Spatial and temporal traction response in human airway smooth muscle cells

Tractions that cells exert on theirsubstrates are essential in cell spreading, migration, and contraction.These tractions can be determined by plating the cells on a flexiblegel and measuring the deformation of the gel by using fluorescent beadsembedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we usethe recently developed method of Fourier transform traction cytometry(FTTC). The ICM and FTTC methods are applied to human airway smoothmuscle cells during stimulation with the contractile agonist histamineor the relaxing agonist isoproterenol. The overall intensity of thecell contraction (the median traction magnitude, the energy transferredfrom the cell to the gel, and the net contractile moment) increasedafter activation with histamine, and decreased after treatment withisoproterenol. Cells exhibited regional differences in the time courseof traction during the treatment. Both temporal evolution and magnitudeof traction increase induced by histamine varied markedly amongdifferent cell protrusions, whereas the nuclear region showed thesmallest response. These results suggest that intracellular mediatorsof cell adhesion and contraction respond to contractile stimuli withdifferent rates and intensities in different regions of the cell.

     

Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells

The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness (G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ((t)) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18(t) + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.     

Micropatterning tractional forces in living cells

Here we describe a method for quantifying traction in cells that are physically constrained within micron-sized adhesive islands of defined shape and size on the surface of flexible polyacrylamide gels that contain fluorescent microbeads (0.2-microm diameter). Smooth muscle cells were plated onto square (50 x 50 microm) or circular (25- or 50-microm diameter) adhesive islands that were created on the surface of the gels by applying a collagen coating through microengineered holes in an elastomeric membrane that was later removed. Adherent cells spread to take on the size and shape of the islands and cell tractions were quantitated by mapping displacement fields of the fluorescent microbeads within the gel. Cells on round islands did not exhibit any preferential direction of force application, but they exerted their strongest traction at sites where they formed protrusions. When cells were confined to squares, traction was highest in the corners both in the absence and presence of the contractile agonist, histamine, and cell protrusions were also observed in these regions. Quantitation of the mean traction exerted by cells cultured on the different islands revealed that cell tension increased as cell spreading was promoted. These results provide a mechanical basis for past studies that demonstrated a similar correlation between spreading and growth within various anchorage-dependent cells. This new approach for analyzing the spatial distribution of mechanical forces beneath individual cells that are experimentally constrained to defined sizes and shapes may provide additional insight into the biophysical basis of cell regulation.     

Myosin IIA Dependent Retrograde Flow Drives 3D Cell Migration

Epithelial cell migration is an essential part of embryogenesis and tissue regeneration, yet their migration is least understood. Using our three-dimensional (3D) motility analysis, migrating epithelial cells formed an atypical polarized cell shape with the nucleus leading the cell front and a contractile cell rear. Migrating epithelial cells exerted traction forces to deform both the anterior and posterior extracellular matrix toward the cell body. The cell leading edge exhibited a myosin II-dependent retrograde flow with the magnitude and direction consistent with surrounding network deformation. Interestingly, on a two-dimensional substrate, myosin IIA-deficient cells migrated faster than wild-type cells, but in a 3D gel, these myosin IIA-deficient cells were unpolarized and immobile. In contrast, the migration rates of myosin IIB-deficient cells were similar to wild-type cells. Therefore, myosin IIA, not myosin IIB, is required for 3D epithelial cell migration.     

Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.     

Cell prestress. II. Contribution of microtubules

The tensegritymodel hypothesizes that cytoskeleton-based microtubules (MTs) carrycompression as they balance a portion of cell contractile stress. Totest this hypothesis, we used traction force microscopy to measuretraction at the interface of adhering human airway smooth muscle cellsand a flexible polyacrylamide gel substrate. The prediction is that ifMTs balance a portion of contractile stress, then, upon theirdisruption, the portion of stress balanced by MTs would shift to thesubstrate, thereby causing an increase in traction. Measurements weredone first in maximally activated cells (10 µM histamine) and thenagain after MTs had been disrupted (1 µM colchicine). We found that after disruption of MTs, traction increased on average by ~13%. Because in activated cells colchicine induced neither an increase inintracellular Ca²⁺ nor an increase in myosin light chainphosphorylation as shown previously, we concluded that the observedincrease in traction was a result of load shift from MTs to thesubstrate. In addition, energy stored in the flexible substrate wascalculated as work done by traction on the deformation of thesubstrate. This result was then utilized in an energetic analysis. Weassumed that cytoskeleton-based MTs are slender elastic rods supportedlaterally by intermediate filaments and that MTs buckle as the cellcontracts. Using the post-buckling equilibrium theory of Euler struts,we found that energy stored during buckling of MTs was quantitativelyconsistent with the measured increase in substrate energy afterdisruption of MTs. This is further evidence supporting the idea thatMTs are intracellular compression-bearing elements.

     

Experimental tests of the cellular tensegrity hypothesis

The tensegrity model depicts the cytoskeleton (CSK) as a prestressed network of interconnected filaments. The prestress is generated by the CSK contractile apparatus and is partly balanced by traction at the cell-substrate interface and partly by CSK internal compression elements such as microtubules (MTs). A key feature of tensegrity is that the shear modulus (G) must increase in proportion with the prestress. Here we have tested that prediction as well as the idea that compression of MTs balance a portion of the cell prestress. Airway smooth muscle cells were studied. Traction microscopy was used to calculate traction. Because traction must be balanced by the stress within the cell, the prestress could be computed. Cell G was measured by oscillatory magnetic cytometry. The prestress was modulated using graded concentrations of contracting (histamine) or relaxing (isoproterenol) agonists and by disrupting MTs by colchicine. It was found that G increased in proportion with the prestress and that compression of MTs balanced a significant, but a relatively small fraction of the prestress. Taken together, these results do not disprove other models of cell deformability, nor they prove tensegrity. However, they do support a priori predictions of tensegrity. As such, it may not be necessary to invoke more complex mechanisms to explain these central features of cell deformability.     

Determining substrate displacement and cell traction fields--a new approach

This paper presents a new approach for the traction force microscopy (TFM) method which determines traction forces exerted by adherent cells on a thin, elastic polyacrylamide gel embedded with fluorescent microbeads. In this enhanced TFM method, a pattern recognition technique is first applied to match the pair of microbead embedded images before and after deformation, which subsequently provides the displacement field of the elastic substrate. Once the displacement field is obtained, the 3-D finite element method (FEM) is used to compute cell traction forces. The new TFM has been applied to determine traction forces of human tendon fibroblasts. Compared to existing TFM methods, the present method has the following advantages: (1) its displacement field obtained is associated with microbead movements; (2) it considers the finite thickness of the thin polyacrylamide gel and is therefore free from the infinite half-space approximation adopted by existing TFM methods; and (3) its computation procedure for determining cell traction forces is fast.     

A predictive model of cell traction forces based on cell geometry

Recent work has indicated that the shape and size of a cell can influence how a cell spreads, develops focal adhesions, and exerts forces on the substrate. However, it is unclear how cell shape regulates these events. Here we present a computational model that uses cell shape to predict the magnitude and direction of forces generated by cells. The predicted results are compared to experimentally measured traction forces, and show that the model can predict traction force direction, relative magnitude, and force distribution within the cell using only cell shape as an input. Analysis of the model shows that the magnitude and direction of the traction force at a given point is proportional to the first moment of area about that point in the cell, suggesting that contractile forces within the cell act on the entire cytoskeletal network as a single cohesive unit. Through this model, we demonstrate that intrinsic properties of cell shape can facilitate changes in traction force patterns, independently of heterogeneous mechanical properties or signaling events within the cell.     

Selected contribution: time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells.

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 microm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G'), loss modulus (friction; G"), and hysteresivity (eta; ratio of G" to G') could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 microM), G' increased by 2.2-fold, G" increased by 3.0-fold, and eta increased transiently from 0.27 to 0.34. By 20 s, eta decreased to 0.25, whereas G' and G" remained above baseline. Comparable results were obtained with bradykinin (1 microM). These changes in G', G", and eta measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G' decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G' could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.     

Contraction of vascular smooth muscle in cell culture

The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells. This limitation has been overcome by the cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane). Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture.     

The dynamics and mechanics of endothelial cell spreading

Cell adhesion to extracellular matrix is mediated by receptor-ligand interactions. When a cell first contacts a surface, it spreads, exerting traction forces against the surface and forming new bonds as its contact area expands. Here, we examined the changes in shape, actin polymerization, focal adhesion formation, and traction stress generation that accompany spreading of endothelial cells over a period of several hours. Bovine aortic endothelial cells were plated on polyacrylamide gels derivatized with a peptide containing the integrin binding sequence RGD, and changes in shape and traction force generation were measured. Notably, both the rate and extent of spreading increase with the density of substrate ligand. There are two prominent modes of spreading: at higher surface ligand densities cells tend to spread isotropically, whereas at lower densities of ligand the cells tend to spread anisotropically, by extending pseudopodia randomly distributed along the cell membrane. The extension of pseudopodia is followed by periods of growth in the cell body to interconnect these extensions. These cycles occur at very regular intervals and, furthermore, the extent of pseudopodial extension can be diminished by increasing the ligand density. Measurement of the traction forces exerted by the cell reveals that a cell is capable of exerting significant forces before either notable focal adhesion or stress fiber formation. Moreover, the total magnitude of force exerted by the cell is linearly related to the area of the cell during spreading. This study is the first to monitor the dynamic changes in the cell shape, spreading rate, and forces exerted during the early stages (first several hours) of endothelial cell adhesion.     

Mechanisms of preserved baseline cardiac systolic function in rats with adrenergic inotropic downregulation

Despite reductions in beta-adrenoreceptor (beta-AR)-mediated inotropic effects induced by sustained sympathetic activation in cardiac disease, whether these changes necessarily result in reductions in systolic function under resting conditions (baseline function) is not clear. Moreover, possible compensatory mechanisms which might contribute to maintaining the baseline systolic function despite reductions in beta-AR-mediated inotropic effects have not been systematically sought. In the present study, 1 month of daily administration of the beta-AR agonist, isoproterenol (0.05 mg/kg/day, i.p.), to rats resulted in an attenuation of left ventricular inotropic responses to isoproterenol over a wide range of concentrations (10(-8)-10(-4) M), whereas a decline of inotropic responses to norepinephrine, an endogenous inotrope, occurred only at high concentrations (10(-5)-10(-4) M). However, chronic isoproterenol administration failed to modify baseline systolic chamber and myocardial function, as determined in vivo using echocardiography (endocardial and midwall fractional shortening), and in isolated, perfused heart preparations (end-systolic chamber and myocardial elastance) Sustained baseline chamber function despite profound beta-AR-mediated inotropic downregulation was not attributed to alterations in cardiac loading conditions, resting heart rate, chamber remodeling, increased myocardial norepinephrine release, or enhanced contractile responses to alternative receptor/signal transduction pathways mediating positive inotropy (as assessed from histamine, serotonin, forskolin, angiotensin II or phenylephrine responsiveness). These findings indicate that baseline cardiac contractile function might be unaltered despite a profound impairment of beta-AR-induced responsiveness, an effect related to a preserved stimulatory influence of low physiological concentrations of endogenous norepinephrine constituting adrenergic tone at rest.     

Modelling Biological Gel Contraction by Cells: Mechanocellular Formulation and Cell Traction Force Quantification

Traction forces developed by most cell types play a significant role in the spatial organisation of biological tissues. However, due to the complexity of cell-extracellular matrix interactions, these forces are quantitatively difficult to estimate without explicitly considering cell properties and extracellular mechanical matrix responses. Recent experimental devices elaborated for measuring cell traction on extracellular matrix use cell deposits on a piece of gel placed between one fixed and one moving holder. We formulate here a mathematical model describing the dynamic behaviour of the cell-gel medium in such devices. This model is based on a mechanical force balance quantification of the gel visco-elastic response to the traction forces exerted by the diffusing cells. Thus, we theoretically analyzed and simulated the displacement of the free moving boundary of the system under various conditions for cells and gel concentrations. This modelis then used as the theoretical basis of an experimental device where endothelial cells are seeded on a rectangular biogel of fibrin cast between two floating holders, one fixed and the other linked to a force sensor. From a comparison of displacement of the gel moving boundary simulated by the model and the experimental data recorded from the moving holder displacement, the magnitude of the traction forces exerted by the endothelial cell on the fibrin gel was estimated for different experimental situations. Different analytical expressions for the cell traction term are proposed and the corresponding force quantifications are compared to the traction force measurements reported for various kind of cells with the use of similar or different experimental devices. This revised version was published online in June 2006 with corrections to the Cover Date.     

Voltage-dependent Vmax blockade in Na+-dependent action potentials after beta 1- and H2-receptor stimulation in mammalian ventricular myocardium

In isolated papillary muscles of guinea pigs, the influence of isoproterenol, histamine, theophylline, and phenylephrine on the maximal rate of rise (Vmax) of Na+-dependent action potentials and on isometric contractile force was studied under rested state conditions. Isoproterenol (1 x 10(-7) mol/L), histamine (2 x 10(-5) mol/L), and theophylline (2 x 10(-3) mol/L) shifted the voltage dependence of Vmax into the hyperpolarizing direction and, consequently, led to a voltage-dependent Vmax blockade. The alpha-adrenoceptor agonist phenylephrine, on the other hand, proved to be ineffective in depressing Vmax. The beta-receptor blocker pindolol (4 x 10(-6) mol/L) or the H2-receptor blocker cimetidine (4 x 10(-5) mol/L) abolished the inhibitory effects of isoproterenol and histamine, respectively, and caused Vmax to return to the initial control value. A concentration-response relationship analysis at -65 mV revealed that isoproterenol exerted only a weak inhibitory effect on Vmax compared with its positive inotropic action. The IC50 value of the former effect amounted to approximately 5 x 10(-6) mol/L, but the EC50 value of the latter effect was 4 x 10(-8) mol/L. It is, therefore, concluded that, in physiologically relevant concentrations, beta-adrenergic agonists are unlikely to significantly modulate Na+-dependent excitability even in partially depolarized myocardium.     

Traction fields, moments, and strain energy that cells exert on their surroundings

Adherent cells exert tractions on theirsurroundings. These tractions can be measured by observing thedisplacements of beads embedded on a flexible gel substrate on whichthe cells are cultured. This paper presents an exact solution to theproblem of computing the traction field from the observed displacementfield. The solution rests on recasting the relationship betweendisplacements and tractions into Fourier space, where the recovery ofthe traction field is especially simple. We present two subcases of thesolution, depending on whether or not tractions outside the observedcell boundaries are set to be zero. The implementation iscomputationally efficient. We also give the solution for the tractionfield in a representative human airway smooth muscle cell contracted by treatment with histamine. Finally, we give explicit formulas for reducing the traction and displacement fields to contraction moments, the orientation of the principal axes of traction, and the strain energy imparted by the cell to the substrate.

     

Effect of the cytoskeletal prestress on the mechanical impedance of cultured airway smooth muscle cells.

We investigated the effect of the cytoskeletal prestress (P) on the elastic and frictional properties of cultured human airway smooth muscle cells during oscillatory loading; P is preexisting tensile stress in the actin cytoskeleton generated by the cell contractile apparatus. We oscillated (0.1 Hz, 6 Pa peak to peak) small ferromagnetic beads bound to integrin receptors and computed the storage (elastic) modulus (G') and the loss (frictional) modulus (G") from the applied torque and the corresponding bead rotation. All measurements were done at baseline and after cells were treated with graded doses of either histamine (0.1, 1, 10 microM) or isoproterenol (0.01, 0.1, 1, 10 microM). Values for P for these concentrations were taken from a previous study (Wang et al., Am J Physiol Cell Physiol, in press). It was found that G' and G", as well as P, increased/decreased with increasing doses of histamine/isoproterenol. Both G' and G" exhibited linear dependences on P: G'(Pa) = 0.20P + 82 and G"(Pa) = 0.05P + 32. The dependence of G' on P is consistent with our previous findings and with the behavior of stress-supported structures. The dependence of G" on P is a novel finding. It could be attributed to a variety of mechanisms. Some of those mechanisms are discussed in detail. We concluded that, in addition to the central mechanisms by which stress-supported structures develop mechanical stresses, other mechanisms might need to be invoked to fully explain the observed dependence of the cell mechanical properties on the state of cell contractility.     

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 μN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 μN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue.     

Sanguinarine Decreases Cell Stiffness and Traction Force and Inhibits the Reactivity of Airway Smooth Muscle Cells in Culture

Airway hyperresponsiveness (AHR) is the cardinal character of asthma, which involves the biomechanical properties such as cell stiffness and traction force of airway smooth muscle cells (ASMCs). Therefore, these biomechanical properties comprise logical targets of therapy. β2-adrenergic agonist is currently the mainstream drug to target ASMCs in clinical practice for treating asthma. However, this drug is known for side effects such as desensitization and non-responsiveness in some patients. Therefore, it is desirable to search for new drug agents to be alternative of β2-adrenergic agonist. In this context, sanguinarine, a natural product derived from plants such as bloodroots, that has been reported to relax gut smooth muscle emerges as a potential candidate. So far, it is unknown whether sanguinarine can regulate the biomechanical properties of ASMCs and reactivity of ASMCs to irritants. Thus, we tested the hypothesis that sanguinarine reduce the contractile potentials of ASMCs in culture. To do so, the primary cultured rat ASMCs were first treated with different concentration of sanguinarine. Then, cell stiffness, traction force, fiber distribution, and calcium signaling of the ASMCs were evaluated by optical magnetic twisting cytometry, Fourier transform traction microscopy, atomic force microscopy, and Fluo-4/AM based fluorescence confocal scanning microscopy, respectively. The results indicated that sanguinarine (0.05 and 0.5 μmol/L) significantly decreased cell stiffness and traction force, inhibited reactivity of ASMCs to histamine, and disrupted the fiber structures in ASMCs in dose-dependent manner. These findings establish that sanguinarine can indeed change the biomechanical properties of ASMCs and may be used to treat AHR in asthma.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.