Beta-Adrnergic receptor and beta-adrenergic sensitive adenylate cyclase were demonstrated in membrane fractions of human placenta. Placental membranes from normal term pregnancies bound the beta-adrenergic antagonist (-)[3H]dihydroalprenolol to a single saturable class of sites (Kd = 2.31 +/- 0.23 nM; n = 9; maximal capacity, 112 +/- 9 fmol/mg). Competition for binding was stereoselective for (-)isomers of propranolol, and beta-adrenergic agonists displayed competition for the placental receptor in the order (-)isoproterenol greater than (-)epinephrine greater than (-)norepinephrine, typical of a beta 2 type receptor. Beta-Adrenergic receptor was present in placental tissue as early as 10 weeks gestational age, and binding capacity decreased slightly with advancing gestation. [3H]Dihydroalprenolol binding was coupled to epinephrine-stimulated adenylate cyclase activity throughout gestation. The subcellular distribution of both beta-adrenergic receptors and epinephrine-stimulated adenylate cyclase suggest their localization primarily in nonbrush border membrane fractions, presumably from plasma membranes more closely related to the fetal rather than to the maternal circulation. Epinephrine-sensitive adenylate cyclase was not present in purified brush border preparations which were directly exposed to maternal blood in the intervillous space.
Summarymore directly proportional to cell protein. The function and affinityThe ontogeny of P-adrenergic receptors and the characteristics of catecholamine sensitive adenylate cyclase activity were demonstrated in rat ventricular myocardium. P-Adrenergic receptors were identified by the binding of (-)-[3HJdihydroalprenolol, , and (f )-[1251]iodohydroxy-benzylpindolol, (+)-[lZ5[IIHYP. Heart weight, protein, and the number of P-adrenergic receptors per cell increased with advancing age; however, binding capacity (B,,,) per mg protein was lower in membrane from the adult rat (220 g body weight) than in membrane from the fetal or neonatal rat, 36.8 + 4.1 and 58.3 + 6.3 fmoles*mg-' protein, respectively, mean + SE, P < 0.01. The age-dependent increase in the numbers of receptors per heart correlated well with the increases in ventricular weight. In contrast, protein content, Na', Kt-ATPase, 5'-nucleotidase, and adenylate cyclase content increased more rapidly than ventricular weight, and the developmental increase in 13H]DHA binding sites (per heart) did not keep up with increases in ventricular protein. Although specific activity of (-)-[3HIDHA binding was lower in the adult ventricle, the characteristics of the P-receptor did not vary with age; the subtype was Dl determined by classical agonist competition in fetal and adult ventricular membranes and the PI-and P2-adrenergic receptor subtypes were present in the ratio of 75% PI and 25% p2 in both fetal and adult samples. Guanine nucleotides decreased agonist affinity for the inhibition of (+)-[12511HYP binding equally in adult and fetal samples and enhanced the activation of adenylate cyclase by catecholamine. Basal adenylate cyclase activity was significantly higher in weanling, 13.4 + 1.42, and adult, 14.2 + 5.8, than in 21-day fetal or newborn ventricular membrane, 8.18 & 1.5 pmoles cyclic adenosine 3':5'-monophosphate produced mg-'a min-' (mean 2 S.D.). The percent activation by catecholamine (with and without GTP) was also slightly higher in samples from the older rats, P < 0.01. In the adult and weanling heart, both guanine triphosphate (GTP) and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] were effective in enhancing catecholamine sensitive activity. Maximal catalytic activation of adenylate cyclase was observed in the presence of (-)-epinephrine and Gpp(NH)p and was significantly higher (fold stimulation) in fetal or neonatal ventricular samples as compared to adult or weanling ventricular samples, P < 0.01. SpeculationThe decreased specific activity (per mg protein) of 8-adrenergic receptors in adult as compared to fetal and neonatal myocardium may therefore be related to the decreasing proportion of sarcolemma1 protein versus total and particulate cell protein which occurs during hypertrophic cardiac growth, supporting the hypothesis that sarcolemmal surface area is a major determinant of P-receptor number. In contrast, sarcolemmal activities which mediate cellular functions by regulating the concentration of various substances within the cell, such as Na+,...
beta-Adrenergic receptors and catecholamine-sensitive adenylate cyclase were identified and partially characterized in membrane fractions of rabbit lungs from day 25 of gestation to adulthood with the beta-adrenergic antagonists (--)-[3H]dihydroalprenolol [(--)-[3H]DHA] and (--)-[125I]iodohydroxybenzylpindolol [(--)-[125I]HYP]. beta-Adrenergic receptor number (Bmax) increased 11.5-fold during this time period, increasing progressively during the latter days of gestation and the early neonatal period, from 37 +/- 10 fmol/mg protein at 25 days gestation to 425 +/- 51 fmol/mg in the adult rabbit lung (mean +/- SD). Receptor affinity for (--)-[3H]DHA (KD = 1.8 nM) or (--)-[125I]HYP (KD - 0.104 nM) and the proportion of beta 1- and beta 2-adrenergic receptor subtypes (60% beta 1 and 40% beta 2) did not change with advancing age. Basal adenylate cyclase activity in lung homogenates decreased significantly with increasing age, whereas the activity in the presence of catecholamine or NaF remained nearly constant. Catecholamines stimulated adenylate cyclase activity at all ages studied supporting a role of the maturation of beta-adrenergic receptors in the regulation of pulmonary function.
Summary AbbreviationsProtein kinase activity that is dependent on 3',5'-Cyclic adenosine monophosphate (CAMP-PK), 13H]cAMP binding, and CAMP-dependent protein phosphorylation were identified and partially characterized in cytosolic preparations of rat lung from day 18 of gestation to adulthood. Major CAMP-dependent phosphoproteins in lung preparations were compared to those in cytosol from purified Type I1 epithelial cells. Both Type I and Type I1 regulatory subunits of CAMP-PK were identified in fetal and adult lung. Inhibition of specific [3H]cAMP binding to lung cytosol (to the regulatory subunit of the CAMP-dependent protein kinase) followed the order of potency: cAMP > cGMP; adenosine, ADP, and ATP were inactive. Scatchard plots of saturation experiments with ["HICAMP and lung cytosol were linear. Dissociation constant (K,) for cAMP binding was approximately 2-3 nM, and did not change significantly with age. In contrast, binding capacity varied significantly during development and agerelated changes in binding capacity were associated with similar changes in CAMP-dependent histone kinase activity. Both I3H] cAMP binding and CAMP-dependent protein kinase activity decreased slightly before birth, reached maximal activity during the suckling period, and decreased in adulthood.cAMP enhanced histone kinase activity in rat lung cytosol at all ages studied, from day 18 of gestation to adulthood. cAMP also specifically enhanced phosphorylation of several endogenous cytosolic proteins that were identified by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major proteins whose phosphorylation was selectively enhanced by cAI41P or inhibited by protein 'kinase inhibitor were approximately Mr = 260,000, 240,000, 97,000, 56,000, 44.000, and 28,000. CAMP-dependent phosphorylation of lung cytosolic proteins was compared to that in cytosol from purified rat Type I1 epithelial cells. Mr = 44,000, the most prominent CAMP-dependent phosphoprotein identified in cytosol from adult lung, comigrated with the major CAMP-dependent phosphoprotein from rat Type I1 epithelial cells. In lung cytosol, phosphorylation of Mr = 44,000 increased dramatically during perinatal development. Phosphorylation of several other major phosphoproteins changed with age, primarily in the perinatal period.Maturation of pulmonary function has been associated previously with marked changes in P-adrenergic receptors and catecholamine-sensitive adenylate cyclase activity which mediate the synthesis of cAMP in lung tissues. cAMP activates protein kinases that catalyze the phosphorylation of regulatory proteins by altering their activity and function. The present work demonstrates developmental changes in CAMP-dependent protein kinase activity and marked ontogenic changes in phosphorylation of several cytosolic lung proteins. Ontogenic changes in specific protein substrates of protein kinases and changes in their state of phosphorylation in response to cAMP are likely to mediate developmental changes in pulmonary responses to h...
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