects on [Ca²⁺]_i. Using this approach, the relative contribution of the AT_1B receptor to the observed changes in [Ca²⁺]_i is estimated to be 35% to 40% in wild-type cells and 100% in cells devoid of AT_1A receptors. In view of the qualitative and quantitative characteristics of [Ca²⁺]_i changes to Ang II stimulation in mutant cells, it is reasonable to postulate that AT_1B receptor expression and function are stimulated. Receptor density and/or the efficiency of signal transduction were probably enhanced in AT_1A–deficient VSMCs, since the overall responses to a given concentration of Ang II were similar in VSMCs with the dominant subtype, either AT_1A in control cells or AT_1B in mutant cells. Such an increased functional role may reflect an upregulation and/or more efficient linkage to second messenger systems in VSMCs lacking AT_1A receptors. Our semiquantitative RT-PCR results support the notion of AT_1B receptor upregulation. Consistent with these conclusions, previous studies show a similar inhibitory efficacy of losartan on Ang II binding to transfected AT_1A and AT_1B receptors.^{22 23} Nevertheless, we cannot exclude the possibility that a losartan-sensitive receptor other than, or in addition to, the AT_1B participates in signal transduction in VSMCs devoid of the AT_1A receptor.

An alternative approach to using pharmacological agents is to transfect cDNA into naive host cells. Such studies provide the basis for potential actions of specific receptor coupling to second messenger systems. It should be emphasized, however, that much of current understanding of AT₁ subtype interactions with intracellular signal transduction pathways is based on insertion of cloned receptors into cells that normally lack Ang II receptors and multiple putative controllers of the various messenger systems. Expression of cloned mammalian AT₁ receptors in surrogate systems such as Chinese hamster ovary cells or COS-7 cells demonstrate similar pharmacological properties with regard to ligand binding and displacement by antagonists. There are no major affinity or antagonist differences in the ability of losartan to displace Ang II binding to rodent AT_1A and AT_1B receptors,^{6 22 24 25} although losartan may be slightly more potent at AT_1B than AT_1A receptors.^{7 23} Losartan can block in vivo and in vitro Ang II stimulation of aldosterone production by the adrenal glomerulosa, cells with a predominance of AT_1B receptors.¹⁵

Transfection of AT_1A receptors reveals that signaling may occur via multiple pathways and that transduction may differ among cell lines.^{23 26 27} Receptor subtype may account for differences in Ang II effects on the shape of temporal [Ca²⁺]_i response. The transfected AT_1A receptor usually elicits a rapid, spikelike [Ca²⁺]_i increase, followed by a sustained plateau phase.^{28 29} Ang II stimulation of the transfected AT_1B receptor is reported to elicit a weaker and less reproducible response. In some cases, only 14% of the preparations display calcium responses.⁶ AT_1B receptor stimulation is characterized by a rapid [Ca²⁺]_i increase that subsequently decays to the baseline without a plateau phase.^{6 7} Another apparent AT₁ subtype difference is noted in steady-state dose-response curves. AT_1A receptors usually trigger a typical sigmoid-shaped concentration-[Ca²⁺]_i response curve indicative of saturation.^{7 30} In contrast, AT_1B receptor stimulation can produce a biphasic or inverted bell-shaped response, with low Ang II concentrations stimulating [Ca²⁺]_i and high concentrations producing less stimulation, suggesting receptor desensitization.^{7 30} The biphasic response to AT_1B receptor stimulation is reminiscent of the dual stimulatory and inhibitory effects of Ang II on proximal tubular reabsorption.²

While informative, it is doubtful that an inserted foreign receptor couples or interacts with the plasma membrane and all intracellular intermediates in naive host cells in a functional manner identical to those stimulated by an endogenous receptor in its natural effector cells. Studies on natural cells are required to establish whether such information about transfected receptors pertains, and to what extent, to specific cell types that normally possess the functional receptors subject to physiological coupling and control. In this regard, animal models with genetic engineering provide an attractive alternative approach to investigate receptor regulation and function in a wide range of normal target cells.

We provide new evidence that endogenous Ang II receptors other than the AT_1A can play a critical role in calcium signaling in VSMCs of rodents. A functional role of non-AT_1A receptors is indicated by the recent observations showing that inhibition of Ang II production by angiotensin-converting enzyme inhibition reduces arterial pressure in AT_1A-deficient mice and that administered Ang II can elicit an acute pressor response after reducing endogenous levels of Ang II.²⁶ This pressor response to Ang II is probably mediated by the AT_1B receptor as it is inhibited by losartan.²⁶ Nevertheless, it is noteworthy that the in vivo pressor response to exogenous Ang II in AT_1A-deficient mice is considerably attenuated, producing smaller effects than might be predicted based on the relatively normal ability of Ang II to stimulate [Ca²⁺]_i in VSMCs in vitro. This apparent discrepancy may reflect one or more differences. One explanation relates to differences in vessel type between conduit arteries and resistance arterioles and the relative strength of AT₁ receptor activation and its coupling on calcium signaling mechanisms and contraction. Resistance VSMCs contribute more importantly in total peripheral vascular resistance, and yet our understanding of the precise mechanisms of calcium signal transduction and calcium-contraction coupling in these microcirculatory cells is less than complete. In addition, receptor subtypes may determine participation of regional vascular beds in the integrated arterial pressure and total peripheral vascular resistance responses to Ang II. Another possibility is that in vitro observations in isolated cells do not accurately predict integrated functional responses in vivo. As a corollary, [Ca²⁺]_i changes in cultured aortic VSMCs may not directly translate to contractile function of resistance vessels. Also, we cannot rule out the possibility that cultured aortic VSMCs undergo phenotypic alterations that are expressed in calcium signaling. For example, culturing may induce or magnify calcium coupling of the AT_1B receptor. However, there is no obvious reason to suspect differential changes in aortic cells cultured from two mouse strains of close genetic background under identical conditions. Further investigations are required to address these issues and provide more insight into extrapolation from changes in [Ca²⁺]_i in aortic VSMCs and cultured arterial or arteriolar VSMCs to functional roles in regulating resistance in specific arterioles.

Previous evidence suggests that humans have no AT_1B receptor. In rodents the AT_1B may be redundant and share functional properties with the AT_1A receptor. Our recent studies on VSMCs from renal resistance arterioles demonstrate parallel regulation of AT_1A and AT_1B receptors in response to changes in salt intake and activity of the renin-angiotensin system.²⁷ On the other hand, several lines of evidence suggest different AT₁ receptor subtype distribution and regulation in rats and mice.^{1 2 3 4 5 6 24 31} In addition, arterial pressure in mice is affected differently by gene targeting of either the AT_1A or AT_1B receptor.^{9 10 32} A pathophysiological role is suggested by cosegregation studies that indicate a link between the AT_1B receptor and models of hypertension.³³ Another potentially important function of an AT_1B or undefined receptor may be proliferation and maturation of renal cells in general and in VSMCs in particular.^{34 35} To the extent that this variation reflects physiological differences, humans may have a heretofore unrecognized receptor that shares similarities with the AT_1B.

In summary, we show that Ang II can activate an endogenous AT_1B receptor to elicit changes in [Ca²⁺]_i in VSMCs isolated from AT_1A knockout mice. This Ang II receptor is functionally coupled to at least two calcium mechanisms leading to increased calcium entry and mobilization. The relative contributions of these two pathways appear to be similar in VSMCs with native AT_1A receptors present or deleted. Losartan blocks Ang II–induced [Ca²⁺]_i increases in mutant VSMCs expressing AT_1B receptors, as well as wild-type VSMCs having AT_1A receptors. Thus, endogenous losartan-sensitive AT_1B as well as AT_1A receptors can be coupled to pathways leading to calcium entry and calcium mobilization in VSMCs.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 = angiotensin II receptor type 1 AT1A = angiotensin II receptor subtype 1A AT1B = angiotensin II receptor subtype 1B AT2 = angiotensin II receptor type 2 [Ca2+]i = cytosolic free calcium concentration RT-PCR = reverse transcriptase–polymerase chain reaction VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by grants-in-aid from the National Institutes of Health Heart, Lung, and Blood Institute (HL-02334 and HL-56122) and a Fellowship Award (NC-96-FW-29) from the American Heart Association, North Carolina Affiliate. The pharmacological agents losartan, PD-123319, and CGP-42112 were generous gifts of DuPont-Merck Pharmaceutical Co, Park Davis Pharmaceutical Co, and Novartis-Ciba Geigy Ltd, respectively.

	Footnotes

 
Reprint requests to William J. Arendshorst, PhD, Department of Physiology, CB #7545, Room 152, Medical Sciences Research Bldg, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545.

Received May 12, 1997; first decision June 17, 1997; accepted December 23, 1997.

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