Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein.

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093- 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711- 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

Enhanced glycosylation and sulfation of secretory proteoglycans is coupled to the expression of a basic secretory protein.

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.

The purification and partial characterization of phospholipase A2, a secretory protein of rabbit parotid gland.

Phospholipase A2 (EC 3.1.1.4), a soluble enzyme present in secretion granule lysates and in the discharged secretion of rabbit parotid gland, has been purified by gel filtration. The enzyme preparations obtained from both lysates and secretion have been found to have identical amino acid compositions, amino terminal residues (Asx), isoelectric points (10.2) and electrophoretic behavior in polyacrylamide gels. The reduced and alkylated protein yields a single band upon electrophoresis in the presence of sodium dodecyl sulfate; the mobility corresponds to an apparent molecular weight of 16000. The enzyme is capable of action on phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol. Activity has been examined using as substrate sonicated vesicles consisting of PC and PE. Rates of hydrolysis have been determined by densitometry of lysophosphatides resolved and charred on thin-layer chromatograms. This approach has been used to follow enzyme purification, to indicate the preferential hydrolysis (approx. 2-fold) of PE vs. PC and to demonstrate that enzyme purified from granule lysates and discharged secretion shows the same heat stability and activity profile as a function of pH. A highly specific Ca2+ requirement for activity also has been identified for substances organized as phospholipid bilayers; the apparent inactivity of this enzyme within a Ca2+-containing storage organelle, the secretion granule, presents an interesting problem for future investigation.

Immunoglobulin-derived polypeptides enter the regulated secretory pathway in AtT-20 cells.

Constitutively secreted proteins have traditionally been believed to be excluded from the regulated secretory pathway. In this work we show that kappa light chain and Fc fragment, two markers of the constitutive pathway, are present in the regulated pathway in AtT-20 cells. They colocalize with the endogenous hormone ACTH and they exhibit stimulus-dependent secretion. The Fc fragment, which undergoes intracellular transport at the same rate as the ACTH precursor POMC, enters the forming secretory granules, however, it is partially lost during granule maturation. These observations show that classic constitutive secretory markers are not excluded from the regulated secretory pathway and that efficient sorting for regulated secretion occurs above a background of proteins which enter the granules without sorting.

A variable stoichiometry model for pH homeostasis in bacteria.

The composition of the proton-motive force of a hypothetical bacterial cell of wide pH tolerance is analyzed according to a model whereby the electron transport chain and various proton-linked sodium and potassium ion transporting modes are responsible for the development of the membrane potential and the chemical potentials of the three cations. Simultaneous use of two or more modes employing the same metal cation, but at a different stoichiometric ratio with respect to protons, produces nonintegral stoichiometry; the modes could represent either different devices or different states of a single device. Cycling of the cation, driven by proton-motive force, results. The relative conductances of the various modes are postulated to be pH-dependent. The pattern of potentials that results is qualitatively in accord with current knowledge and may reflect the mechanism of pH homeostasis in bacteria. The membrane potential is outwardly directed (positive inside) at extremely acid pH, becoming inwardly directed as the pH increases; the pH gradient across the membrane is large and inwardly directed (alkaline inside) at acid pH, becoming smaller and eventually inverting at alkaline pH values; the transmembrane potassium gradient is outwardly directed (high concentration inside) at all pH values; the transmembrane sodium gradient is inwardly directed at all pH values, following the pH gradient from acid through neutral pH, but then diverging at alkaline pH.

Sorting and secretion of salivary proteins.

Most salivary proteins are stored in secretion granules prior to export from acinar cells in response to neural stimuli. A small subset of these proteins undergo unstimulated secretion without apparent storage. This pathway probably comprises vesicles that bud from maturing storage granules and carries proteins that do not aggregate efficiently at the storage site. Expression of a parotid proline-rich protein (and deletion mutants) in pituitary AtT-20 cells has shown that an N-terminal domain is necessary for storage in secretion granules. Evidence suggests that self-aggregation of proline-rich protein mediated by this domain may function in both efficient intracellular transport and storage. Thus selective aggregation may be an important secretory sorting mechanism.

Novel secretory proline-rich proteoglycans from rat parotid. Cloning and characterization by expression in AtT-20 cells.

We have isolated two cDNAs that encode backbones of proline-rich proteoglycans identified previously in parotids of isoproterenol-treated rats (Blair, A. E., Castle, A. M., and Castle, J. D. (1991) Am. J. Physiol. 30, C897-C905). The sequences and domain structure of these cDNAs are characteristic of acidic proline-rich proteins, and potential glycosaminoglycan attachment sites are present within the acidic N-terminal domain. When the cDNAs were individually expressed in a mouse pituitary cell line, AtT-20, the expressed proteins were modified to sulfated proteoglycans. As in parotid acinar cells, only a fraction of the larger backbone (M(r) = 38,000) is modified by glycosaminoglycan addition; however, most of the smaller backbone (M(r) = 30,000) appears modified. Both proteoglycans expressed in AtT-20 cells contain heparan sulfate and chondroitin sulfate, whereas the proteoglycan in parotid contained chondroitin sulfate exclusively. Both proteoglycans are targeted to the regulated secretory pathway in AtT-20 cells where they are stored more efficiently than a parotid basic proline-rich protein.

Two regulated secretory pathways for newly synthesized parotid salivary proteins are distinguished by doses of secretagogues.

Low doses of the muscarinic agonist pilocarpine (0.1-1 microM) and the beta-adrenergic agonist isoproterenol (0.5-4 nM) stimulate a minor regulated secretory pathway for salivary proteins in rat parotid lobules. Newly synthesized proteins (labeled biosynthetically) are selectively discharged, and they are secreted in the same relative proportions as observed in constitutive-like unstimulated secretion but different from the proportions of older proteins that are discharged by granule exocytosis in response to higher doses of secretagogue. The response to low doses of agonists is transient and involves output of no more than 1-2% of tissue-associated amylase. The same increase in output of pulse-labeled proteins is observed when agonist is added at various chase times (1.5-6 hours), implying that release occurs from a post-Golgi storage pool. Stimulation for 40 minutes significantly depletes the storage pool as a second stimulation elicits smaller output. Stimulation also partially depletes labeled proteins from subsequent constitutive-like secretion after the agonist is removed implying that the constitutive-like and low dose agonist mediated pathways draw on the same pool of secretory proteins. While these results indicate that acinar cells have a second regulated secretory pathway, this new pathway is unlikely to contribute uniquely to the protein composition of parotid secretion. Rather it may serve a different role in secretion at the apical cell surface.

Ammonium chloride alters secretory protein sorting within the maturing exocrine storage compartment.

Addition of 20 mM ammonium chloride during in vitro chase incubation of [35S]methionine pulse-labeled parotid tissue does not perturb the magnitude or radiochemical composition of secretion stimulated by isoproterenol. An apparent inhibition of stimulated output of radiolabeled secretory proteins that was observed when ammonium chloride was added immediately postpulse (but not at later time points prior to stimulation) could be accounted for by slowdown in Golgi transit of exocrine secretory protein at a stage prior to completion of terminal glycosylation. Thus, ammonium chloride does not block entry of newly synthesized secretory proteins into the secretagogue-releasable storage granule compartment. By contrast, ammonium chloride increases the output and substantially alters the relative composition of newly synthesized protein in unstimulated secretion. The latter effects could be assigned to stages of intracellular transport that normally occur at chase times greater than 60 min postpulse and thus are focused within the maturing acinar storage granule. Notably, the compositional alterations cannot reflect the preferential exocytosis of immature granules. Taken together, these results suggest that the sorting of exocrine secretory proteins into the secretagogue-regulated pathway may not involve positive selection by a pH-based process initiated in a pregranule compartment. Rather, unstimulated secretion may arise by a negative sorting (or exclusion) process that occurs during compaction of proteins for storage within maturing granules and that is perturbed by weak base addition. Sorted (or excluded) proteins would appear to follow the vesicular (nongranular) secretory pathway that originates in maturing granules (von Zastrow, M., and Castle, J.D. (1987) J. Cell Biol. 105, 2675-2684).

Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance.

The relationship between the steady-state sodium gradient (delta pNa) and the protonmotive force developed by endogenously respiring Escherichia coli cells has been studied quantitatively, using 23Na NMR for measurement of intracellular and extracellular sodium concentrations, 31P NMR for measurement of intracellular and extracellular pH, and tetraphenylphosphonium distribution for measurement of membrane potential. At constant protonmotive force, the sodium concentration gradient was independent of extracellular concentrations over the measured range of 4-285 mM, indicating that intracellular sodium concentration is not regulated. The magnitude of delta pNa was measured as a function of the composition and magnitude of the protonmotive force. At external pH values below 7.2, delta pNa was parallel to delta pH but showed no simple relationship to the membrane potential; above pH 7.2 the parallel relationship began to diverge, with delta pH continuing to decrease but delta pNa starting to level off or increase. Although plots of delta pNa versus delta pH had slopes of close to 1, the value of delta pNa consistently exceeded that of delta pH by approximately 0.4 units, indicating a partially electrogenic character to the putative H+/Na+ antiport. The apparent stoichiometry was 1.13 +/- 0.01 at external pH below 7.2. The possible significance of this nonintegral stoichiometry is discussed according to a model in which two distinct integral stoichiometries (possibly 1H+/1Na+ and 2H+/1Na+) are available with some relative probability; the model predicts futile cycling of sodium ions and a dissipative proton current. In the course of this study, we discovered that the magnitude of the pH gradient developed by the cells was osmolarity-dependent, yielding steady-state intracellular pH values that varied from 7.1 at 100 mosm to 7.7 at 800 mosm.

Measurement of intracellular sodium concentration and sodium transport in Escherichia coli by 23Na nuclear magnetic resonance.

Escherichia coli is known to actively extrude sodium ions, but little is known concerning the concentration gradient it can develop. We report here simultaneous measurements, by 23Na NMR, of intracellular and extracellular Na+ concentrations of E. coli cells before and after energization. 23Na spectra in the presence of a paramagnetic shift reagent (dysprosium tripolyphosphate) consisted of two resonances, an unshifted one corresponding to intracellular Na+ and a shifted one corresponding to Na+ in the extracellular medium, including the periplasm. Extracellular Na+ was found to be completely visible despite the presence of a broad component in its resonance; intracellular Na+ was only 45% visible. Measurements of Na+ were made under aerobic and glycolytic conditions. Na+ extrusion and maintenance of a stable low intracellular Na+ concentration were found to correlate with the development and maintenance of proton motive force, a result that is consistent with proton-driven Na+/H+ exchange as a means of Na+ transport. In both respiring and glycolyzing cells, at an extracellular Na+ concentration of 100 mM, the intracellular Na+ concentration observed (4 mM) corresponded to an inwardly directed Na+ gradient with a concentration ratio of about 25. The kinetics of Na+ transport suggest that rapid extrusion of Na+ against its electrochemical gradient may be regulated by proton motive force or intracellular pH.

A 13-amino acid n-terminal domain of a basic proline-rich protein is necessary for storage in secretory granules and facilitates exit from the endoplasmic reticulum.

We have investigated the role of different domains of a salivary basic proline-rich protein in intracellular transport and sorting of proline-rich proteins to the secretory granules. We have cloned a full-length cDNA of a basic proline-rich protein from the rat parotid and expressed it in AtT-20 cells. It was correctly sorted into secretory granules as shown by EM immunolocalization and by its presence in 8-bromocyclic AMP-stimulated secretion. Deletion of the N-terminal thirteen amino acid domain upstream from the proline-rich domain eliminated storage whereas deletion of the C-terminal 20-amino acid domain downstream from the proline-rich domain had no effect. Intracellular transport of full-length and mutant proline-rich proteins was unusually slow due to slow exit from the endoplasmic reticulum. However, the rate of transport increased with increasing level of expression for the full-length protein and the C-terminal deletion mutant. In contrast, the rate of transport of the N-terminal deletion mutant was independent of the level of expression. These results imply that the N-terminal domain is necessary for both storage and efficient intracellular transport. Moreover, interactions (self-aggregation?) that mediate sorting may begin as early as the endoplasmic reticulum.

Proteoglycan sulfation and storage parallels storage of basic secretory proteins in exocrine cells.

Increased storage of basic proline-rich secretory proteins induced in rat parotid acinar cells by isoproterenol is accompanied by increased storage of a chondroitin sulfate-containing proteoglycan. Amino acid analysis of the purified proteoglycan and the chondroitinase digestion products reveals that the polypeptide backbone is a proline-rich protein. Most sulfation occurs in Golgi elements; however, a small fraction of the proteoglycan can be labeled by incubating isolated secretion granules with [35S]phosphoadenosine phosphosulfate ([35S]PAPS), and the amount of sulfate incorporation decreases with increased granule maturity. In vitro incorporation is sensitive to inhibitors of PAPS transport and occurs in intact granules as shown by radioautography. Both the increased production of a chondroitin sulfate proteoglycan following isoproterenol treatment and its sulfation at sites of secretory condensation and storage suggest that sulfation may aid secretory packaging by reducing the known fixed positive charge that stems from the large concentration of basic secretory proteins.

Characterization of common salivary protein 1, a product of rat submandibular, sublingual, and parotid glands.

We have isolated and characterized cDNA clones derived from a developmentally regulated neonatal rat submandibular gland salivary protein gene called "common salivary protein 1" (CSP1). Identical clones were also identified in cDNA libraries from adult male parotid, submandibular, and sublingual glands. CSP1 transcripts are at least 10-fold more abundant in the sublingual gland than in the submandibular or parotid glands. In situ hybridization and immunocytochemical localization demonstrated the presence of CSP1 transcripts and proteins in sublingual gland serous demilune cells, parotid and submandibular gland intercalated duct cells, and in the type III (proacinar) cells of the neonatal submandibular gland. This cell-type distribution is similar to that described by Ball and colleagues (Ball, W. D., Hand, A. R., and Johnson, A. O. (1988) Dev. Biol. 125, 265-279) for the developmentally regulated submandibular gland B1-immunoreactive proteins. Immunoblotting of salivary secretion identified proteins of M(r) 20,000 in sublingual, 16,000 in submandibular and 22,000 and 16,000 in parotid gland. The M(r) 20,000 sublingual and 22,000 parotid proteins represent N-glycosylated forms of a M(r) 16,000 apoprotein, suggesting that these salivary proteins arise by post-translational modification of a common precursor.

Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells.

Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types.

The unique proline-rich domain of parotid proline-rich proteins functions in secretory sorting.

When expressed in pituitary AtT-20 cells, parotid proline-rich proteins enter the regulated pathway. Because the short N-terminal domain of a basic proline-rich protein is necessary for efficient export from the ER, it has not been possible to evaluate the role of this polypeptide segment as a sorting signal for regulated secretion. We now show that addition of the six-amino acid propeptide of proparathyroid hormone to the proline-rich protein, and especially to a deletion mutant lacking the N-terminal domain, dramatically accelerates intracellular transport of these polypeptides. Under these conditions the chimeric deletion mutant is stored as effectively as the full-length protein in dense core granules. The propeptide does not function as a sorting signal in AtT-20 cells as it does not reroute a constitutively secreted reporter protein to the regulated pathway. During transit, the propeptide is cleaved from the chimeric polypeptides such that the original structures of the full-length and the deletion mutant proline-rich proteins are reestablished. We have also found that the percentage stimulated secretion of the proline-rich proteins increases incrementally (almost twofold) as their level of expression is elevated. The increase reflects an enrichment of these polypeptides in the granule pool and its incremental nature suggests that sorting of proline-rich proteins involves an aggregation-based process. Because we can now rule out contributions to sorting by both N- and C-terminal segments of the proline-rich protein, we deduce that the unique proline-rich domain is responsible for storage. Thus at least some of the determinants of sorting for regulated secretion are protein-specific rather than universal.

Differential targeting of recombinant fibronectins in AtT-20 cells based on their efficiency of aggregation.

In pituitary-derived AtT-20 cells, recombinant fibronectin containing the N-terminal matrix assembly domain and the C-terminal half of fibronectin does not follow the regulated secretory pathway but instead concentrates in distinct organelles prior to secretion. These organelles are larger than the dense-core granules and localize to the cell body at sites that differ from lysosomes, endosomes and endoplasmic reticulum. Unlike the dense-core granules, their discharge is not stimulated by 8-bromo-cyclic-AMP or phorbol esters. The kinetics of intracellular transport and secretion of the recombinant fibronectin suggest that it is present in a post-Golgi pool that turns over more slowly than constitutive vesicles. Indeed, the fibronectin-containing organelles disappear with a half-time of 3 hours after inhibiting protein synthesis. Presence of the organelles correlates with intracellular aggregation of dimeric fibronectin polypeptides. The organelles are absent in cells expressing monomeric recombinant fibronectin (lacking C-terminal dimerization sites) or the C-terminal half of fibronectin (which dimerizes but lacks the N-terminal matrix assembly domain), both of which aggregate less efficiently than dimeric fibronectin. Instead, the latter polypeptides enter the dense-core granules. Thus while the formation of the fibronectin-containing organelles may require efficient aggregation, it may not require a specific structural signal. Moreover, efficient aggregation is not necessarily a prerequisite for following the regulated pathway.

Effects of pH and repellent tactic stimuli on protein methylation levels in Escherichia coli.

Intracellular pH (pH(int)) and extracellular pH (pH(ext)) of Escherichia coli were measured at 12-s time resolution by (31)P-nuclear magnetic resonance: a sudden neutral-to-acid shift in pH(ext) (e.g., from 7.0 to 5.6) caused a transient failure of homeostasis, with pH(int) decreasing by about 0.4 unit in ca. 30 s and then returning to its original value (ca. 7.5) over a period of several minutes. Membrane proton conductance was estimated to be 20 pmol s(-1) cm(-2) pH unit(-1). Addition of the membrane-permeant weak acid benzoate at constant pH(ext) also caused a lowering of pH(int); at high concentrations it generated an inverted transmembrane pH gradient (DeltapH). The buffering capacity of the cells was estimated by such experiments to be ca. 50 mM per pH unit. Effects of pH-related stimuli on the methyl-accepting chemotaxis proteins (MCPs) were examined: the steady-state methylation of MCP I was found to decrease when pH(int) was lowered by weak acid addition or when pH(ext) was lowered. The extent of demethylation in the latter case was too great to be explained by imperfect steady-state homeostasis; a small but reproducible undershoot in methylation level correlated with the observed short-term homeostatic failure. MCP II underwent smaller and more complex changes than MCP I, in response to pH-related stimuli. The methylation level of MCP I could not, by any condition tested, be driven below a limit of ca. 15% of the control level (unstimulated cells at pH(ext) 7.0). The weak-acid concentration needed to reach that limit was dependent on pH(ext), as would be expected on the basis of DeltapH-driven concentrative effects. The potency ranking of weak acids was the same with respect to lowering pH(int), demethylating MCP I, and causing repellent behavioral responses. The data are consistent with a model whereby MCP I and hence tactic behavior are sensitive to both pH(int) and pH(ext). Evidence is presented that pH(int) may also have a direct (non-MCP-related) effect on motor function. Comparison of methyl-(3)H- and (35)S-labeled MCP I revealed that in both unstimulated and repellent-stimulated cells the major species did not carry methyl label, yet it had an electrophoretic mobility that indicated that it was more positively charged than the unmethylated form observed in methyltransferase mutants, and it was susceptible to base hydrolysis. This suggests that a substantial fraction of MCP I molecules is methylated or otherwise modified but neither exchanges methyl label nor undergoes reverse modification by repellent stimuli.

An enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells.

Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24-25 degrees C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17-37 degrees C. Both radioactive linoleic and palmitic acids were incorporated selectively at the beta-position of the glycerol backbone of PC. PC labeling by [3H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced beta-acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A2 that favors PC as a substrate. Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [3H]palmitate-labeled PC. Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly.