Quantitative spectroscopy of single molecule interaction times.

Single molecule fluorescence tracking provides information at nanometer-scale and millisecond-temporal resolution about the dynamics and interaction of individual molecules in a biological environment. While the dynamic behavior of isolated molecules can be characterized well, the quantitative insight is more limited when interactions between two indistinguishable molecules occur. We address this aspect by developing a theoretical foundation for a spectroscopy of interaction times, i.e., the inference of interaction from imaging data. A non-trivial crossover between a power law to an exponential behavior of the distribution of the interaction times is highlighted, together with the dependence of the exponential term upon the microscopic reaction affinity. Our approach is validated with simulated and experimental datasets.

A functional genetic variation of SLC6A2 repressor hsa-miR-579-3p upregulates sympathetic noradrenergic processes of fear and anxiety.

Increased sympathetic noradrenergic signaling is crucially involved in fear and anxiety as defensive states. MicroRNAs regulate dynamic gene expression during synaptic plasticity and genetic variation of microRNAs modulating noradrenaline transporter gene (SLC6A2) expression may thus lead to altered central and peripheral processing of fear and anxiety. In silico prediction of microRNA regulation of SLC6A2 was confirmed by luciferase reporter assays and identified hsa-miR-579-3p as a regulating microRNA. The minor (T)-allele of rs2910931 (MAFcases = 0.431, MAFcontrols = 0.368) upstream of MIR579 was associated with panic disorder in patients (pallelic = 0.004, ncases = 506, ncontrols = 506) and with higher trait anxiety in healthy individuals (pASI = 0.029, pACQ = 0.047, n = 3112). Compared to the major (A)-allele, increased promoter activity was observed in luciferase reporter assays in vitro suggesting more effective MIR579 expression and SLC6A2 repression in vivo (p = 0.041). Healthy individuals carrying at least one (T)-allele showed a brain activation pattern suggesting increased defensive responding and sympathetic noradrenergic activation in midbrain and limbic areas during the extinction of conditioned fear. Panic disorder patients carrying two (T)-alleles showed elevated heart rates in an anxiety-provoking behavioral avoidance test (F(2, 270) = 5.47, p = 0.005). Fine-tuning of noradrenaline homeostasis by a MIR579 genetic variation modulated central and peripheral sympathetic noradrenergic activation during fear processing and anxiety. This study opens new perspectives on the role of microRNAs in the etiopathogenesis of anxiety disorders, particularly their cardiovascular symptoms and comorbidities.

All-optical microscope autofocus based on an electrically tunable lens and a totally internally reflected IR laser.

Microscopic imaging at high spatial-temporal resolution over long time scales (minutes to hours) requires rapid and precise stabilization of the microscope focus. Conventional and commercial autofocus systems are largely based on piezoelectric stages or mechanical objective actuators. Objective to sample distance is either measured by image analysis approaches or by hardware modules measuring the intensity of reflected infrared light. We propose here a truly all-optical microscope autofocus taking advantage of an electrically tunable lens and a totally internally reflected infrared probe beam. We implement a feedback-loop based on the lateral position of a totally internally reflected infrared laser on a quadrant photodetector, as an indicator of the relative defocus. We show here how to treat the combined contributions due to mechanical defocus and deformation of the tunable lens. As a result, the sample can be kept in focus without any mechanical movement, at rates up to hundreds of Hertz. The device requires only reflective optics and can be implemented at a fraction of the cost required for a comparable piezo-based actuator.

Increased fear learning, spatial learning as well as neophobia in Rgs2-/- mice.

Anxiety disorders result from a complex interplay of genetic and environmental factors such as stress. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety. However, there is limited knowledge about the role of Rgs2 in fear learning and reactivity to stress. In this study, Rgs2-/- mice showed increased fear learning, male mice displayed increased contextual and cued fear learning, while females showed selectively enhanced cued fear learning. Male Rgs2-/- mice displayed increased long-term-contextual fear memory, but increased cued fear extinction. Learning in spatial non-aversive paradigms was also increased in Rgs2-/- mice. Female, but not male mice show increased spatial learning in the Barnes maze, while male mice showed enhanced place preference in the IntelliCage, rendering enhanced cognitive function non-specific for aversive stimuli. Consistent with the previous results, Rgs2 deletion resulted in increased innate anxiety, including neophobic behavior expressed as hypolocomotion, in three different tests based on the approach-avoidance conflict. Acute electric foot shock stress provoked hypolocomotion in several exploration-based tests, suggesting fear generalization in both genotypes. Rgs2 deletion was associated with reduced monoaminergic neurotransmitter levels in the hippocampus and prefrontal cortex and disturbed corresponding GPCR expression of the adrenergic, serotonergic, dopaminergic and neuropeptide Y system. Taken together, Rgs2 deletion promotes improved cognitive function as well as increased anxiety-like behavior, but has no effect on acute stress reactivity. These effects may be related to the observed disruption of the monoaminergic systems.

Antibodies to cardiac receptors.

Inflammation of cardiac tissue is generally associated with an activation of the host's immune system. On the one hand, this activation is mandatory to protect the heart by fighting the invading microbial agents or toxins and by engaging myocardial reparation and healing processes. On the other hand, uncontrolled activation of the immune defense has the risk of an arousal of auto- or cross-reactive immune cells, which in some cases bring more harm than good. Dependent on the individual genetic predisposition, such heart-directed autoimmune reactions most likely occur as a result of myocyte apoptosis or necrosis and subsequent liberation of self-antigens previously hidden to the immune system. During the past two decades, evidence for a pathogenic relevance of autoimmunity in human heart disease has substantially increased. Conformational cardiac (auto)antibodies affecting cardiac function and, in particular, (auto)antibodies that target G protein-coupled cardiac membrane receptors are thought to play a key role in the development of heart failure. Clinical pilot studies even suggest that such antibodies negatively affect survival in heart failure patients. However, the true prevalence and clinical impact of many cardiac (auto)antibodies in human heart diseases are still unclear, as are the events triggering their formation, their titer course, and their patterns of clearance and/or persistence. The present article summarizes current knowledge in the field of cardiac receptor (auto)antibodies including recent efforts to address some of the aforementioned gaps of knowledge, thereby attempting to pave the way for novel, more specific therapeutic approaches.

Temporally resolved cAMP monitoring in endothelial cells uncovers a thrombin-induced [cAMP] elevation mediated via the Ca²+-dependent production of prostacyclin.

The barrier function of the endothelium is controlled by the second messengers Ca2+ and cAMP that differentially regulate the permeability of endothelial cells. The Ca2+-elevating agent thrombin has been demonstrated to increase endothelial permeability and to decrease cAMP levels as detected via enzyme immunoassays. To study the effects of thrombin on cAMP with high temporal resolution, we utilised the FRET-based cAMP sensor Epac1-camps in single intact human umbilical vein endothelial cells (HUVECs). In these cells, thrombin induced a delayed increase in [cAMP], initiating after about 40 s, with maximum cAMP levels after 130 s of thrombin application. This increase of cAMP levels was Ca2+-dependent, but did not require calmodulin (CaM). Pharmacological approaches revealed that phospholipase A2 (PLA2) activity and cyclooxygenase (COX)-mediated synthesis of prostaglandins was required for the thrombin-induced elevation of [cAMP]. Furthermore, preincubation of HUVECs with a prostacyclin-receptor antagonist significantly reduced the thrombin-induced increase in [cAMP]. We conclude that thrombin causes the synthesis of prostacyclin in endothelial cells and that the subsequent stimulation of Gs-coupled prostacyclin receptors then results in an increase in [cAMP].

Real-time monitoring of cAMP levels in living endothelial cells: thrombin transiently inhibits adenylyl cyclase 6.

The crosstalk between Ca(2+) and cAMP signals plays a significant role for the regulation of the endothelial barrier function. The Ca(2+)-elevating agent thrombin was demonstrated to increase endothelial permeability and to decrease cAMP levels. Since Ca(2+) and cAMP signals are highly dynamic, we aimed to study the temporal resolution between thrombin-evoked Ca(2+) signals and subsequent changes of cAMP levels. Here we conduct the first real-time monitoring of thrombin-mediated regulation of cAMP signals in intact human umbilical vein endothelial cells (HUVECs) by utilising the Ca(2+)-sensitive dye Fluo-4 and the fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps. We calibrated in vitro FRET responses of Epac1-camps to [cAMP] in order to estimate changes in intracellular [cAMP] evoked by thrombin treatment of HUVECs. After increasing [cAMP] to 1.2 +/- 0.2 microm by stimulation of HUVECs with isoproterenol (isoprenaline), we observed a transient decrease of cAMP levels by 0.4 +/- 0.1 microm which reached a minimum value 30 s after thrombin application and 15 s after the thrombin-evoked Ca(2+) peak. This transient decrease in [cAMP] was Ca(2+)-dependent and independent of a G(i)-mediated inhibition of adenylyl cyclases (ACs). Instead the knock down of the predominant subtype AC6 in HUVECs provided the first direct evidence that the Ca(2+)-mediated inhibition of AC6 accounts for the thrombin-induced decrease in cAMP levels.

Imaging cytoplasmic cAMP in mouse brainstem neurons.

BACKGROUND: cAMP is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular Ca2+ levels. While imaging of calcium is commonly used in neuroscience applications, probing for cAMP levels has not yet been performed in living vertebrate neuronal tissue before. RESULTS: Using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-Bötzinger complex, constituting the rhythm-generating part of the respiratory network. Fluorescent cAMP sensor Epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of cAMP, its absolute levels and time-dependent changes in response to physiological stimuli. We recorded [cAMP]i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of G-protein-coupled metabotropic receptors. [cAMP]i levels increased after membrane depolarisation and release of Ca2+ from internal stores. The effects developed slowly and reached their maximum after transient [Ca2+]i elevations subsided. Ca2+-dependent [cAMP]i transients were suppressed after blockade of adenylate cyclase with 0.1 mM adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. During paired stimulations, the second depolarisation and Ca2+ release evoked bigger cAMP responses. These effects were abolished after inhibition of protein kinase A with H-89 pointing to the important role of phosphorylation of calcium channels in the potentiation of [cAMP]i transients. CONCLUSION: We constructed and characterized a neuron-specific cAMP probe based on Epac1-camps. Using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. Strong fluorescence, resistance to photobleaching and possibility of direct estimation of [cAMP] levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. Epac1-camps was applied to examine the crosstalk between Ca2+ and cAMP signalling and revealed a synergism of actions of these two second messengers.

Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes.

Several lines of evidence suggest that G-protein-coupled receptors can adopt different active conformations, but their direct demonstration in intact cells is still missing. Using a fluorescence resonance energy transfer (FRET)-based approach we studied conformational changes in alpha(2A)-adrenergic receptors in intact cells. The receptors were C-terminally labeled with cyan fluorescent protein and with fluorescein arsenical hairpin binder at different sites in the third intracellular loop: N-terminally close to transmembrane domain V (I3-N), in the middle of the loop (I3-M), or C-terminally close to transmembrane domain VI (I3-C). All constructs retained normal ligand binding and signaling properties. Changes in FRET between the labels were determined in intact cells in response to different agonists. The full agonist norepinephrine evoked similar FRET changes for all three constructs. The strong partial agonists clonidine and dopamine induced partial FRET changes for all constructs. However, the weak partial agonists octopamine and norphenephrine only induced detectable changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced FRET-signals were approximately 1.5-fold slower than those for norepinephrine in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that the different ligands induced conformational changes in the receptor that were sensed differently in different positions of the third intracellular loop. This agrees with X-ray receptor structures indicating larger agonist-induced movements at the cytoplasmic ends of transmembrane domain VI than V and suggests that partial agonism is linked to distinct conformational changes within a G-protein-coupled receptor.

Kinetics of G-protein-coupled receptor signals in intact cells.

G-protein-coupled receptors (GPCRs) are the largest group of cell surface receptors. They are stimulated by a variety of stimuli and signal to different classes of effectors, including several types of ion channels and second messenger-generating enzymes. Recent technical advances, most importantly in the optical recording with energy transfer techniques--fluorescence and bioluminescence resonance energy transfer, FRET and BRET--, have permitted a detailed kinetic analysis of the individual steps of the signalling chain, ranging from ligand binding to the production of second messengers in intact cells. The transfer of information, which is initiated by ligand binding, triggers a signalling cascade that displays various rate-controlling steps at different levels. This review summarizes recent findings illustrating the speed and the complexity of this signalling system.

Conformational changes in G-protein-coupled receptors-the quest for functionally selective conformations is open.

The G-protein-coupled receptors (GPCRs) represent one the largest families of drug targets. Upon agonist binding a receptor undergoes conformational rearrangements that lead to a novel protein conformation which in turn can interact with effector proteins. During the last decade significant progress has been made to prove that different conformational changes occur. Today it is mostly accepted that individual ligands can induce different receptor conformations. However, the nature or molecular identity of the different conformations is still ill-known. Knowledge of the potential functionally selective conformations will help to develop drugs that select specific conformations of a given GPCR which couple to specific signalling pathways and may, ultimately, lead to reduced side effects. In this review we will summarize recent progress in biophysical approaches that have led to the current understanding of conformational changes that occur during GPCR activation.

Kinetics of G-protein-coupled receptor signalling and desensitization.

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor-arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.

Hypoglycemia-dependent beta2-adrenoceptor downregulation: a contributing factor to hypoglycemia unawareness in patients with Type-1 diabetes?

To evaluate the influence of the incidence and unawareness of hypoglycemia on lymphocyte beta2-adrenoceptor densities, we measured beta2-adrenoceptor density using [125I]-iodocyanopindolol and CGP 12177 before and after 1 week of treatment optimization in 33 adults with type-1 diabetes mellitus. Diabetes treatment of all patients was modified to improve their glycemic control. During this week, all patients had to complete a protocol with 7 daily glucose measurements, one of which was at night. The subjective symptoms were evaluated in case of hypoglycemia. A significant correlation between a hypoglycemia incidence below (but not above) the threshold of 2.75 mmol/l (50 mg/dl) and beta2-adrenoceptor densities on lymphocytes was found after the study week (r = -0.72, p < 0.00001). Nine patients suffering from hypoglycemia unawareness had a significantly higher incidence of hypoglycemia (p < 0.002) and lower beta2-adrenoceptor densities on lymphocytes compared to 24 patients who recognized all of their hypoglycemic episodes (p < 0.004). We conclude that downregulation of beta2-adrenoceptor densities on lymphocytes occurs as a result of recurrent hypoglycemia defined as glucose levels of < 2.75 mmol/l. Beta2-adrenoceptor densities are decreased in patients with subjective hypoglycemia unawareness and might contribute to the reduced beta-adrenergic sensitivity in this subgroup of patients.

Comparative pharmacology of human beta-adrenergic receptor subtypes--characterization of stably transfected receptors in CHO cells.

Although many beta1-receptor antagonists and beta2-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of beta-adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human beta-adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three beta-adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist 125I-cyanopindolol as a radioligand. Furthermore, we analyzed the beta-receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three beta-receptor subtypes. In particular, a number of beta2- or beta3-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, beta1-receptor antagonists with agonistic activity at beta2- and beta3-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.

Effects of two Gbetagamma-binding proteins--N-terminally truncated phosducin and beta-adrenergic receptor kinase C terminus (betaARKct)--in heart failure.

Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated phosducin (nt-del-phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.

Vascular hypertrophy and increased P70S6 kinase in mice lacking the angiotensin II AT(2) receptor.

BACKGROUND: Angiotensin II activates 2 distinct G protein-coupled receptors, the AT(1) and AT(2) receptors. Most of the known cardiovascular effects of angiotensin II are mediated by the AT(1) receptor subtype. The aim of the present study was to test whether deletion of the AT(2) receptor gene in mice (AT(2)-KO mice) leads to long-term functional or structural alterations in the cardiovascular system. METHODS AND RESULTS: In vivo pressure responses to angiotensin II or the alpha(1)-adrenergic receptor agonist phenylephrine were greatly enhanced in AT(2)-KO mice. Deletion of the angiotensin AT(2) receptor did not lead to a compensatory increase of the activity of the circulating renin-angiotensin system, and arterial blood pressure was identical in wild-type control mice (WT) and AT(2)-KO mice. Cardiac contractility as assessed by LV catheterization and by rapid MRI also did not differ between AT(2)-KO and WT mice. Isolated femoral arteries from AT(2)-KO mice, however, showed enhanced vasoconstriction to angiotensin II, norepinephrine, and K(+) depolarization compared with WT. Morphometric analysis of large and small femoral arteries revealed a significant hypertrophy of media smooth muscle cells. Phospho-P70S6 kinase levels were significantly increased in aortas from AT(2)-KO mice compared with WT mice. Treatment of mice with an ACE inhibitor for 8 weeks abolished the increased pressure responsiveness, vascular hypertrophy, and enhanced P70S6 kinase phosphorylation in AT(2)-KO mice. CONCLUSIONS: These results indicate that vascular AT(2) receptors inhibit the activity and, hence, hypertrophic signaling by the P70S6 kinase in vivo and thus are important regulators of vascular structure and function.

Activation and deactivation kinetics of alpha 2A- and alpha 2C-adrenergic receptor-activated G protein-activated inwardly rectifying K+ channel currents.

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from alpha(2A)-adrenergic receptor knockout mice suggested that the alpha(2A)-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the alpha(2C)-adrenergic receptor. Here we investigated whether these functional differences between presynaptic alpha(2)-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. alpha(2A)- and alpha(2C)-receptors were stably expressed in HEK293 cells at moderate ( approximately 2 pmol/mg) or high (17-24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K(+) (GIRK) channels was similar in extent and kinetics for alpha(2A)- and alpha(2C)-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. alpha(2C)-Receptor-activated GIRK currents returned much more slowly to base line than did alpha(2A)-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine alpha(2C)- than at the alpha(2A)-receptor subtype and may explain why alpha(2C)-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the alpha(2A)-receptor subtype.

Receptor-ligand interactions studied with homogeneous fluorescence-based assays suitable for miniaturized screening.

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and beta(2)-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1 microl without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Early impairment of calcium handling and altered expression of junctin in hearts of mice overexpressing the beta1-adrenergic receptor.

Chronic stimulation of cardiac beta1-adrenergic receptors contributes to disease progression and mortality in patients and animal models of heart failure. To search for the mechanism of adrenergic impairment of cardiac function in vivo, we studied transgenic mice with cardiac-specific overexpression of beta1-adrenergic receptors. Transgenic mice with cardiac overexpression of beta1-adrenergic receptors showed progressive left ventricular fibrosis starting at 4 months of age. Left ventricular catheterization revealed a modest enhancement of contractility and relaxation at 2 months of age, followed by progressive dysfunction in both parameters and ultimately cardiac failure. When the effects of endogenous catecholamines were blocked by the b-receptor antagonist propranolol, maximal rate of contractility (dp/dtmax) and maximal rate of relaxation (dp/dtmin) were significantly blunted in 2-month-old beta1-receptor transgenic mice. Isolated cardiomyocytes from these animals displayed markedly altered calcium transients with significant prolongation of the intracellular calcium transient compared with nontransgenic littermates. We determined the expression of sarcoplasmic reticulum proteins involved in calcium handling by RNase protection assay and by immunoblotting. Although the expression of calsequestrin, triadin, and phospholamban was not altered, we observed a progressive decrease in junctin abundance in beta1-receptor transgenic mice (Pbeta1-adrenergic receptors.