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Am J Physiol Regul Integr Comp Physiol 283: R1236-R1244, 2002. First published August 29, 2002; doi:10.1152/ajpregu.00409.2002
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Vol. 283, Issue 5, R1236-R1244, November 2002

beta -Adrenoceptor control of G protein function in the neonate: determinant of desensitization or sensitization

J. T. Auman, F. J. Seidler, and T. A. Slotkin

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neonatal beta -adrenoceptors (beta -ARs) are resistant to agonist-induced desensitization. We examined the functioning of Gi and Gs after repeated administration of beta -AR agonists to newborn rats. Isoproterenol (beta 1/beta 2 agonist) obtunded Gi function in the heart but not the liver; in contrast, terbutaline, a beta 2-selective agonist, enhanced Gi function. Isoproterenol, but not terbutaline, increased membrane-associated Gsalpha , which would enhance receptor function. In addition, isoproterenol increased and terbutaline maintained the proportion of the short-splice (S) variant of Gsalpha in the membrane fraction; Gsalpha S is functionally more active than the long-splice variant. Either isoproterenol or terbutaline treatment increased Gsalpha in the cytosolic fraction, a characteristic usually associated with desensitization in the adult. Decreased Gi activity, coupled with increased membrane-associated Gsalpha concentrations and maintenance or increases in membrane Gsalpha S, provide strong evidence that unique effects on G protein function underlie the ability of the immature organism to sustain beta -AR cell signaling in the face of excessive or prolonged stimulation; these mechanisms also contribute to tissue selectivity of the effects of beta -agonists with divergent potencies toward different beta -AR subtypes.

development; heart; isoproterenol; liver; terbutaline


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RECEPTOR DESENSITIZATION REPRESENTS the major mode for cellular homeostasis in the presence of continued stimulation (12). In the case of beta -adrenoceptors (beta -ARs) and their signaling mediated through adenylyl cyclase (AC), attenuation of receptor function is especially important: prolonged, excessive beta -AR stimulation can lead to cell damage (5, 9, 29, 36). It is therefore critical to note that, in all mammalian species that have been examined, the ability of beta -agonists to elicit desensitization is absent in the fetus or neonate and is acquired during postnatal development (34, 35, 42, 44). This anomaly has both physiological and therapeutic implications. The perinatal transition requires a coordinated series of cardiovascular, respiratory, and metabolic adjustments (17). These are triggered by intense catecholaminergic stimulation (17) so that maintenance of beta -AR signaling is critical to perinatal survival and indeed to trophic effects on general somatic growth (16, 38). Nevertheless, the deficiency in beta -AR desensitization renders developing cells vulnerable to disruption by beta -AR agonists (5, 8, 9, 31). These effects are likely to account for a number of adverse consequences noted after fetal exposure to drugs such as terbutaline or ritodrine, beta 2-AR agonists that are used to arrest preterm labor but that also cross the placenta to stimulate fetal beta -ARs (5, 7, 9, 14, 19, 24).

Studies exploring the resistance of immature tissues to beta -AR desensitization uncovered a number of unique features. In neonatal rats given repeated injections of either isoproterenol or terbutaline, cardiac or hepatic beta -AR/AC signaling is not desensitized, but rather shows agonist-induced sensitization (2, 4, 37). One main factor accounting for the anomalous response is the induction of AC, leading to heterologous sensitization of all signals mediated through this signaling pathway (3, 4, 43, 45); thus administration of beta -AR agonists augments the response to glucagon, which shares the same effector, AC (3, 42, 45). In addition, alterations in G protein expression and/or function may also participate in the production of sensitization instead of desensitization. We recently found that repeated beta -AR agonist administration decreased neonatal cardiac Gi expression and enhanced Gs function (42, 44), a response pattern opposite to that typically seen in the adult (11, 25, 26).

The current study addresses two key issues in beta -AR control of G protein function in the neonate. First, does the beta -agonist-induced decrease in Gi expression (44) elicit impairment of the ability of this protein to control AC activity? There is a relative excess of G proteins compared with neurotransmitter or hormone receptors or with AC (23), so that demonstrating a loss of Gi function is essential. Accordingly, we treated neonatal rats with beta -AR agonist drugs and then evaluated the ability of pertussis toxin (PTX) to affect AC responses mediated by the beta -AR or by forskolin, a direct AC stimulant whose activity is influenced by the association of AC with Gs or Gi (28). The second issue was to determine how neonatal beta -agonist treatment elicits an increase in Gs function (42, 44). Overexpression of Gsalpha protects beta -ARs from agonist-induced desensitization (39), so that an increase in the concentration of Gsalpha could provide a ready explanation for enhanced receptor-Gs coupling. Gsalpha is in equilibrium between the cell membrane and cytosol (21), and in the mature cell, beta -AR activation displaces Gsalpha from membrane to cytosol, contributing to desensitization (41). Similarly, during development, there are major shifts both in the expression of specific long- and short-splice variants of Gsalpha (Gsalpha L and Gsalpha S, respectively) and in the relative proportions of each variant in the membrane-bound and cytosolic fractions (21). Accordingly, we evaluated the relative proportions of Gsalpha L and Gsalpha S in membrane and cytosol after neonatal beta -agonist administration to determine the potential role of these factors in the ability of the neonate to resist desensitization.

In designing these studies, we used models based on our earlier work that delineated tissue- and beta -AR-subtype selectivity for the balance between agonist-induced neonatal sensitization and desensitization (3, 4, 42-45). First, we compared the effects of isoproterenol, a mixed beta 1/beta 2-AR agonist, to those of terbutaline, which is more selective for beta 2-ARs. Second, we contrasted effects on the heart to those in the liver; these two tissues differ both in their relative expression of beta -AR subtypes (beta 1 predominant in heart, beta 2 in liver) and in their ontogenetic patterns of receptor expression, because the heart acquires beta -ARs during neonatal development (18), whereas the liver shows developmental decrements in beta -AR expression (13).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal treatments. Studies were carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed pregnant female Sprague-Dawley rats were shipped by climate-controlled truck (transit time 12 h) and housed with free access to food and water. The day after birth, pups were randomized and redistributed to the nursing dams with a litter size of 10; randomization was repeated daily and, in addition, dams were reassigned to different litters to distribute any maternal differences equally. Equivalent numbers of males and females were assigned to each treatment group. On postnatal days 2-5, pups were given daily subcutaneous injections of L-isoproterenol hydrochloride (1.25 mg/kg), terbutaline hemisulfate (10 mg/kg), or an equivalent volume (1 ml/kg) of isotonic saline vehicle. These regimens elicit robust beta -AR downregulation in the adult (2) but produce sensitization of beta -AR/AC signaling in the neonate (3, 4, 37, 42-45). Twenty-four hours after the final injection, one animal was selected from each litter, and hearts and livers were frozen in liquid nitrogen and stored at -45°C.

PTX treatment of membranes. PTX treatment of membranes was adapted from an earlier study (15). Tissues were thawed and homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in 39 vol of ice-cold buffer containing 145 mM NaCl, 2 mM MgCl2, and 20 mM Tris (pH 7.5) and sedimented at 40,000 g for 15 min. The pellets were washed once by resuspension (Polytron) in homogenization buffer followed by resedimentation and were then dispersed with a homogenizer (smooth glass fitted with a Teflon pestle) to achieve a protein concentration of 2-4 mg/ml in a buffer consisting of 250 mM sucrose, 1 mM EGTA, and 10 mM Tris (pH 7.4). Aliquots of membrane preparations containing 0.4-0.8 mg protein were incubated for 30 min at 30°C in the presence or absence of 1 ng/µl PTX (activated by preincubation with 10 mM dithiothreitol for 30 min at 30°C), and with final concentrations of 50 mM nicotinamide adenine dinucleotide, 2.5 mM ATP, 4 mM GTP, 10 mM thymidine, and 10 mM dithiothreitol. Membranes were then sedimented at 40,000 g for 15 min and then resuspended (smooth glass fitted with a Teflon pestle) to achieve a final protein concentration of 0.5-1.0 mg/ml in a buffer consisting of 250 mM sucrose, 1 mM EGTA, and 10 mM Tris (pH 7.4).

AC activity. Aliquots of membrane preparation containing 25-50 µg protein were incubated for 30 min at 30°C with final concentrations of 100 mM Tris · HCl (pH 7.4), 10 mM theophylline, 1 mM ATP, 2 mM MgCl2, 1 mg/ml bovine serum albumin, and a creatine phosphokinase-ATP-regenerating system consisting of 10 mM sodium phosphocreatine and 8 IU/ml phosphocreatine kinase, with 10 µM GTP in a total volume of 250 µl. The enzymatic reaction was stopped by placing the samples in a 90-100°C water bath for 5 min, followed by sedimentation at 3,000 g for 15 min; the supernatant solution was assayed for cAMP using radioimmunoassay kits. Preliminary experiments showed that the enzymatic reaction was linear well beyond the assay period and was linear with membrane protein concentration; concentrations of cofactors were optimal and, in particular, the addition of higher concentrations of GTP produced no further augmentation of activity. In addition to measuring basal AC activity, we assessed the response to beta -AR stimulation by addition of L-isoproterenol (100 µM), as well as the response to the direct AC stimulant forskolin (100 µM). Gi function was then determined by comparing the activity in membranes that had been preincubated with PTX to those that had undergone the same preincubation without PTX.

Gsalpha isolation and quantitation. To separate membrane-bound from cytosolic Gsalpha , tissues were homogenized (Polytron) in 9 vol of ice-cold buffer containing 145 mM NaCl, 1 mM EDTA, and 20 mM Tris (pH 7.5), with freshly added protease inhibitor (0.5 mM phenylmethylsulfonyl fluoride). Homogenates were sedimented at 600 g for 5 min, and the supernatant solution was then sedimented at 50,000 g for 30 min to separate cell membranes from the cytosol. Pellets were dispersed with a Polytron in one-half the original volume of buffer and aliquots of supernatants and resuspended pellets were stored at -80°C.

Gsalpha isoforms were determined by Western immunoblot analysis essentially as described previously (44). Aliquots containing 40 µg of protein were diluted in buffer containing 2% sodium dodecyl sulfate, 10% glycerol, 0.1% bromphenol blue, 100 mM dithiothreitol, and 50 mM Tris (pH 6.8) and denatured for 5 min at 65°C. Samples were then separated by electrophoresis, after which proteins were transferred from the gels to nitrocellulose membranes at 100 V for 1.5 h. The membranes were shaken for 1 h at room temperature in blocking solution, consisting of 200 mM CaCl2, 800 mM NaCl, 0.0025% sodium azide, 0.2% NP-40, 5% nonfat dry milk, and 200 mM Tris (pH 7.7). Antibody specific to Gsalpha (diluted 1:10,000) was then added for a further 1 h incubation, after which there were three 10-min washes with blocking solution. The membranes were incubated with goat anti-rabbit IgG (Fc) alkaline phosphatase conjugate (1:7,500) for 1 h, followed by three washes in blocking solution, two rinses in 200 mM CaCl2, 800 mM NaCl, and 200 mM Tris (pH 7.7), and three 2-min washes in 150 mM NaCl, 0.05% Tween 20, and 50 mM Tris (pH 7.7). The blots were developed in 100 mM NaCl, 5 mM MgCl2, 100 mM Tris, 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, and 0.33 mg/ml nitroblue tetrazolium (pH 9.5), and images were digitized and quantitated.

As we evaluated subcellular fractions containing different populations of proteins, it was not feasible to standardize the preparations against a housekeeping protein such as beta -actin, especially as the study involved drugs that specifically alter cardiac contractile proteins. Accordingly, we ensured standardization of the Western blots in several ways. First, protein concentrations were measured before blotting to ensure that exactly the same amount of protein was applied to each lane. Second, in addition to the samples, a standard preparation from the same adult heart was run on every blot to enable normalization of values between blots. Furthermore, a sample of authentic Gsalpha L and Gsalpha S was included both to identify the bands and to standardize the hybridization of these specific proteins from blot to blot. Finally, each blot contained a protein ladder to verify molecular weights of the Gsalpha bands. Values were calculated in relative units by dividing the reading for each band by the value of the standard preparation run on the same blot. Thus, although the actual measurement units are arbitrary, the values maintained their relative proportions and could be contrasted among ages, treatments, and tissues.

Data analysis. Data are presented as means and SEs, with intergroup differences established by ANOVA (data log-transformed whenever variance was heterogeneous), incorporating all relevant variables: treatment, specific agonist, and tissue; for AC studies, values with vs. without PTX; for Gsalpha distribution studies, Gsalpha L vs. Gsalpha S, and cytosol vs. membrane. Fisher's protected least significant difference was used post hoc to establish differences among individual treatments for each variable; this was carried out only where the global test indicated an interaction between treatment and the other variables; in the absence of significant interactions, only main treatment effects were compiled. Significance was assumed at the level of P < 0.05 for main effects; however, for interactions at P < 0.1, we also examined whether lower-order main effects were detectable after subdivision of the interactive variables (33).

Materials. Rats were obtained from Zivic Laboratories (Pittsburgh, PA). cAMP radioimmunoassay kits were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Gsalpha S, Gsalpha L, and Gsalpha antibody were gifts from Dr. P. J. Casey (Duke University, Durham, NC) and goat anti-rabbit IgG (Fc) alkaline phosphatase conjugate was purchased from Promega (Madison, WI). All other reagents were obtained from Sigma Chemical (St. Louis, MO).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Before evaluating Gi function with PTX, we assessed the effect of preincubation of the cardiac and hepatic membranes with the reagents required for PTX-induced ADP ribosylation, but conducted in the absence of PTX itself. The preincubation led to a loss of ~50% of basal AC activity (pmol · min-1 · g tissue-1) in control preparations: heart, 432 ± 17 without preincubation, 196 ± 9 with preincubation (n = 12, P < 0.0001); liver, 311 ± 13 without preincubation, 160 ± 9 with preincubation (n = 12, P < 0.0001). However, the specific beta -AR-mediated cardiac response (isoproterenol-stimulated/basal AC) was not reduced and was actually increased over the unincubated condition (2.43 ± 0.08 without preincubation, 3.80 ± 0.21 with preincubation, n = 12, P < 0.0001). In the liver, the preincubation led to the loss of about one-third of the net beta -AR response, but robust stimulation was still evident (2.57 ± 0.05 without preincubation, 1.93 ± 0.18 with preincubation, n = 12, P < 0.005). Similarly, the forskolin response (forskolin-stimulated/basal AC) remained robust despite the preincubation: heart, 44 ± 2 without preincubation, 49 ± 3 with preincubation (n = 12, not significant); liver, 16.1 ± 1.0 without preincubation, 12.2 ± 0.6 with preincubation (n = 12, P < 0.005). The loss of AC activity entailed by the preincubation required for ADP ribosylation agrees with an earlier report (1).

Next, we determined the effects of the neonatal beta -agonist treatments on AC in the membrane preparations preincubated for ADP ribosylation but without addition of PTX (Table 1). Neither isoproterenol nor terbutaline administration had any significant effect on basal AC activity in heart and liver. In accord with earlier results (3, 4, 45), the beta -AR-mediated AC response did not exhibit pronounced desensitization in animals treated with either of the beta -agonists. Animals given isoproterenol displayed sensitization (10-15% increase), whereas those given terbutaline showed a small (5-10%) decrement in the response. Similarly, the response to forskolin showed significant enhancement in the animals given isoproterenol (10% increase in the heart, 25% increase in the liver) but was unchanged by neonatal terbutaline treatment.

                              
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  		Table 1.   Effects of neonatal terbutaline or isoproterenol treatment on cardiac and hepatic adenylyl cyclase measured in vitro

beta -AR agonists affect Gi signaling. Before examining effects of PTX on individual components of AC signaling, we performed a global ANOVA incorporating all treatments, both tissues, and the three different AC measures (basal, beta -AR response, forskolin response). This initial test indicated a significant overall increase in AC activity evoked by PTX (main effect, P < 0.0001) and significant interactions of treatment × tissue × PTX (P < 0.009) and treatment × PTX × AC measure (P < 0.0004). Accordingly, we evaluated the three AC measures separately across the two different tissues. For basal activity, PTX failed to cause an overall stimulation of AC (Fig. 1). Although a tissue-selective effect was seen (treatment × tissue interaction for the response to PTX), the only individually significant change was a small (5%) increment in the effect of PTX in the liver of isoproterenol-treated animals; other differences of similar magnitude were inconsistent and did not achieve statistical significance.


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  		Fig. 1.   Effect of pertussis toxin (PTX) pretreatment on basal adenylyl cyclase (AC) activity and the AC response mediated by beta -adrenoceptors (beta -ARs) (isoproterenol-stimulated/basal AC). Animals were given isoproterenol or terbutaline on postnatal days (PN) 2-5 and membrane preparations were evaluated on PN6. Data represent means and SEs obtained from 6-12 animals in each group. For basal AC, ANOVA indicates an interaction of treatment × tissue × PTX (P < 0.05). For the beta -AR response, ANOVA indicates a main effect of PTX (P < 0.0001), as well as interactions of treatment × PTX (P < 0.0001), and treatment × tissue × PTX (P < 0.0004). * Significant differences from control; dagger significant difference from control values (P < 0.05) and a significant difference between terbutaline and isoproterenol groups.

In contrast to the relatively minor effect of PTX on basal AC, the beta -AR-mediated response showed robust overall enhancement when the membranes were preincubated with PTX (Fig. 1). Treatment of neonates with beta -agonists had a significant effect on the PTX response (treatment × PTX interaction) that differed between the two tissues (treatment × tissue × PTX interaction). In the heart, PTX elicited an increase in beta -AR-mediated AC stimulation in controls, whereas the response to PTX was completely absent in animals given isoproterenol treatment; in contrast, terbutaline treatment elicited a significant increase in the PTX response. In the liver, PTX elicited a small overall enhancement of the beta -AR-mediated response, with little or no alteration evoked by isoproterenol or terbutaline treatment.

Preincubation of cardiac and hepatic membranes with PTX also increased the AC response to forskolin (forskolin/basal AC activity; main effect of PTX, P < 0.03). However, neither isoproterenol nor terbutaline treatment evoked any significant alterations in the PTX effect. Values for the ratio of forskolin response with/without PTX were heart: control 1.02 ± 0.02, isoproterenol 1.02 ± 0.02, terbutaline 0.99 ± 0.03; liver: 1.05 ± 0.03, 1.11 ± 0.04, and 1.02 ± 0.06, respectively.

beta -AR agonists affect Gsalpha subcellular distribution and splice variants. Western blot analysis of Gsalpha detected both Gsalpha L (52 kDa) and Gsalpha S (45 kDa) isoforms in the membrane and cytosolic fractions of neonatal tissues (Fig. 2). Quantitation was conducted on a relative basis because of incompatibilities in measuring the absolute quantities present in the membrane vs. cytosolic fractions: determinations were conducted relative to a fixed amount of protein loaded onto the gel, but absolute concentrations of membrane and cytosolic proteins in intact cells are not equivalent; additionally, the membrane fraction required solubilization and attendant recovery corrections, factors that do not operate for the cytosolic fraction. We did, however, include standards to ensure blot-to-blot comparability (see METHODS).


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  		Fig. 2.   Representative Western blot of Gsalpha splice variants in neonatal liver, demonstrating the presence of long and short splice variants (Gsalpha L and Gsalpha S, respectively) in both membrane and cytosolic fractions of control neonates and in animals treated with isoproterenol or terbutaline. Each lane contained 40 µg of protein (see METHODS).

Neonatal isoproterenol treatment increased the levels of membrane-associated Gsalpha , evaluated as the total of Gsalpha L and Gsalpha S, with a prominent effect in the liver (approx 2.5-fold above control values) and a more modest effect (approx 30% increase) in the heart (Fig. 3A). In contrast, treatment with terbutaline did not affect membrane Gsalpha levels. Changes in cytosolic Gsalpha were quantitatively and qualitatively different from those in the membrane fraction (Fig. 3B). Isoproterenol evoked a marked increase (approx 2.5-fold) that was equivalent for both the heart and liver; terbutaline also caused significant elevations of cytosolic Gsalpha . Selectivity of the shift toward cytosolic Gsalpha was readily evident from the ratio of cytosol/membrane values (Fig. 3C): terbutaline preferentially and significantly increased cytosolic Gsalpha , whereas isoproterenol was much less effective (not statistically significant). In addition, the liver showed a much higher relative pool of cytosolic Gsalpha than the heart (note different scales for Fig. 3C).


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  		Fig. 3.   Effects of isoproterenol or terbutaline treatment on total Gsalpha in membrane (A) and cytosolic (B) fractions and on the ratio of cytosolic/membrane Gsalpha (C); total Gsalpha was assessed as the sum of Gsalpha L and Gsalpha S. Data represent means and SEs obtained from 5-12 animals for each group. ANOVA across all treatments and both tissues appears at the top of each panel. Where a treatment × tissue interaction was found (membrane Gsalpha ), ANOVA for each tissue appears at the bottom of the panel and asterisks denote significant differences from controls; without an interaction, only the main effect was evaluated. Note different scales for heart and liver in C.

Different Gsalpha splice variants also influence the effectiveness of beta -AR signal transduction (6). In the neonatal heart, Gsalpha S was a minor species of the membrane fraction, accounting for only ~5% of membrane Gsalpha (Fig. 4A). In the neonatal liver, however, Gsalpha S represented nearly 40% of membrane-associated Gsalpha . Isoproterenol treatment, but not terbutaline treatment, substantially increased the proportion of Gsalpha S in both tissues. In contrast to the membrane fractions, nearly all of the cytosolic fraction was Gsalpha S (Fig. 4B). Isoproterenol treatment had little or no effect on the proportion of cytosolic Gsalpha S in the heart but evoked a significant decrement in the liver. Terbutaline treatment decreased the proportion of Gsalpha S in the cardiac cytosol; although the hepatic effect was not significant compared with control values, it also could not be distinguished from the effect in the heart (treatment × tissue interaction was not significant for terbutaline), and the main effect of terbutaline was significant (P < 0.02) when compiled across both tissues.


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  		Fig. 4.   Effects of isoproterenol or terbutaline treatment on Gsalpha representing Gsalpha S in the membrane (A) and cytosolic (B) fractions. Data represent means and SEs obtained from 5-12 animals for each group. ANOVA across all treatments and both tissues appears at the top of each panel, with subdivision by tissue at the bottom of the panel. * Significant differences from control.

Finally, we evaluated whether the effects of neonatal beta -agonist treatment on Gsalpha distribution and isoforms were unique to development. Adult male rats (275 g body wt, 6 animals per treatment group) were given the same isoproterenol regimen as that used in neonates. Twenty-four hours after the last dose, we evaluated the characteristics of hepatic Gsalpha . Membrane Gsalpha increased ~35% after isoproterenol exposure (control, 2.5 ± 0.3 units; isoproterenol, 3.4 ± 0.5), a much smaller effect than had been seen in the neonate (treatment × age, P < 0.04). In the adult, isoproterenol treatment did not produce a significant increase in cytosolic Gsalpha (control, 2.0 ± 0.2 units; isoproterenol, 2.3 ± 0.2), and again this was statistically distinguishable from the increase seen in the neonate (treatment × age, P < 0.05). The proportion of membrane Gsalpha S was unaffected by isoproterenol treatment in the adult liver (control, 55 ± 2%; isoproterenol, 52 ± 2%), whereas the same treatment evoked a robust increase in the neonate (treatment × age, P < 0.03). Finally, in the adult, isoproterenol treatment did not alter the proportion of cytosolic Gsalpha representing the short-splice variant (control, 84 ± 2%; isoproterenol, 79 ± 3%), whereas it had elicited a significant reduction in the neonate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Repeated isoproterenol administration to neonatal rats increased the AC response to beta -AR stimulation in both the heart and liver, instead of uncoupling receptors from the signaling pathway. In previous work, we showed that induction of AC is responsible, in part, for agonist-induced sensitization in the neonate (43). In addition, unique adaptations at the level of G proteins have been hypothesized to contribute to the response pattern: isoproterenol administration reduces the concentration of Gi and enhances beta -AR coupling to Gs (42, 44). Results obtained here indicate that the isoproterenol-induced reduction in Gi produces a decrement in the function of this inhibitory G protein: PTX increased the cardiac AC response to beta -AR stimulation in membrane preparations from control animals but failed to do so in membranes from isoproterenol-treated animals. In contrast, in mature cardiac cells, isoproterenol increases Gi expression and activity, contributing to desensitization (26). A decrease in Gi function thus helps produce the opposite response, heterologous sensitization of beta -AR signaling, seen in the neonate.

Isoproterenol treatment elicited an ~30% reduction in the concentration of Gi, yet the inhibitory contribution of Gi to the net beta -AR signal was completely lost. Thus, although the G proteins are in stoichiometric excess compared with beta -ARs or AC (23), loss of a relatively minor proportion nevertheless is sufficient to compromise the response. Recent evidence indicates that beta -AR function is determined by restriction of signaling elements to caveolae containing the receptor juxtaposed to its target proteins (22) and our results suggest that the loss of Gi is likely to involve decrements in protein colocalized with beta -ARs. Furthermore, the isoproterenol-induced decrement in Gi signaling may contribute ultimately to adverse effects on neonatal cardiac function. Vagal parasympathetic control of heart rate and contractility involve cholinergic receptors operating through Gi, and these are only weakly established in the neonatal period (20). The same isoproterenol treatment found here to interfere with Gi function, elicits a decrement in cardiac m2-cholinergic receptor expression (10), so that the combination of downregulation of the Gi-linked m2-receptor, downregulation of Gi and loss of Gi function can cumulate to produce impairment of vagal cardiac signaling.

Our results for effects of isoproterenol in the liver and for terbutaline in both heart and liver provide a third corollary: loss of the PTX-related component of AC signaling was not seen in the liver after neonatal isoproterenol treatment, nor in either tissue when the beta 2-selective agonist terbutaline was substituted for isoproterenol. Given the predominance of beta 1-ARs in the heart and beta 2-ARs in the liver (2, 32), these results suggest that the suppression of Gi expression and function are specifically related to stimulation of the beta 1-AR subtype. In fact, terbutaline tended to increase the inhibitory actions mediated by Gi, as evidenced by an augmented cardiac AC response to treatment of the membranes with PTX; this resembles the homeostatic response that is seen ordinarily in mature cells (26). The importance of Gi in determining the net response, heterologous sensitization vs. desensitization, is illustrated by the fact that terbutaline, unlike isoproterenol, did not sensitize the AC response to beta -AR stimulation.

Although our results indicate that Gi-mediated signaling responds differently to beta -AR stimulation in neonates compared with adults, this factor cannot totally explain why agonist administration elicits sensitization instead of desensitization, as the PTX-sensitive component of AC activity represented no more than 15% of the total AC signal. Accordingly, we also examined effects on Gs. Earlier work indicated that cardiac beta -AR coupling to Gs was enhanced after neonatal isoproterenol administration, instead of exhibiting the uncoupling typical of the mature cell (42). In the current study, we found a modest (30%) increase in the Gsalpha concentration in cardiac membranes but a massive (2.5-fold) increase in hepatic membranes; because Gsalpha overexpression is known to protect cells from beta -AR desensitization (39), our findings provide a ready explanation for the ability of hepatic cells to maintain their signaling capabilities despite the fact that they did not display a loss of Gi function. Again, this was seen with isoproterenol treatment but not with terbutaline. Nevertheless, we found increased Gsalpha expression in the cytosolic fractions of both cardiac and hepatic cells after neonatal treatment with either of the beta -agonist drugs. In the mature cell, beta -AR-mediated heterologous desensitization involves a shift of Gsalpha from the membrane to the cytosol, where it is incapable of coupling to the membrane-associated beta -ARs (41). Our findings indicate that this component of desensitization is intact in immature cells. However, as isoproterenol induced Gsalpha by the same proportion in both the membrane and cytosolic fractions, the removal of Gsalpha from the membrane was offset, so that membrane signaling was sustained. With terbutaline administration, the membrane component was maintained (but not enhanced), whereas the cytosolic fraction showed the increase characteristic of desensitization; accordingly, beta -AR/AC signaling was preserved with the terbutaline model, but did not show the enhancement that was characteristic of the isoproterenol treatment paradigm. For these effects, relative contributions of beta 1-ARs and beta 2-ARs cannot explain the differences in effects between isoproterenol and terbutaline. Isoproterenol had a much greater proportional effect on membrane-associated Gsalpha in the liver, which expresses the beta 2-subtype, than in the heart, which has a beta 1-AR majority (2, 32). If receptor subtype dictated the tissue difference, then terbutaline should have been even more efficacious, whereas it actually had a smaller effect. It is thus likely that the differences in responsiveness with the two treatments reflect another factor; as terbutaline is longer lasting than isoproterenol, it is possible that episodic stimulation of beta -ARs elicits greater Gsalpha induction or, alternatively, that continuous stimulation by terbutaline provides for neonatal upregulation of Gsalpha but combined with the internalization that is characteristic of adult-type desensitization. The greater contribution of desensitization components to the terbutaline response is likely to explain why heterologous sensitization of the beta -AR/AC pathway is less notable after terbutaline than after isoproterenol (4, 37). Regardless of the differences in details of the effects of isoproterenol and terbutaline, our findings for the effects on the expression and subcellular distribution of Gs indicate an additional unique mechanism not present in the adult that contributes to the resistance of immature cells to agonist-induced desensitization.

We also evaluated the effects of neonatal beta -agonist administration on splice variants of Gsalpha . The lower molecular weight subtype, Gsalpha S, has greater functional activity than the longer splice variant, Gsalpha L (6, 40). Neonatal isoproterenol treatment increased membrane Gsalpha S in both the heart and liver but by differing mechanisms. In the heart, the increase in membrane-associated Gsalpha S occurred without a corresponding decrease in the cytosolic fraction, thus representing net induction of the protein. In fact, neonatal isoproterenol treatment shifted the proportion of the Gsalpha S splice variant in neonates to approximate the higher value in the normal adult heart (41) or liver (this study), suggesting that neonatal isoproterenol treatment accelerates the maturational profile of Gsalpha splice variants. This actually matches the functional effect, which is to shift beta -AR association with G proteins from the lower efficacy of the neonate to the higher coupling characteristic of the adult (42). In the liver, the increase in membrane-associated Gsalpha S was juxtaposed to a decrease in the cytosolic fraction, thus implying redistribution rather than (or in addition to) induction. Regardless of the mechanism, induction or redistribution, either effect would contribute to preservation or enhancement of beta -AR/AC signaling. Furthermore, both effects represent actions that are unique to development: no such changes were seen in adult hepatic cells in the present study, nor in earlier work with mature cardiac cells (41). As before, the response to terbutaline differed from isoproterenol and resembled those associated with desensitization (i.e., no change in membrane-associated Gsalpha S but a decrease in the cytosolic fraction), effects that are likely to offset agonist-induced sensitization of AC as seen with terbutaline (2-4).

Superimposed on the disparities in effects between isoproterenol and terbutaline, some of the differences in G protein expression or function may reflect selectivities dictated by the types of cells or tissues over and above any contribution from different beta -AR subtypes. Although they both express beta -ARs and G proteins, cardiac and hepatic cells obviously bear little resemblance to each other, either in their repertoires of other proteins or in their differentiation characteristics. As just one example, cardiac cells undergo terminal differentiation and lose the ability to replicate, whereas hepatic cells maintain mitotic capabilities into adulthood (27). Thus terbutaline treatment evoked an increase in Gi function in the heart (augmented beta -AR stimulation in PTX-treated membranes) but not in the liver, despite the fact that hepatic cells express a higher proportion of beta 2-ARs. Similar tissue disparities are likely to contribute to differential effects of beta -agonists on G protein splice variants and their subcellular distributions. Indeed, recent studies demonstrated disparate patterns of terbutaline-induced sensitization vs. desensitization in different brain regions, with the outcome dictated by the maturational timetable for each region (30). Factors dictated by the cellular milieu may thus contribute to some of the differences in the effects of isoproterenol and terbutaline on beta -AR-mediated responses in cardiac and hepatic cells, but obviously, future work with other tissues will be needed to clarify this issue.

In conclusion, we found changes in G protein concentrations, subcellular distribution, and functional activity that provide mechanistic explanations for the resistance of neonatal beta -ARs to desensitization: loss of inhibitory actions mediated through Gi, enhancement of membrane-associated Gsalpha , and a shift to expression of the more active, short-splice Gsalpha variant. Differences in the relative contributions of each of these factors explain the disparities of effects seen for neonatal isoproterenol vs. terbutaline treatments on beta -AR/AC signaling in the heart and liver. However, for either treatment, the net effect of these mechanisms, superimposed on induction of AC (43) and resistance to agonist-induced beta -AR downregulation (2, 4, 37), combines to preserve and enhance cell signaling mediated by beta -ARs during the critical period of the perinatal transition (17).


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grant HD-09713.


    FOOTNOTES

Address for reprint requests and other correspondence: T. A. Slotkin, Box 3813 DUMC, Dept. of Pharmacology & Cancer Biology, Duke Univ. Med. Ctr., Durham, NC 27710 (E mail: t.slotkin{at}duke.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 29, 2002;10.1152/ajpregu.00409.2002

Received 9 July 2002; accepted in final form 9 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 283(5):R1236-R1244
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