INTRODUCTION

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₂-ADRENOCEPTOR (₂-AR) agonists, such as terbutaline, are widely used to prevent preterm delivery, a leading cause of infant morbidity and mortality in the United States (3). In addition to blocking uterine contractions, terbutaline penetrates the placenta to stimulate fetal -ARs (2, 16). To a large extent, these effects are likely to be beneficial if preterm delivery occurs, as catecholaminergic stimulation promotes cardiovascular, respiratory, and metabolic adaptations that are necessary for perinatal survival (9). However, neonates from mothers receiving tocolytic therapy also exhibit adverse effects, including tachycardia, alterations in glucose metabolism (4), and elevated incidence of cardiac anomalies (12, 17).

Both the positive and negative effects of prenatal terbutaline exposure are likely to reflect the result of prolonged, excessive -AR stimulation. In adult tissues, -AR responses are limited by receptor downregulation and receptor uncoupling from cell signaling, best exemplified by desensitization of the adenylyl cyclase (AC) signaling cascade (25). However, immature tissues appear to be resistant to desensitization (23, 24, 27, 30), and we recently found that -AR/AC responses are maintained in the face of repeated terbutaline administration in fetal rats (1). The current study extends this work into the neonatal period, a developmental stage in rats that corresponds more closely to the status of sympathetic innervation of peripheral tissues in the third trimester human fetus (9), the period in which terbutaline would most likely be used. We assessed the ability of terbutaline to induce receptor downregulation and desensitization of -AR signaling after treatment on postnatal (PN) days 2-5, before the onset of sympathetic function, and on PN 11-14, the period in which innervation shows its most rapid development (19). We compared effects in the heart and liver, tissues that differ in the predominant -AR subtype (₁ in the heart, ₂ in the liver). These tissues also represent major targets for the catecholamines in the perinatal transition, mediating essential cardiovascular and metabolic adjustments (9). In addition to assessment of -ARs and -AR-mediated responses, we determined AC responses to stimulants that bypass the receptors to act on G proteins or on AC itself, so as to dissect the heterologous, downstream mechanisms underlying resistance to -AR desensitization (1, 24).

	METHODS

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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 (Zivic-Miller Laboratories, Allison Park, PA) were shipped by climate-controlled truck (transit time, 12 h) and housed with free access to food and water. After birth, pups were randomized and redistributed to the nursing dams with litter sizes maintained at 10 pups; dams were periodically reassigned to different litters to distribute any maternal differences equally. Within each litter, equal numbers of males and females were assigned to each treatment group. Pups were given daily subcutaneous injections of 10 mg/kg of terbutaline hemisulfate or an equivalent volume (1 ml/kg) of isotonic saline vehicle on PN 2-5 or on PN 11-14. This regimen has been shown previously to elicit robust ₂-AR downregulation in the adult and to evoke cardiac activation and enhancement of lung surfactant synthesis in the fetus (1, 8, 16). Twenty-four hours after the final injection, hearts, and livers were dissected, frozen in liquid nitrogen, and stored at 45°C until assayed. Determinations on PN 6 required two hearts for each assay.

Membrane preparation. Tissues were thawed and homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in 39 volumes of ice-cold buffer containing 145 mM NaCl, 2 mM MgCl₂, and 20 mM Tris (pH 7.5) strained through several layers of cheesecloth when necessary to remove connective tissue and sedimented at 40,000 g for 15 min. The pellets were washed twice 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 final protein concentration (determined with Folin reagent) of 0.5-1 mg/ml in a buffer consisting of 250 mM sucrose, 1 mM EGTA, and 10 mM Tris (pH 7.4).

-AR binding. Receptor binding capabilities were assessed by methods described in earlier publications (7, 15). The overall strategy was to examine binding of [¹²⁵I]iodopindolol at a single, subsaturating ligand concentration (67 pM) in preparations from each animal; changes can thereby be detected regardless of whether they result from alterations in receptor dissociation constant or maximal binding (B_max). This approach was necessitated by the requirement to measure binding in hundreds of membrane preparations in the study; earlier work indicates that receptor downregulation elicited by terbutaline, administered to either fetal or adult rats, reflects a decrease in B_max (1). Binding was determined in samples containing 200 µg of membrane protein in 250 µl of 145 mM NaCl, 2 mM MgCl₂, 1 mM sodium ascorbate, 20 mM Tris (pH 7.5); samples were incubated for 20 min at ambient temperature, and labeled membranes were trapped by vacuum filtration onto glass fiber filters. Nonspecific binding was determined by displacement with 100 µM D,L-isoproterenol and ranged from 7 to 25%, depending on age and tissue.

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 or without 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, and 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.

Data analysis. Data are presented as means and SE. For convenience, some data are presented as the percent change from control values, but statistical differences were always established using the original data. To establish treatment differences in receptor binding or AC activity, a global ANOVA (data log transformed whenever variance was heterogeneous) was first conducted across the in vivo treatment groups, age, sex, tissue, and for AC, all in vitro conditions under which AC was determined; the in vitro stimulant conditions were repeated measures, because each membrane preparation was used for the multiple types of AC determinations. As justified by significant interactions of treatment × age and treatment × tissue (see RESULTS), data were then subdivided to permit testing of individual treatments and AC measures that differed from control values; these were conducted by lower-order ANOVAs, followed, where appropriate, by Fisher's protected least-significant difference to identify specific ages at which the terbutaline group differed from the corresponding control. However, in situations where there was no interaction of treatment × age, only main treatment effects are reported without conducting separate tests for each age. Tests of drug effects on body and tissue weights and on tissue membrane protein concentration were evaluated by similar procedures. For all tests, significance for main treatment effects was assumed at P < 0.05; however, for interactions at P < 0.1, we also examined whether lower-order main effects were detectable after subdivision of the interactive variables (22).

Materials. cAMP radioimmunoassay kits were purchased from Amersham (Chicago, IL) and [¹²⁵I]iodopindolol (specific activity 2,200 Ci/mmol) was obtained from New England Nuclear (Boston, MA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

	RESULTS

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Development of control rats. Cardiac -AR binding increased between PN 6 and PN 15, paralleled by a large augmentation of the AC response to -AR stimulation by isoproterenol (Table 1). There was no change in basal AC activity, but the coupling of G proteins to AC showed a substantial rise over this period, evidenced by a greater response to the addition of GTP (15% increase on PN 6, 180% increase on PN 15). Although the response to maximal activation of all G proteins with NaF also increased over this period, the degree of increase was smaller than that for GTP alone (age × stimulant, P < 0.0001). At both age points, forskolin produced massive stimulation of AC, much greater than the response to Mn²⁺ (main effect of stimulant, P < 0.0001); the preference for forskolin over Mn²⁺ increased significantly between PN 6 and PN 15 (age × stimulant, P < 0.0001).

View this table: [in this window] [in a new window]   Table 1.   -Adrenoceptor binding and AC activities in control tissues

In contrast to the heart, -AR binding in the liver decreased by one-third between PN 6 and PN 15, consistent with earlier reports (7, 15). Indexes of AC activity also showed an overall decrease during this period (main effect of age, P < 0.0001). The proportional stimulation evoked by addition of GTP remained similar (double basal activity), as did the response to isoproterenol (double the activity compared with +GTP) or NaF (7 times +GTP), so that all of them decreased in parallel. Although the liver, like the heart, displayed a preferential stimulation by forskolin compared with Mn²⁺ (main effect of stimulant, P < 0.0001), the preference ratio was substantially smaller than seen in the heart (tissue × stimulant, P < 0.0001) and did not show a significant change with age (stimulant × age, not significant).

General effects of terbutaline. Neonatal terbutaline treatment on PN 2-5 or PN 11-14 did not cause any mortality and failed to alter body weight, heart weight, or liver weight nor did it affect the concentration of membrane proteins (Table 2). Accordingly, results for -ARs and AC are compared on the basis of binding or activity per milligram of membrane protein, so as to correct for any sample-to-sample variability in the recovery of membranes. Given the lack of significant effect on the membrane protein concentration, differences in -ARs and AC were also detectable when values were determined per gram of tissue.

Effects of terbutaline on the heart. When neonatal rats were given terbutaline on PN 2-5 or PN 11-14, there was a 15-20% decrement in cardiac -AR binding, comparable to what is seen in fetal or adult heart after terbutaline treatment (1; Fig. 1). Despite the significant -AR downregulation, neither treatment regimen elicited desensitization of -AR-mediated AC activity, as assessed by the response to isoproterenol, the response to isoproterenol relative to GTP alone (isoproterenol/+GTP ratio), or the response to isoproterenol relative to maximal G protein stimulation with NaF (isoproterenol/NaF ratio). For other AC stimulants, however, there were distinct disparities in the effects of the two regimens. On PN 6, 24 h after the last injection of the early treatment regimen, there were no changes in any of the components of AC activity. However, with later terbutaline treatment (PN 11-14) there was significant induction of basal AC activity and of the responses to NaF or forskolin. The induction was selective for forskolin compared with the other direct AC stimulant, Mn²⁺, as evidenced by a significant decrease in the Mn²⁺/forskolin ratio.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of neonatal terbutaline treatment [10 mg/kg sc daily on postnatal day (PN) 2-5 or PN 11-14] on cardiac -adrenergic receptor (-AR) binding and adenylyl cyclase (AC) activity. Data represent means and SE obtained from 8-16 determinations in each treatment group at each age, presented as the percentage change from control values (Table 1). ANOVA across all AC measures appears at the top of A, with subdivision by measure at the bottom of A and of B. *Individual values for which the terbutaline group differs from the corresponding control, evaluated only where ANOVA indicated a significant interaction of treatment × age; otherwise, only main effects are reported. Rx, treatment; Iso, isoproterenol.

Effects of terbutaline on the liver. Neonatal terbutaline had a much greater effect on hepatic -AR binding than on cardiac -ARs (treatment × tissue, P < 0.0001; Fig. 2). Values were decreased by 40% regardless of whether treatment occurred in the early neonatal period (PN 2-5) or in the second postnatal week (PN 11-14). Despite the marked decline in -ARs, the ability of isoproterenol to stimulate AC activity was only slightly reduced (10-15%), an effect clearly distinguishable from the robust receptor downregulation (treatment × measure, P < 0.0001). Examination of the effects on the AC response to stimulants downstream from the -AR indicated that heterologous sensitization of the signaling pathway offset the effects of homologous desensitization of receptor-mediated signaling. On PN 6, the AC responses to NaF, forskolin, or Mn²⁺ all were elevated (main effect of treatment, P < 0.0001). The activity ratios confirmed that the increases in G protein-mediated and total AC catalytic activity were able to offset homologous desensitization: there were significant decreases in the isoproterenol/+GTP and isoproterenol/NaF activity ratios (main effect of treatment, P < 0.0001), with a greater effect on the latter ratio (treatment × measure, P < 0.0001), as would be expected from differential effects on the -AR signaling component. With treatment on PN 11-14, the same directional changes were seen except for smaller net effects on the responses to forskolin and Mn²⁺ (treatment × age, P < 0.04).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of neonatal terbutaline treatment (10 mg/kg sc daily on PN 2-5 or PN 11-14) on hepatic -AR binding and AC activity. Data represent means and SEs obtained from 8 determinations in each treatment group at each age, presented as the percentage change from control values (Table 1). ANOVA across all AC measures appears at the top of A, with subdivision by measure at the bottom of A and of B. *Individual values for which the terbutaline group differs from the corresponding control, evaluated only where ANOVA indicated a significant interaction of treatment × age; otherwise, only main effects are reported.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of neonatal terbutaline treatment (10 mg/kg sc daily on PN 2-5 or PN 11-14) on hepatic AC responses to clonidine (500 µM), measured in the presence of isoproterenol (A) or forskolin (B). Data represent means and SE obtained from 8 determinations in each treatment group at each age, presented as the percent change from values obtained without addition of clonidine. ANOVA across all measures appears at the top of A and of B; subdivision into separate ages was not carried out because of the absence of interactions of treatment × age.

	DISCUSSION

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Signaling mediated by the -AR/AC pathway undergoes major changes in the neonatal period. In the fetus or in the adult, the cardiac AC response to isoproterenol is much smaller than that seen with global activation of G proteins by NaF (1), whereas in the current study, we found that the two stimulants were equally effective in the neonate. Accordingly, the relative efficiency of -AR coupling to AC peaks in this period. In contrast, hepatic AC responses to -AR stimulation are highest in the fetus (1) and decline sequentially through the neonatal period into adulthood, paralleling the ontogenetic decline in the expression of -ARs (7, 15). The two tissues also differ substantially in their developmental patterns for catalytic properties of AC (1): throughout development, the heart displays a preferential response to forskolin vs. Mn²⁺, just as seen here in the neonate, whereas in the liver, Mn²⁺ is more effective than forskolin in the fetus, less effective in the neonate, and equally effective in the adult. Accordingly, the effects of neonatal terbutaline treatment can be expected to differ in the two tissues not only because of disparate proportions of ₁- and ₂-subtypes but also because of inherent discrepancies in receptor coupling and in the ontogenetic patterns of G proteins and AC itself.

In mature cells, prolonged or excessive stimulation of -ARs elicits receptor downregulation as a key component of desensitization. However, previous reports indicate that -ARs in the neonate are resistant to the downregulation elicited by administration of isoproterenol, a mixed ₁/₂-agonist (10, 24). In an earlier study (1), we found that in the fetus, terbutaline, a ₂-selective drug, did evoke a small degree of cardiac -AR downregulation and a much more robust decrease of hepatic -ARs. The current results resolve the apparent discrepancy between the downregulation caused by fetal terbutaline and lack of downregulation seen with isoproterenol in the neonate: terbutaline administered to neonatal rats evoked cardiac and hepatic -AR downregulation similar in magnitude (15 and 40%, respectively) to that seen in the fetus or the adult (1); the relatively greater effect in the liver is expected, given the higher proportion of hepatic ₂-ARs (1). Obviously, immature cells contain the machinery necessary to elicit receptor downregulation, yet are resistant to doing so when isoproterenol is administered instead of terbutaline. There are two possible explanations. First, terbutaline and isoproterenol differ in their pharmacokinetics, with terbutaline providing a much more prolonged effect. Accordingly, immature cells may be resistant to, albeit not absolutely incapable of, -AR downregulation, requiring much more prolonged stimulation to elicit the effect; thus there may be a relative (but not absolute) deficiency in components necessary for downregulation. Alternatively, specificity toward ₁- vs. ₂-subtypes may elicit different trophic actions on cardiac cell development that offset receptor downregulation, producing an active response that prevents the loss of receptors from the cell surface; if this interpretation is correct, it would imply a specific role for ₁-ARs in resistance to downregulation.

Regardless of the presence or absence of -AR downregulation, and notwithstanding the marked difference in the degree of downregulation between heart and liver, we did not observe desensitization of AC signaling mediated through -ARs, the same result found after fetal terbutaline exposure (1) or neonatal isoproterenol treatment (30). Again, the issue comes down to two possibilities: is there something missing from developing cells that is necessary to receptor desensitization, or are there active processes that are unique to developing cells, which offset the effects of desensitization? Our results provide evidence for contributions from both processes. In the heart, terbutaline administered on PN 11-14 produced induction of AC catalytic activity, evidenced by a significant increase in the enzymatic response to forskolin, with a parallel increase in the response to NaF, which operates through G proteins; we also detected a shift in catalytic properties of AC, a decrease in the Mn²⁺/forskolin stimulation ratio, indicative of promotion of stimulatory G protein actions and/or induction of a different AC isoform (28). As -ARs also operate through G proteins to stimulate AC, the induction of responses at these two downstream signaling loci, an active compensatory process that is not seen in mature cells, clearly could offset desensitization at the level of the -AR. However, terbutaline treatment on PN 2-5 did not elicit induction of the responses to forskolin or NaF, but nevertheless the AC response to isoproterenol was maintained in the face of receptor downregulation, suggesting that, at this earlier stage, there is a deficiency in cellular components required for desensitization.

Of course, a third possibility is simply that the degree of cardiac -AR downregulation is simply too small to elicit a corresponding loss of signaling capabilities, especially given the predominance of the ₁-subtype in this tissue. This latter possibility is addressed by our results in the liver, a tissue in which virtually all the receptors are of the ₂-subtype (1) and in which we observed robust downregulation after neonatal terbutaline administration. Neonates given terbutaline on PN 2-5 showed statistically significant desensitization of AC responses to -AR stimulation, but the loss of the response was far smaller than would be expected from the 40% decrease in receptor numbers. Just as in the heart, we observed induction at the levels of AC (responses to forskolin and Mn²⁺) and G proteins (response to NaF). The heterologous sensitization of signaling elements downstream from the receptor clearly contribute to the protection from desensitization: when we compared the response to isoproterenol with that to NaF, we detected homologous desensitization, but the net effect was offset by induction at the level of G proteins and AC. Notably, however, terbutaline administration on PN 11-14 produced the same protection of the -AR-mediated AC response, despite much smaller induction of AC. So again, the resistance of the developing cells to agonist-induced desensitization resides in a combination of active, heterologous processes that offset homologous receptor downregulation and desensitization, as well as a deficiency in the processes that mediate desensitization. Future work needs to address what specifically prevents developing cells from efficient desensitization of receptor signals. Prolonged agonist exposure activates G protein receptor kinase 2 (GRK2), which translocates to the membrane to phosphorylate -ARs, resulting in recruitment of -arrestin to initiate receptor internalization (14). Neonatal hearts contain an excess of GRK2 relative to the adult (27), yet still do not show -AR desensitization, even before induction of AC (27). It is not yet known if -AR stimulation effectively recruits GRK2 to the plasma membrane in the neonatal heart or whether there might be deficiencies in -arrestin expression or function. However, regardless of the actual mechanism responsible for the deficient ability of neonatal tissues to elicit -AR desensitization, the important net result is that -AR-mediated signaling is maintained regardless of the presence or absence of reductions in -ARs themselves.

Earlier work with neonatal isoproterenol administration indicated that -AR stimulation of immature cardiac cells evokes unique changes in the expression and function of stimulatory vs. inhibitory G proteins, marked by an increase in G_s effect and suppression of G_i expression (27, 29). In the current study with terbutaline, several of our findings also indicated the targeting of G proteins. If the only effect of terbutaline were to induce total AC activity or a new AC isoform, then basal activity and activity in the presence of GTP would simply follow suit. Instead, we found differential effects on basal AC and activity in the presence of GTP, differences of these two measures from the effects on the isoproterenol or NaF response, as well as decreases in the Mn²⁺/forskolin activity ratio (28, 29). We therefore examined the responses mediated by ₂-ARs, which ordinarily act as a negative modulators of AC activity through coupling to G_i. We found that stimulation of neonatal hepatic ₂-ARs potentiated isoproterenol-stimulated cAMP production, an effect previously observed in the fetal liver (1) and in the pregnant rat myometrium (11). Neonatal terbutaline treatment did not alter this pattern. Surprisingly, when we used forskolin as the AC stimulant, we observed a different ontogenetic pattern for ₂-AR-mediated effects: on PN 6, we found a "normal" inhibitory effect of clonidine but on PN 15 we again found a stimulatory effect. In this case, neonatal terbutaline administration enhanced the inhibitory component of the ₂-AR effect, leading to greater decreases in AC on PN 6 and smaller increases on PN 15. The results indicate a complex relationship of neonatal -AR stimulation to the expression and/or function of -ARs and their coupling to G_i, but the exact nature of this interaction is not clear at this time. Previously, we found crosstalk of receptor expression between -ARs and -ARs in developing heart and liver, effects not seen in mature cells (24). Additionally, ₂-ARs couple to both G_s and G_i (26), so that differential effects of terbutaline on the development of the two G protein classes could produce the results seen here (29).

The failure of terbutaline to produce effective -AR desensitization in the neonatal rat is an important consideration in its use as a tocolytic, because the neonatal rat resembles a third trimester human fetus with respect to sympathetic innervation (9). The effective linkage of the -ARs to AC, combined with heterologous sensitization downstream from the receptor, means that terbutaline administration will produce profound and prolonged stimulation of fetal target tissues. This is a favorable outcome in that catecholamine actions at -ARs are necessary for the cardiovascular and metabolic events that enable the successful transition to extrauterine life (9). At the same time, the fact that -AR stimulation elicits changes in target cell differentiation or even apoptosis (5, 6, 21) means that the failure to desensitize the receptors in the face of continued stimulation may confer long-term liabilities. Indeed, abnormalities of neonatal cardiac control and glucose metabolism have been noted after tocolytic therapy (4), along with an increased incidence of cardiac structural anomalies (12, 17).

In conclusion, neonatal terbutaline exposure produces -AR downregulation without reducing the net ability of -AR stimulation to elicit an increase in cAMP production. -AR signaling is maintained because of deficiencies in the ability of immature cells to uncouple receptors from response elements, but also because of heterologous sensitization of signaling components downstream from the receptor. Although these processes enable -AR function to be maintained during the perinatal period, the same processes may promote abnormalities in the development of -AR target tissues when ₂-AR agonists are used to arrest preterm labor.