Neurite Extension Occurs in the Absence of Regulated Exocytosis in PC12 Subclones

We have investigated the process leading to differentiation of PC12 cells. This process is known to include extension of neurites and changes in the expression of subsets of proteins involved in cytoskeletal rearrangements or in neurosecretion. To this aim, we have studied a PC12 clone (trk-PC12) stably transfected with the nerve growth factor receptor TrkA. These cells are able to undergo both spontaneous and neurotrophin-induced morphological differentiation. However, both undifferentiated and nerve growth factor-differentiated trk-PC12 cells appear to be completely defective in the expression of proteins of the secretory apparatus, including proteins of synaptic vesicles and large dense-core granules, neurotransmitter transporters, and neurotransmitter-synthesizing enzymes. These results indicate that neurite extension can occur independently of the presence of the neurosecretory machinery, including the proteins that constitute the fusion machine, suggesting the existence of differential activation pathways for the two processes during neuronal differentiation. These findings have been confirmed in independent clones obtained from PC12-27, a previously characterized PC12 variant clone globally incompetent for regulated secretion. In contrast, the integrity of the Rab cycle appears to be necessary for neurite extension, because antisense oligonucleotides against the neurospecific isoform of Rab-guanosine diphosphate-dissociation inhibitor significantly interfere with process formation.     

Difference in the Effectiveness of Ca2+ to Evoke Catecholamine Secretion Between Adrenaline-and Noradrenaline-Containing Cells of Bovine Adrenal Medulla

Abstract: Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca²⁺ (0.8-160 μM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 μM), high K⁺ (14-56 μM), and bradykinin (0.1-1,000 μM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca²⁺-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K⁺ in perfused adrenal glands. These results indicate that the effectiveness of Ca²⁺ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Neuronal differentiation of PC12 cells abolishes the expression of membrane androgen receptors

Sex steroids affect adrenal chromaffin cell function. In the present work, we have examined the expression and functional significance of membrane androgen receptor sites in normal rat adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell line which can differentiate to either a neuronal or to an epithelial phenotype and expresses membrane estrogen receptor sites. Our data are as follows: (a) no cytosolic androgen receptors were found in both normal chromaffin and PC12 cells; (b) both types of chromaffin cells expressed high affinity membrane testosterone binding sites; (c) activation of these sites increased cytosolic Ca²⁺, decreased catecholamine secretion and induced apoptosis; (d) NGF-induced neuronal differentiation of PC12 cells resulted in the suppression of the number of membrane testosterone sites. In conclusion, our data provide evidence for the existence of specific membrane testosterone receptors on adrenal chromaffin cells via which androgens, (some of them originating in the cortex) modulate their function. Neuronal differentiation of chromaffin cells results in a significant attenuation of these effects, via suppression of the expression of membrane androgen receptors suggesting, that the latter are specific for epithelioid chromaffin cells.     

The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well-established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.     

Phosphorylation of Adrenal Medulla Cell Proteins in Conjunction with Stimulation of Catecholamine Secretion

Abstract: Enhanced phosphorylation of two specific protein bands accompanied catecholamine secretion from cultured bovine adrenal medulla cells stimulated by different secretagogues. Cells preincubated with ³²P_i were treated with nicotine, veratridine, Ionomycin, or barium. Each of these secretagogues stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 95,000. Phosphorylation of the 60,000 M.W. protein band was two- to threefold higher than that of the 95,000 M.W. band on stimulation with nicotine, veratridine, or barium, but Ionomycin stimulated phosphorylation of each protein band to the same extent. In general, the increase in phosphorylation was most rapid during the first minute of stimulation and occurred prior to detectable secretion. Phosphorylation reached a relatively constant level within 5 min after onset of stimulation at a time when catecholamine release was still proceeding at a rapid rate. Nicotine-stimulated phosphorylation and catecholamine secretion were calcium-dependent and blocked by d -tubocurarine, whereas tetrodotoxin inhibited veratridine-stimulated secretion and phosphorylation. We conclude that catecholamine secretion and protein phosphorylation occur under similar conditions and that Ca²⁺-dependent incorporation of phosphate into specific proteins may be a link in stimulus-secretion coupling.     

Human growth hormone co-transfection assay to study molecular mechanisms of neurosecretion in PC12 cells

The recent introduction of transient co-transfection assays, with human growth hormone (hGH) as a marker of transfection, has allowed us to study the function of transfected proteins in neurosecretion using intact PC12 cells and adrenal chromaffin cells [J. Biol Chem. 268 (1993) 10983]. Since this assay requires several non-trivial steps (transfection of the genes, triggering of secretion, and measurement of hGH), care should be taken before attributing any observed effect to the transfected proteins. In this article, we describe a detailed procedure for the hGH transfection assay and show that this approach can be used to study the molecular events involved in neurosecretion.     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Nerve growth factor-stimulated neuronal differentiation induces changes in P2 receptor expression and nucleotide-stimulated catecholamine release

Extracellular nucleotides modulate synaptic transmission and neuronal communication by activating purinergic 2 (P2) (nucleotide) receptors. Here, we assessed changes in the regulation by nucleotides and their receptors of an important physiological response - release and uptake of catecholamines - that accompanies sympathoadrenal neuronal differentiation. Nerve growth factor (NGF)-promoted differentiation of pheochromocytoma 12 (PC12) cells enhanced the ability of the non-hydrolyzable ATP analog, ATPgammaS, to stimulate catecholamine (norepinephrine, NE) release and this enhancement occurred without a significant alteration in NE uptake. In addition to ATPgammaS, 2-MeSATP and alphabetaMeATP, P2X receptor-selective agonists, caused greater NE release from NGF-differentiated than from undifferentiated PC12 cells. NGF-differentiated PC12 cells had altered mRNA expression of several P2Y and P2X receptors but protein expression was only increased for P2X, in particular P2X(1-4,) receptors and P2X, but not P2Y, receptor inhibitors blunted the NGF-promoted enhancement in nucleotide-regulated catecholamine release. Surprisingly, siRNA directed against P2X(2), the receptor with the highest expression, failed to alter NE release by ATPgammaS. These findings indicate that sympathetic neuronal differentiation by NGF increases both the expression of P2X receptor sub-types and their regulation of catecholamine release. NGF-promoted increased expression of P2X receptors thus appears to be a physiologically important response that characterizes sympathetic neuronal differentiation.     

Regulatory mechanisms of nerve growth factor synthesis in vitro

Astroglial cells and various types of non-neuronal cells in the peripheral nervous system, such as epithelial, Schwann and fibroblast cells synthesize and secrete nerve growth factor (NGF) in culture. NGFmRNA contents are well-correlated with the density of axonal projection from NGF-sensitive neurons, suggesting that NGF synthesis in vivo tissues is regulated by neuronal environments. We investigated neuronal regulations of NGF synthesis using cultured mouse astroglial cells and rat pheochromocytoma PC12 cells. It was found that astroglial NGF synthesis was enhanced by the addition of catecholamine into the cultured medium or the co-culture with differentiated PC12 cells. These results suggest that NGF synthesis in the in vivo tissues is increased by the release of catecholamine as neurotransmitters and/or the contact of NGF-producing cells with differentiated cell bodies and neurites of NGF-sensitive neurons.     

Neurotoxicity of 25-OH-Cholesterol on NGF-Differentiated PC12 Cells

PC12 cells induced to differentiate with nerve growth factor were used to study the neurotoxicity of 25-OH-cholesterol. This agent induced a dose- and time-dependent cell death in neuronal PC12 cells. Cells treated with this agent showed condensed nuclei, a morphology similar to that of cells dying of programmed cell death. However, agents known to prevent neuronal programmed cell death (cyclic AMP, KCl, aurintricarboxylic acid, and cycloheximide) failed to prevent the 25-OH-cholesterol-mediated cytotoxicity. On the other hand, cell death induced by 25-OH-cholesterol was prevented by treatment with vitamin E and methyl-beta-cyclodextrin. In contrast to observations made in other cell types, whole-cell patch clamp recording of neuronal PC12 cells revealed that treatment with 25-OH-cholesterol did not significantly alter calcium influx through voltage-dependent channels. These results provide the first characterization of the toxicity of cholesterol oxides toward neuronal PC12 cells, which should be useful in future studies on the interactions between cholesterol oxides and cells from the nervous system.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Calmodulin- and protein phosphorylation-independent release of catecholamines from PC-12 cells

Catecholamine secretion from PC-12 cells can be triggered by agents that increase intracellular Ca2+ and is enhanced by phorbol esters and agents that elevate intracellular cAMP concentrations. In mutant PC-12 cells lacking cAMP-dependent protein kinase (PK-A) in which protein kinase C (PK-C) was down-regulated, Ca2+-dependent secretion occurred normally but was no longer enhanced by cAMP or phorbol esters. In digitonin-permeabilized PC-12 cells that lacked PK-C and PK-A, a range of calmodulin (CaM) inhibitors failed to block Ca2+-triggered catecholamine release. Moreover, Mn2+, a CaM activator, failed to trigger catecholamine release whereas Ba2+, which does not activate CaM, supported secretion. These results indicate that the basic mechanism of stimulus/secretion coupling in PC-12 cells does not absolutely require a regulated protein phosphorylation- or calmodulin-dependent step.     

Synergistic enhancement of histamine release from rat peritoneal mast cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate is not reflected by corresponding changes in phospholipid turnover

In an attempt to elucidate further the relationship between changes in phospholipid metabolism in, and histamine secretion from, purified rat peritoneal mast cells, the effects of the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) on these responses in stimulated and unstimulated cells was investigated. TPA caused a dose-dependent increase in the incorporation of 32PO4(3-) into the mast cell phospholipids; phosphatidic acid (PA) and phosphatidylcholine (PC), but not phosphatidylinositol (PI). TPA synergistically enhanced histamine release from cells stimulated by anti-immunoglobulin E (IgE) and the calcium ionophore A23187, reducing its ED50 from 150 nM to 40 nM, but did not alter histamine release from cells stimulated by compound 48/80. The effect of TPA on the changes in 32PO4(3-) incorporation into phospholipids associated with the above secretagogues did not, however, correlate well with the observed effects on histamine secretion induced by the same secretagogues. These observations are discussed in relation to the known effects of phorbol esters upon both secretory processes and phospholipid metabolism in other tissues.     

Inhibition of the RhoA/Rho kinase system attenuates catecholamine biosynthesis in PC 12 rat pheochromocytoma cells

The small GTPase, RhoA, and its downstream effecter Rho-kinase (ROK) are reported to be involved in various cellular functions, such as myosin light chain phosphorylation during smooth muscle contraction and exocytosis. Indeed, growing evidence suggests that the RhoA/Rho-kinase pathway plays an important role in regulating exocytosis in these cells. However, it is not known whether the RhoA/Rho-kinase pathway has an effect on catecholamine synthesis. Using the rat pheochromocytoma cell line, PC12, we examined the effects of either Rho-kinase inhibitor (Y27632) or RhoA inhibitor (C3 toxin) on nicotine-induced catecholamine biosynthesis. We show that nicotine (10 microM) induces a significant, though transient, increase in RhoA activation in these cells. Treatment with either Y27632 (1 microM) or C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH) mRNA and the corresponding enzyme activity. TH catalyzes the rate-limiting step in the biosynthesis of catecholamine. Y27632 significantly inhibited nicotine-induced phosphorylation of TH at Ser40 as well as Ser19, which are known to be phosphorylated by Ca(2+)/calmodulin kinase II. Furthermore, Y27632 (10 microM) as well as C3 toxin (10 microg/ml) significantly inhibited the nicotine-induced increase of TH at the protein level. Thus, we propose that activation of RhoA, and its downstream effecter Rho-kinase, is a prerequisite for catecholamine biosynthesis in PC12 cells. At the concentrations used in our experiments, Y27632 does not affect cAMP/PKA activity or PKC activity, indicating that the inhibitory effect of Y27632 can be attributed to the inhibition of Rho-kinase activity as observed in chromaffin cells. In contrast, neither Y27632 (10 microM) nor C3 toxin (10 microg/ml) significantly altered catecholamine secretion in PC12 cells. In conclusion, we have demonstrated that inhibition of the Rho/Rho-kinase pathway in chromaffin cells lowers TH activity, probably through CaMKII inhibition. By contrast, neither Y27632 nor C3 toxin affect the secretion of catecholamine.     

Fish scales as Biosensors for Catecholamines

Certain fish scales contain specialized cells (chromatophores) with pigment granulas, which can be dispersed or aggregated in the cells. The degree of aggregation is determined by a transmitter substance, nonadrenaline, released by the sympathetic nerve endings in the skin. Isolated scales from, for example, cuckoo wrasse (Labrus ossifagus) retain a large sensitivity to externally applied nonadrenaline (or more generally catecholamines) for several weeks. The degree of aggregation in isolated scales can be measured objectively by simple photometric techniques. We demonstrate in this paper how fish scales can be used to monitor catecholamine levels in human blood plasma. A discussion of other potential biosensor applications of this intact biological receptor-effector system is also given.     

Laminin-1 and the RKRLQVQLSIRT Laminin-1 α1 Globular Domain Peptide Stimulate Matrix Metalloproteinase Secretion by PC12 Cells

Here we have investigated the ability of laminin-1 and specific laminin-1-derived synthetic peptides to stimulate neuronal cell matrix metalloproteinase secretion. Zymographic analysis of conditioned media from laminin-1-treated PC12 and NG108-15 cells revealed a 72-kDa matrix metalloproteinase which was not secreted by untreated cells. Laminin-1 α1 chain-derived synthetic peptides, AASIKVAVSADR (LAM-L) and RKRLQVQLSIRT (AG-73), also stimulated PC12 cell secretion of a 72-kDa matrix metalloproteinase. We further investigated the structural requirements of AG-73 for cell attachment, neurite outgrowth, and matrix metalloproteinase secretion using a series of AG-73 analogs that had single amino acids substituted with alanine. At the substrate levels tested, the AG-73 peptide promoted the adhesion of 67% of the PC12 cells and neurite outgrowth in 71% of the PC12 cells. Substitutions in any one of the amino acids within the central LQVQ sequence resulted in a large reduction in cell attachment whereas substitution in the carboxyl terminal proximal amino acids L, S, and R had little effect on attachment. Alanine substitution of any of the amino terminal proximal LQV amino acids and the carboxyl terminal L, I, and R residues resulted in a 65–91% reduction in neurite outgrowth. These data demonstrate that the sequence requirements for cell attachment and neurite outgrowth were not necessarily coupled but that the sequence requirements for neurite outgrowth and matrix metalloproteinase secretion were identical. We conclude that laminin-1 is able to stimulate neuronal cells to secrete a matrix metalloproteinase. Further, this study identifies the LQVXLXIR laminin-1 α1 globular domain peptide to be capable of stimulating both neurite outgrowth and matrix metalloproteinase secretion.     

Synaptotagmin VII is targeted to secretory organelles in PC12 cells, where it functions as a high-affinity calcium sensor

Synaptotagmin (syt) I is thought to act as a Ca2+ sensor that regulates neuronal exocytosis. Fifteen additional isoforms of syt have been identified, but their functions are less well understood. Here, we used PC12 cells to test the idea that different isoforms of syt impart cells with distinct metal (i.e., Ca2+, Ba2+, and Sr2+) requirements for secretion. These cells express syt's I and IX (syt IX sometimes referred to as syt V), which have low apparent metal affinities, at much higher levels than syt VII, which we show has a relatively high apparent affinity for metals. We found that syt I and VII partially colocalize on large dense core vesicles and that upregulation of syt VII produces a concomitant increase in the divalent cation sensitivity of catecholamine release from PC12 cells. Furthermore, RNA interference-mediated knockdown of endogenous syt VII reduced the metal sensitivity of release. These data support the hypothesis that the complement of syt's expressed by a cell, in conjunction with their metal affinity, determines the divalent cation sensitivity of exocytosis.     

Regulation of SP-B and SP-C secretion in rat type II cells in primary culture

Secretion of lung surfactant phospholipids is a highly regulated process. A variety of physiological and pharmacological agents stimulate surfactant phospholipid secretion in isolated type II cells. Although the lipid and hydrophobic protein components of surfactant are believed to be secreted together by exocytosis of lamellar body contents, regulation of surfactant protein (SP) B and SP-C secretion has not previously been examined. To address the question of whether secretion of SP-B and SP-C is stimulated by the same agonists that stimulate phospholipid secretion, we measured secretion of all four SPs under the same conditions used to measure phosphatidylcholine secretion. Freshly isolated rat type II cells were cultured overnight and exposed to known surfactant phospholipid secretagogues for 2.5 h, after which the amounts of SP-A, SP-B, SP-C, and SP-D in the medium were measured with immunoblotting. Secretion of SP-B and SP-C was stimulated three- to fivefold by terbutaline, 5'-(N-ethylcarboxyamido)adenosine, ATP, 12-O-tetradecanoylphorbol 13-acetate, and ionomycin. Similar to their effects on phospholipid secretion, the stimulatory effects of the agonists were abolished by Ro 31-8220. Secretion of SP-A and SP-D was not stimulated by the secretagogues tested. We conclude that secretion of the phospholipid and hydrophobic protein components of surfactant is similarly regulated, whereas secretion of the hydrophilic proteins is regulated differently.