Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.

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1 The effect of methylene, thio, and imido substituted analogues of adenosine 5'-triphosphate (ATP) on surfactant phospholipid secretion and calcium mobilization in rat isolated alveolar Type II cells was studied. 2 ATP was the most potent secretagogue of adenine nucleotides studied. The rank order of agonist potency for [3H]-phosphatidylcholine secretion was ATP > adenosine 5'-O-(3-thiotriphosphate) (,S-ATP) > P, 'y-imido adenosine 5'-triphosphate (AMPPNP) > P, y-methylene adenosine 5'-triphosphate (f, 7-CH2-ATP) > a, P-methylene adenosine 5'-triphosphate (a, P-CH2-ATP). The respective EC5os were 10-6M, 2 x 10-6M, 2 x 10-M, 5 x 10-5M, and >2.5 x 10-4 M. 3 Exogenous ATP also induced a rapid mobilization ofintracellular calcium monitored by changes in Fura 2 fluorescence. The rank order of agonist potency for calcium mobilization was similar to the rank order ofagonist potency for surfactant secretion: ATP = yS-ATP> AMPPNP> a, P-CH2-ATP. 4 There was no effect of EGTA on ATP-induced calcium mobilization, consistent with the hypothesis that exogenous ATP induces release of calcium from intracellular stores.5 These data are consistent with a P21-purinoceptor regulating surfactant secretion from isolated Type II cells via mobilization of intracellular calcium, since: (a) non-hydrolyzed analogues of ATP are potent secretagogues, (b) P, -y-CH2-ATP was a more potent secretagogue than a, P-CH2-ATP and (c) the rank orders of agonist potency for calcium mobilization and phospholipid secretion were the same.

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